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International Buffalo Information Center (IBIC)

Buffalo Bulletin

ISSN: 0125-6726 (Print), 2539-5696 (Online)

Aims

IBIC is a specialized information center on water buffalo. Established in 1981 by Kasetsart University

(Thailand) with an initial financial support from the International Development Research Center (IDRC) of

Canada. IBIC aims at being the buffalo information center of buffalo research community throughout the world.

Main Objectives

1. To be world source on buffalo information.

2. To provide literature search and photocopy services.

3. To disseminate information in newsletter.

4. To publish occasional publications such as an inventory of ongoing research projects.

Buffalo Bulletin is published quarterly in January-March, April-June, July-September and October-

December. Contributions on any aspect of research or development, progress reports of projects and news on

buffalo will be considered for publication in the bulletin. Manuscripts must be written in English and follow the

instruction for authors which describe at inside of the back cover.

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International Buffalo Information Center, Office of the University Library, Kasetsart University

Online available

http://ibic.lib.ku.ac.th/e-Bulletin

Advisory Board

Prof. Dr. Charan Chantalakhana Thailand

Prof. Dr. John Lindsay Falvey Faculty of Veterinary and Agricultural Science, University

of Melbourne, Australia

Prof. Dr. Metha Wanapat Department of Animal Science, Faculty of Agriculture,

Khon Kaen University, Thailand

Mr. Antonio Borghese International Buffalo Federation, Italy

Dr. Aree Thunkijjanukij International Buffalo Information Center, Office of the

University Library, Kasetsart University, Thailand

Miss Supanee Hongthong International Buffalo Information Center, Office of the


Editorial Member

Dr. Pakapan Skunmun Thailand

Dr. Kalaya Bunyanuwat Department of Livestock Development, Thailand

Prof. Dr. Federico Infascelli Department of Veterinary Medicine and Animal Science,

University of Naples Federico II, Italy

Dr. Rafat Al Jassim School of Agriculture and Food Sciences, Faculty of Science,

The University of Queensland, Australia

Prof. Dr. Nguyen Van Thu Department of Animal Sciences, Faculty of Agriculture and

Applied Biology, Can Tho University, Vietnam

Prof. K. Sarjan Rao Department of Livestock Production and Management,

College of Veterinary Science, India

Prof. Dr. Masroor Ellahi Babar Virtual University of Pakistan, Pakistan

Asst. Prof. Dr. Asif Nadeem Institute of Biochemistry and Biotechnology, University of

Veterinary and Animal Sciences, Pakistan

Prof. Dr. Raul Franzolin Departamento de Zootecnia, Universidade de São Paulo, Brazil

Editor

Dr. Sunpetch Sophon Thailand

Journal Manager

Mr. Chalermdej Taterian International Buffalo Information Center, Office of the


Assistant Journal Manager

Miss Kanchana Anuphan International Buffalo Information Center, Office of the


Miss Jirawadee Wiratto International Buffalo Information Center, Office of the


Buffalo Bulletin

IBIC, Kasetsart University,

P.O. BOX 1084, Bangkok 10903, Thailand

E-mail: [email protected]

Tel: 66-2-9428616 ext. 344

Fax: 66-2-9406688

Buffalo Bulletin (January-March 2017) Vol.36 No.1

CONTENTS Page Review Article

Cryopreservation of buffalo (Bubalus bubalis) semen-limitations and expectations Dawar Hameed Mughal, Ahmad Ijaz, Muhammad Shahbaz Yousaf, Fazal Wadood and Umer Farooq..........................................................................................................1

Strouble shooting off-flavor (bad odor) and bad taste in milk Ghulam Muhammad, Imaad Rashid, Sehrish Firyal and Muhammad Saqib.................................15

Original Article

Mimosa tenuiflora extract reduces the number of Staphylococcus aureus with low toxicity in Bubalus bubalis with mastitis Andréia Vieira Pereira, Marcelo Biondaro Gois, Fabiana Nabarro Ferraz, Jozinete Vieira Pereira, Sérgio Santos Azevedo, Ednaldo Queiroga de Lima, Onaldo Guedes Rodrigues, Elizabeth Sampaio de Medeiros, Rinaldo Aparecido Mota and Maria do Socorro Vieira Pereira.....................................................21

Effect of two management systems and mineral feeding on age at puberty in Nili-Ravi buffalo heifers Muhammad Tariq Bodla, Muhammad Anwar, Ejaz Ahmad, Zahid Naseer and Umair Ahsan.......................................................................................................................................27

Biochemical profile and methane emission during controlled thermal stress in buffaloes (Bubalus bubalis) Alok K. Wankar, Gyanendra Singh and Brijesh Yadav........................................................................35

Effects of microcystins toxins contaminated drinking water on hepatic problems in animals (cows and buffalos) and toxins removal chemical method M. Badar, Fatima Batool, Safder Shah Khan, Irshad Khokhar, M.K. Qamar and Ch. Yasir......................................................................................................................43

Haemato-biochemical response to lumbar epidural anaesthesia using xylazine, ketamine alone and its combination in buffalo calves P.K. Pandey, S.K. Tiwari, Deepak Kumar Kashyap, Govina Dewangan and Devesh Kumar Giri...........................................................................................................................57


CONTENTS Page Original Article Effect of pre and post-partum supplementation to buffaloes on body condition, lactation and reproductive performance B.K. Ojha, Narayan Dutta, S.K. Singh, A.K. Pattanaik and Amit Narang......................................63

Study of buffalo husbandry practices in rural area of central Gujarat in India B.S. Khadda, Kanak Lata, Brijesh Singh and Raj Kumar...................................................................75

Return to fertility in postpartum Mehsana buffaloes after therapeutic approach for large ovarian cysts and inactive ovaries using a short-term progestin-based regime under higland field conditions in Thailand T. Moonmanee, S. Tangtaweewipat, J. Jitjumnong and P. Yama.......................................................89

Study the responses of progesterone administration on resumption of cyclicity on post-partum anestrus buffaloes Deepak Suvarn, C. Singh and M.M. Ansari...........................................................................................97

Testicular biometry and its correlation with body weight and semen output in Murrah bull Sanjeet Kumar and Sushant Srivastava..................................................................................................105

Effect of nongenetic factors on semen production charecteristics of Murrah buffalo bulls at organized semen station Pushp Raj Shivahre, A.K. Gupta, A. Panmei, A.K. Chakravarty, M. Bhakat, S.K. Dash, S.K. Sahoo, V. Kumar and M. Singh.................................................................................115

Comparative evaluation of functional activity of neutrophil in high and low yielding Murrah buffaloes during peripartum period M.M. Pathan, M. Kaur, A.K. Mohanty and A.K. Dang.......................................................................123

Screening for genetic disorders in Indian Murrah and Surti buffalo (Bubalus bubalis) bulls K.P. Ramesha, Akhila Rao, Rani Alex, G.R. Geetha, M. Basavaraju, M.A. Kataktalware, D.N. Das and S. Jeyakumar................................................................................133


CONTENTS Page Original Article Incidence of nasal schistosomiasis in graded Murrah buffaloes Hareesh Didugu and Ch. E. Narasimha Reddy....................................................................................143

Assessment of diagnostic efficacy of various methods in detection of Trypanosoma evansi infection in buffaloes A.P. Singh, A.K. Tripathi, Ajit Singh, A. Srivastava and Rakesh Singh...........................................147

Prenatal development of buffalo major salivary glands: Gross morphological and biometrical studies A.D. Singh and Opinder Singh.................................................................................................................155

Body condition scoring by visual and digital methods and its correlation with ultrasonographic back fat thickness in transition buffaloes Randhir Singh, Sarnarinder Singh Randhawa and Charanjit Singh Randhawa...........................169

Karyotype of Mazani water buffalo from Iran M. Pournourali, A. Tarang and F. Mashayekhi....................................................................................183

Economics of rumen bypass fat feeding on cost of milk production, feeding and realizable receipts in lactating Jaffrabadi buffaloes H.H. Savsani, K.S. Murthy, P.U. Gajbhiye, P.H. Vataliya, K.S. Dutta, M.R Gadariya and A.R. Bhadaniya.................................................................................193

Concurrent sarcoptic and psoroptic mange complicated with Staphylococcus aureus in a Murrah buffalo (Bubalus bubalis) R.L. Rakesh, K. Mahendran, K. Karthik, Priyanka, A.G. Bhanuprakash and V.K. Gupta..........................................................................................................................................199

Analysis of constraints faced by beneficiaries of integrated Murrah development scheme (IMDS) in Haryana Y.S. Jadoun, S.K. Jha, Pragya Bhadauria and Rajiv Baliram Kale.................................................207


CONTENTS Page Original Article

Seasonal variation in the characteristics of the swamp buffalo semen of northeast India G.C.Das, P.K.Das, S. Deori, H. Mazumdar, B.N. Bhattacharyya and Arundhati Phookan...........................................................................................................................215

Cryopreservation induces capacitation-like changes of the swamp buffalo spermatozoa D.J. Talukdar, K. Ahmed, S. Sinha, S. Deori, G.C. Das and Papori Talukdar.................................................................................................................................221

A response of in vitro, in sacco and in vivo digestibility and rumen parameters of swamp buffaloes supplemented Sesbania grandiflora leaves Nguyen Van Thu and Nguyen Thi Kim Dong.........................................................................................231

Varicosity and pregnancy induced blood flow changes in the cranial tibial vein in buffaloes: B-mode and doppler ultrasound study Vandana Sangwan, Jitender Mohindroo, Ashwani Kumar and Shashi Kant Mahajan.......................................................................................................................239

Effect of different antibiotic combinations in extender on bacterial load and seminal characteristics of Murrah bulls G.S. Meena, M. Bhakat, V.S. Raina, A.K. Gupta, T.K. Mohanty and R. Bishist...........................251


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ABSTRACT

Artificial insemination with cryopreserved semen is the most viable biotechnology for faster and increased genetic improvement in many species allowing for improved herd performance and productivity. Pakistan has 34.6 million buffalo which have major share in total milk produced in the country. Rapid increase in human population and demand for animal products motivated the researchers to increase per animal milk production using this biotechnology. In buffalo, natural breeding practice is common in the country compared to artificial insemination, low fertility rate with cryopreserved semen is main hindrance in its propagation. It is need of the hour to disseminate the knowledge of various factors and components contributing in buffalo semen cryopreservation to improve milk and fertility rate of this breed.

Keywords: buffalo, cryopreservation, extender, processing, semen

INTRODUCTION

The genetic improvement and disease

control in livestock have primary importance in the success of agri-food industry. In this contribution, artificial insemination (AI) is possibly the most decisive tool for the progression of modern animal production. By means of AI, each ejaculate collected from genetically superior male is used to inseminate females at a large scale and also controls sexually transmitted diseases. The development of cryopreservation protocols in dairy industry began in 1950s. Bratton et al. (1955) demonstrated that bovine sperm frozen to -79oC and packed on dry ice could still yield high fertility. Since then, cattle and buffalo industry have seen progressive improvement in tropical developing countries including Pakistan and AI gained wide acceptance in the development of the livestock resources (Cheema and Samad, 1986). The development and application of AI in the last four to five decades especially in cattle has been striking. Extensive research has been stimulated by the rapid expansion of this practice (Andrabi et al., 2001; Anzar et al., 2003). Semen cryopreservation is a complex process which involves many steps: extension, cooling, freezing, storage and thawing. During each step sperm structure and function are affected (Bailey et al., 2003) resulting in reduced sperm

CRYOPRESERVATION OF BUFFALO (BUBALUS BUBALIS) SEMEN-LIMITATIONS AND EXPECTATIONS

Dawar Hameed Mughal1,*, Ahmad Ijaz2, Muhammad Shahbaz Yousaf2, Fazal Wadood3 and Umer Farooq4

Review Article

1Directorate of Quality Enhancement Cell, University of Veterinary and Animal Sciences, Lahore, Pakistan, *E-mail: [email protected] of Physiology, University of Veterinary and Animal Sciences, Lahore, Pakistan3Department of Theriogenology, University of Veterinary and Animal Sciences, Lahore, Pakistan.4University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan


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motility (Tuli et al., 1981), acrosomal damage and alteration in sperm membrane integrity (Rasul et al., 2001).A thorough knowledge of each step is vital in order to attain a maximum conception rate. Successful cryopreservation varies highly among species, individuals within species and even within ejaculates of individuals, which is largely attributed to the differences in biophysical characteristics among cell types (Thurston et al., 2001; Waterhouse et al., 2006). In general, the plasma membrane is considered to be the primary site of cryo-injury and the principal damage occurs during freezing and thawing (Parks and Graham, 1992) resulting in substantial loss of viable spermatozoa. This vulnerability has been documented to be higher for buffalo bull semen owing to a higher oxidative stress though higher lipid oxidation rate, reduced activity of naturally present antioxidant enzymes, higher membrane contents of unsaturated fatty acids and lower osmotic pressure of buffalo semen (Raizada et al., 1990; Khan and Ijaz, 2008). Comparatively, AI in buffalo with cryopreserved semen has been limited due to poor semen freezability (Kumaresan et al., 2006; Andrabi et al., 2008) and conception rate (Chohan et al., 1992; Bhosrekar et al., 2001). The purpose of this article is to point out various factors affecting the efficacy and fertility using cryopreserved semen.

EXTENDER COMPONENTS

Buffer A buffer solution resists changes in pH when small quantity of an acid or an alkali is added to it. In various species considerable work has already been done on this aspect for extender preparation. The composition of the extender is decided on the basis of duration and storage

temperature of the semen and need a suitable buffer for this purpose (Rasul et al., 2000). Ideally, a buffer should possess following characteristics (i) Maximum water solubility (ii) pH between 6 to 8, favorably 7 (iii) Minimum salt/temperature effects/buffer concentration (iv) Well behaved cations interaction(v) Better ionic strengths and chemical stability (Andrabi, 2009). Efforts are being made to develop a most suitable buffering system for buffalo bull semen cryopreservation which should have composition close to natural medium and help in maintaining fertility of the frozen semen. Starting from the use of various organic buffers for the cryopreservation of the bull semen (Foote, 1970), chemically defined buffers gotattention. Matharoo and Singh (1980) found a least loss of post-thawed motility with Tris-based extender. However, Chinnaiya and Ganguli (1980) reported that spermatozoa cryopreserved in citrate, citric acidor Tris-based extender had similar acrosomal damage, while Dhami and Kodagali (1990) reported improved freezability using Tris-based extender. Similarly, in buffalo bull spermatozoa, Singh et al. (1991) reported least release of sorbitol dehydrogenase and lactic dehydrogenase during cryopreservation using Tris-based extender followed by citrate and citric acid extenders. Singh et al. (2000) reported better results with Tris-based buffer as compared to Laiciphos and Biociphos. While, Rasul et al. (2000) also reported improved motility rate using Tris citrate as compared to tri-sodium citrate, Tris-Tes or Tris-Hepes. On the other hand, Oba et al. (1994) and Chachur et al. (1997) reported similar effects on motility rate, acrosomal and plasma membrane integrity using Tesor Tris-based extender. All these studies clearly document the use of zwitterions buffers as a better option for bubaline


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semen extension and Tris-citric acid seems to be the most appropriate buffering system for the freezability of buffalo bull spermatozoa. The pH of zwitterions buffers is closer to the pKa (acid dissociation constant) and it is least affected by temperature (Graham et al., 1972). As compared to cattle spermatozoa, the prone inclination of buffalo spermatozoa towards freezing stress makes it a dire need of time to study the effects of buffers on spermatozoa pre and post-cryogenic membrane stability at molecular and biochemical levels.

Antibiotics The viability and fertility potential of cryopreserved spermatozoa is highly affected by presence of bacteria (Thibier and Guerin, 2000) either directly or indirectly. Hence, control of these bacteria through the use of various antibiotics in the extenders becomes one of the vital measures to attain in maxima results of AI. Benzyl penicillin alone or in combination with streptomycin sulphate is generally added in semen extenders of buffalo bulls (Akhter et al., 2008). However, these antibiotics have not been effective in controlling bacteriospermia of buffalo semen (Aleem et al., 1990). Ahmed and Greesh (2001) reported norfloxacin (200 µg / ml) or gentamicin or amikacin (500 µg / ml) as drug of choice for efficient preservation of buffalo bull semen. In the recent years, a new combination of gentamicin tylosin and lincospectin, GTLS has shown maximum capacity to control bacteria present in buffalo bull semen (Hasan et al., 2001; Akhtar et al., 2008). The testing of a wider range of antibiotics alone and in combinations is recommended to enhance the quality of cryopreserved buffalo bull semen.

Cryoprotectants In order to protect the spermatozoa from

the cryoinjuries, various cryoprotectants are added in the extenders (Purdy, 2006). On a broad based level, these cryoprotectants are classified into two categories (permeable and non permeable). Permeable cryoprotectants include glycerol, methanol, butanediol, 1,2-proparediol, ethylene glycol, dimethylsulfoxide (DMSO) and propylene glycol. These cryoprotectants have ability to pass through the sperm plasma membrane and act intra-cellularly and extra-cellularly, rearrange the membrane proteins, reduce formation of intracellular ice and thus protect the sperm from damage (Holt, 2000). Most penetrating cryoprotectants serve as both solvent and a solute. Non permeable cryoprotectantsinclude egg yolk, amino acids, trehalose, dextran, sucrose, xylose, fructose, lactose. mannose, raffinose, synthetic polymers such polyvinyl pyrollidone (PVP), amides and skimmed milk (without fat) which do not penetrate the sperm membrane and act outside the sperm (Aisen et al., 2000). Cryoprotectants have a property to lower the freezing temperature and ultimately reduce extracellular ice formation (Kundu et al., 2002). Glycerol (6 to 7%) is commonly used as cryoprotectant for buffalo bull semen. However, addition of glycerol at 2 to 3% or more than 7% reduced post-thaw sperm motility (Ramakrishan and Ariff, 1994; Nastri et al., 1994). Similarly, Fahy, (1986) reported 2.25 to 9% glycerol to be safe. Rasul et al. (2007) observed synergistic outcome of dimethyl sulfoxide (DMSO) and glycerol on the post-thaw buffalo sperm quality in terms of motion characteristics, plasma membrane integrity and acrosome morphology using tris citric acid extender differing in glycerol and DMSO concentrations. Glycerol is beneficial for the sperm as its freezing point is much lower than water. Hence, 5 to 7% concentration of glycerol in the


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semen extender may be suitable for buffalo bull semen cryopreservation. Development of less toxic and more efficient cryoprotectant will further make an ample contribution in improving buffalo bull semen characteristics. Glycerol and egg yolk are used in combination as cryoprotectants in cryopreservation. Proteins and lecithin are found in egg yolk which maintain and preserve the lipoprotein sheath of spermatozoa (Kumar et al., 1992). Egg yolk possess ability to stimulate enzymes found in the spermatozoa which cause deamination of amino acids causing damage to spermatozoa during storage period (Sahni and Mohan, 1990). To avoid production of hydrogen peroxide, egg yolk must be dialyzed before adding it to the extender. In past, limited attention was levied upon the concentration of egg yolk for freezing buffalo bull semen. Most of the researchers have used 20% concentration (Sansone et al., 2000; Andrabi et al., 2008) while Sahni and Mohan (1990) reported that the egg yolk concentration could be reduced to 5% in tris glycerol medium without affecting the post-thaw sperm motility. Andrabi et al. (2008) reported improved freezability of buffalo bull spermatozoa by adding duck egg yolk in extender as compared to other avian egg yolks. Cheshmedjieva et al. (1996) studied the effect of addition of polyethylene glycol (PEG 20) to egg yolk based freezing medium and concluded that addition of PEG 20 to semen extenders preserves the lipids of frozen buffalo bull spermatozoa. Further studies on PEG20 in future may find it a better choice for semen cryopreservation. Sugars like xylose, galactose, raffinose, fructose, maltose, glucose and sucrose are non permeable cryoprotectants, these are also added in semen extender (Nagase et al., 1964). Sugars and polyols have the ability to replace water

molecules with normal hydrated polar groups and hence stabilize the spermatozoa membrane by protecting the sperm from damage during cryopreservation (Woelders et al., 1997). As sugars have high molecular weights, which changes the cell membrane permeability and maintains the electrolyte balance during cryopreservation. The newer international trends in disease control consider the ingredients of animal origin (egg yolk) to be a source of contamination to the semen (Bousseau et al., 1998), hence emerges the need of using non-animal sources.

Other additives Attempts have been made to ameliorate the freezability of buffalo bull semen by adding different substance like antioxidants, metabolic stimulants, detergents and chelating agents. Bhosrekar et al. (1990) added tri ethanolamine laurel sulphate in extender having Tris-citric acid base and reported to improve post thaw spermatozoa motility. Detergents are believed to exert directly their protective effect on the membrane of the spermatozoa, or by emulsifying the egg yolk lipids which become readily available to spermatozoa membrane during cryopreservation (Arriola and Foote, 1987; Buhr and Pettitt, 1996). The addition of cysteine or ethylenediamine tetraacetic acid (EDTA) at 0.1% in semen diluents during buffalo bull semen freezing did not improve semen quality in terms of release of lactate dehydrogenize (Dhami and Sahni 1993). Singh et al. (1996) reported that addition of 2.5 mM ascorbic acid in buffalo bull semen diluents significantly improve post-thaw spermatozoa motility and livability. Whereas, Kolev (1997) reported 0.3 mg/ml addition of vitamin E in extender had best effects on buffalo bull spermatozoa motility, acrosomal integrity and survivability during cryopreservation.


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Report from Fabbrocini et al. (2000) revealed that sodium pyruvate at 1.25 mM concentration in the extender significantly improved post-thaw progressive motility and viability of buffalo bull semen. It is also documented that by adding oviductal proteins of different stages of estrous cycle also effect post-thaw semen characteristics by improving functions and reducing lipid per-oxidation during cryopreservation (Kumaresan et al., 2006). Bradykinin (2 mg/ml) in tris-based extender is also reported to improve the buffalo bull semen quality by Shukla and Misra (2007). Butylatedhydroxytoluene (BHT) at concentration 1.0 and 2.0 mM in tris citrate egg yolk extender gave best results for cryopreservation of buffalo bull semen (Ijaz et al., 2009).

Osmotic pressure All solutes or colloids present within or outside spermatozoa contribute towards the osmotic properties of the solutions and when the concentration of the solutes become high, the osmotic pressure is raised. In this way, number of solute particles in a solution not only affect the osmotic pressure but also change the freezing point of the solvents. When spermatozoa are exposed to hyperosmolal solution, more extra cellular ice crystals are formed (Watson, 1995) and vice versa. So, any change in spermatozoa volume during extension, freezing and thawing processes result in sperm injury (Mazur, 1985). As water and polyhydric alcohols have high osmotic permeability coefficient which enhances the shifting of these substance across the cell membrane of the spermatozoa during freezing (Noiles et al., 1993). Under hypotonic/hypertonic solution, spermatozoa either swell or shrink (DU et al., 1994; Gilmore et al., 1996) and change its size. Changes in osmotic pressure

during cryopreservation exert stress and damage spermatozoa plasma membrane. Therefore, osmotic pressure plays a vital role during cryopreservation of the buffalo bull semen and ultimately it affects the frozen semen quality (Khan and Ijaz, 2008). Different osmotic pressures of the buffalo bull semen have been reported, which include 293.33 mOsm/kg (Sansone et al., 2000), 268.81 mOsm/L (Khan and Ijaz, 2008) and 289.4 mOsm/kg (Mughal et al., 2013) From these findings, it is clear that buffalo bull semen has osmotic pressure lower than cattle semen. Therefore, buffalo bull semen diluents should have osmotic pressure close to their original values.

SEMEN PROCESSING

Initial processing Methods of semen processing after collection are different from organization to organization. Changes in the sperm motility, morphology and freezability were not observed when ejaculate was processed within an hour of collection (Fabbrocini et al., 1995). Usually semen dilution is done at 30oC to 37oC with a media having all necessary constituents. Vale et al. (1991) recommended that ejaculates should be kept for 10 to 15 minutes, sometimes under these circumstances semen of buffalo bulls may show agglutination. To avoid this condition in such cases addition of diluents soon after the collection prevents irreversibly agglutination and also maintains sperm motility. Whenever there is delay in semen processing, then immediately after semen collection it should be diluted with freezing medium and stored at 5oC (Televi et al., 1994). Under these circumstances spermatozoa motility up to 6 h at 5oC remains constant in such diluents.


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Dilution of collected semen depends on the demand and concentration of the semen of the sire.

Semen dilution One step or two steps dilution methods are being used. Del Sorbo et al. (1994) compared both of these methods using tris-egg yolk extender and reported better results with two step method with equilibration duration of 6 h. One step method needs shorter equilibration time (2 to 4 h) before freezing. Addition of glycerol in two step dilutions method showed higher sperm motility when glycerol was added 1 h before freezing. (Fabbrocini et al., 1995). Del Sorbo et al. (1995) also suggested two step dilution methods using sodium pyruvate with second dilution 1h prior to freezing the semen.

Cooling rates, equilibration time and freezing Slow and fast cooling rates are being used for semen cryopreservation. D during slow cooling process spermatozoa are exposed to high salt concentration and osmolality with changes in PH is observed. While during fast cooling, intracellular water may not pass out of membrane, resulting in intracellular ice crystals formation (Mazur et al., 1972; Mazur, 1977). Osmolality of the medium and rate of cooling also had a significant interaction (Woelders et al., 1997). Singh et al. (1989) and Sahi and Mohan (1990) also compared similar cooling rates. No significant difference in terms of post-thaw sperm motility was reported between these two cooling rates. In another study Anzar et al. (2010) used different cooling rates for buffalo bull spermatozoa and reported higher motion characteristics, acrosomal morphology and plasma membrane integrity at high freezing rates of -30oC/minute. However researchers prefer slow cooling rate of 0.2 to 0.4oC/minute for the pre-freezing processing of the buffalo bull semen.

Slow cooling rate of diluted semen to 5oC is considered beneficial (Ennen et al., 1976; Gilbert and Almquist, 1978) due to rapid penetration of glycerol in the cell membrane. Martin (1965) and De Leeuw et al. (1993) reported that glycerol can be added at any time during the cooling period. Investigators reported different equilibration durations. Short equilibration periods of 2 to 4 h is recommended by Singh et al., 1989; Del Sorbo et al., 1995) while other researchers preferred longer duration of about 6 h (Haranath et al., 1990; Televi et al., 1994). For cryopreservation of buffalo bull semen, it is generally believed that semen should be kept at 5oC for not less than 2 h and not more than 6 h before freezing. Semen is filled and sealed usually at 5oC in the straws of 0.25 ml or 0.5 ml capacity (Cassou, 1964), Straws of 0.25 ml are commonly used because of their cost and storage space. Filled and sealed straws are placed in horizontal position 1 to 4 cm above liquid nitrogen gas for 10 to 20 minutes before plunging into liquid nitrogen gas at -196oC.

Thawing rate and temperature Thawing rate and temperature had a direct

effect on the interior temperature of the semen. Different thawing temperature and time were used by the scientists. Mazur (1984) reported that rapid thawing of semen prevents formation of re-crystallization of water. Thawing of semen straws at 40oC for 30 S was suggested by Dhami et al. (1994); Vale (1997). Slower thawing rates have also been tried by Kumar et al. (1993); Ramakrishnan and Ariff (1994); Fabbrocini et al. (1995) and reported various suitable times and temperatures: 37oC for 30 S, 35oC for 30 S and 39oC for 30 S, respectively. Thawing for shorter period may keep the internal temperature of the straw at level below


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freezing while thawing for longer duration can also affect the acrosomal integrity. Some of the worker reported that buffalo bull semen quality can be enhanced using longer thawing time while Ziada et al. (1992) found no significant different when thawing was done at 35oC for 30 S or at 50oC for 15 S. El-Amrawi (1997) used different thawing procedure and got best fertility rates when semen was thawed at 35oC for 60 seconds.

Post-thaw spermatozoa appraisal Different researches used different

parameters to access the morphological status of the spermatozoa after thawing which include spermatozoa motility, viability, acrosomal/DNA/plasma membrane integrity. Fabbrocini et al. (1996) studied the acrosomalintegrity using fluoresceinated lecithin, FITC-labeled Maclurapomifera Agglutinin (MPA) and suggested this technique for evaluating non lethal damage to spermatozoa.

Other protocols include analysis of enzymes that play role in fertility. Akhtar et al. (1990) reported significantly elevated hyaluronidase activity in semen after thawing. Previous studies conclude that after thawing, level of different enzymes increases significantly in extra-cellular medium as a result of leakage from spermatozoa. Therefore, enzyme leakage is another marker to evaluate the sperm freezability. The sperm motility and acrosomal integrity had negative correlation with enzyme like hyaluronidase amino transferaseand aspartate amino transferase, whereas, acid phosphatase (ACP), lactic dehydrogenase (LDH), alkaline phosphatase (AKP) had positive correlation. Kaker and Anand (1984) reported that glycerol concentration, cold shock, cooling and freezing rate influence on the release of glutamate oxaloacetate transaminase (GOT) and glutamic

pyruvic transaminase (GPT) in seminal plasma. Dhami and Kodagali (1990) and Dhami and Sahni (1994) also measured post-thaw levels of GOT, GTP, LDH, ACP and AKP and recorded negative correlation of these enzymes with fertility.

Fertility evaluation The changes in the morphology of

spermatozoa are not reflected by the fertility rate. However, just laboratory aid can assess the severity of damage during cryopreservation and thawing. Fertility rate is the most suitable parameter to evaluate frozen thaw semen quality (Vale, 1997). Despite of all efforts, buffalo’s hygienic conditions, estrus detection and time of insemination are also main contributor in poor fertility results (Vale, 1997). Buffalo rarely show behavioral sings, so estrus detection is a serious problem (Ohashi, 1994). Similar problems have also been reported by Danell et al. (1984); Drost et al. (1985). It is also believed that low conception rates in buffaloes using cryopreserved semen is due to its small uterus size as compared to cattle, so it is believed that during AI, semen is deposited in the uterine horn rather in its body. In contrast to this belief, Zicarelli et al. (1997) compared two semen deposit sites: cervical and cranial end of the horn, better fertility results were obtained when insemination was carried out at the cranial end of the horn. Pregnancy rate more than 50% using cryopreserved semen of buffalo bulls is believed to be a good result (Vale, 1997).

FUTURE DIRECTIONS

Future research for cryopreserved semen should emphasize on freezing protocols up-gradation for lower spermatozoa damage during cryopreservation. To achieve this goal, diluents


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composition, freezing protocols along with the up gradation of presently used extenders must be focused for buffalo semen cryopreservation. Commercially available extenders i.e. Triladyl (Minitub Germany), Biociphos (IMV, France) and Laciphos (IMV, France), etc. should also be tried for cryopreservation of buffalo bull semen.

REFERENCES

Ahmed, K. and M. Greesh. 2001. Effect of antibiotics on the bacterial load and quality of semen of Murrah buffalo bulls during preservation. Indian J. Anim. Reprod., 22: 79-80.

Aisen, E.G., H.L. Alvarez, A. Venturino and J.J. Garde. 2000. Effect of trehalose and EDTA on cryoprotective action of ram semen diluents. Theriogenology, 53: 1053-1061.

Akhtar, T., R.A. Chaudhry, I.H. Khan and T.M. Khan. 1990. Extracellular release of hyaluronidase and acrosin from buffalo bull spermatozoa extended in different extenders. Recent Advances in Buffalo Research, 3: 75-79.

Akhter, S., M.S. Ansari, S.M.H. Andrabi, N. Ullah and M. Qayyum. 2008. Effect of Antibiotics in extender on bacterial and spermatozoal quality of cooled buffalo (Bubalus bubalis) bull semen. Reprod. Domest. Anim., 43: 272-278.

Aleem, M., R.A. Chaudhry, N.U. Dhan, A.U.R. Rizvi and R. Ahmad. 1990. Occurrence of pathogenic bacteria in buffalo bull semen and their antibiotic sensitivity. In Acharya, R.M., R.R. Lokeshwar and S. Kumar. (eds). Recent Advances in Buffalo Research, 3: 100-104.

Andrabi, S.M.H. 2009. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod. Domest. Anim., 44: 552-569.

Andrabi, S.M.H., M.S. Ansari, N. Ullah, M. Anwar, A. Mehmood and S. Akhter. 2008. Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa. Anim. Reprod. Sci., 104: 427-433.

Andrabi, S.M.H., N. Ahmad, A. Abbas and M. Anzar. 2001. Effect of two different antibiotic combinations on fertility of frozen buffalo and Sahiwal bull semen. Pak. Vet. J., 21: 166-169.

Anzar, M., Z. Rasul, T.A. Ahmed and N. Ahmad. 2010. Response of buffalo spermatozoa to low temperatures during cryopreservation. Reprod. Fert. Develop., 22: 871-880.

Anzar, M., U. Farooq, M.A. Mirza, M. Shahab and N. Ahmad. 2003. Factors affecting the efficiency of artificial insemination in cattle and buffalo in Punjab, Pakistan. Pak. Vet. J., 23: 106-113.

Arriola, J. and R.H. Foote. 1987. Glycerolation and thawing effects on bull spermatozoa frozen in detergent-treated egg yolk and whole egg extenders. J. Dairy Sci., 70: 1664-1670.

Bailey, J., A. Morrier and N. Cormier. 2003. Semen cryopreservation: Successes and persistent problems in farm species. Can. J. Anim. Sci., 83: 393-401.

Bhosrekar, M.R., J.R. Purohit and B.R. Mangurkar. 1990. Studies on the effect of additives to semen diluent. Indian J. Anim. Reprod., 11: 85-88.

Bhosrekar, M.R., R.S. Rane and R.C. Mazokari. 2001. Fertility of Murrah buffalo bulls in Bhilawadi area of Sangli District. In Proceedings of the 17th Annual


9

Convention and National Seminar on Fertility Management of Farm Animals Under Adverse Agro-Climatic Conditions, Jodhpur, India.

Bousseau, S., J.P. Brillard, L. Marquant, B. Guienne, B. Guerin, A. Camus and M. Lechat. 1998. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology, 50: 699-706.

Bratton, R.W., R.H. Foote and J.C. Cruthers. 1955. Preliminary fertility results with frozen bovine spermatozoa. J. Dairy Sci., 38: 40-46.

Buhr, M.M. and M.J. Pettitt. 1996. Frozen-thawed boar sperm: isolation of membranes and fluidity measurement. Reprod. Domest. Anim., 31: 147-152.

Cassou, R., 1964. La methode de paillettes en plastique adaptee a la generalisation de la congelation. In Proceedings of 5th International Congress on Animal Reproduction and Artificial Insemination, Trento, Italy. 4: 540-546.

Chachur, M.G.M., E. Oba and C.I.M. Gonzales. 1997. Equilibrium time influence on motility, vigourand membrane integrity of thawed buffalo semen using triladyl, glycine-egg yolk and tes extenders. In Borghese, A., S. Failla and V.L. Barile (eds). Proceedings of 5th World Buffalo Congress, Caserta, Italy. International Buffalo Federation, Roma, Italy.

Cheema, A.A. and H.A. Samad. 1986. Performance traits of imported Holstein-Friesian Cows in Quetta (Pakistan). Pak. Vet. J., 6: 6.

Cheshmedjieva, S.B., C.N. Vaisberg and S.I. Kolev. 1996. On the lipid composition of

buffalo spermatozoa frozen in different cryoprotective media. Bulgarian J. Agric Sci., 2: 23-26.

Chinnaiya, G.P. and N.C. Ganguli. 1980. Freezability of buffalo semen in different extenders. Zbl. Vet. Med. A., 27: 563-568.

Chohan, K.R., J. Iqbal, A.A. Asgar and M.A. Chaudhry. 1992. Fertility of liquid and frozen semen in Nili Ravi buffaloes. Pak. Vet. J., 12: 4-5.

Danell, B., N. Gopakumar, M.C.S. Nair and K. Rajagopalan. 1984. Heat symptoms and detection in Surti buffalo heifers. Indian J. Anim. Reprod., 5: 1-7.

De Leeuw, F.E., A.M. De Leeuw, J.H.G. Den Daas, B. Colenbrander and A.J. Verkleij. 1993. Effects of various cryoprotective agents and membrane stabilizing compounds on bull sperm membrane integrity after cooling and freezing. Cryobiology, 30: 32-44.

Del Sorbo, C., G. Fasano, A. Fabbrocini, S.L. Lavadera and G. Sansone. 1995. Piruvato quale substrato energetico in extenders crioprotettivi. Effetti sulla motilitaallo scongelamento di spermatozoi bufalini Bubalus bubalis. In Proceedings of 7th

Meeting Nazionale “Studio Sulla Efficienza Riproduttiva Degli Animali Diinteresse Zootecnico.” Bergamo, Italy. l: 585-588.

Del Sorbo, C., G. Fasano, A. Fabbrocini, M.J.F. Nastri and G. Sansone. 1994. Studio preliminare su effetti di vari agenti crioprotettivi sulla motilita di spermatozoi bufalini B. bubalis allo scongelamento. In Proceeding of 6th meeting nazionale “Studiodella efficienza riproduttiva deli animali di interesse zootecnico.” Bergamo, Italy. 1: 63-67.

Dhami, A.J. and K.L. Sahni. 1993. Effect of


10

extenders, additives and deep freezing on the leakage of lactic dehydrogenase from cattle and buffalo spermatozoa. Indian J. Anim. Sci., 63: 251-256.

Dhami, A.J., V.R. Jani, G. Mohan and K.L. Sahni. 1994. Effect of extenders and additiveson freezability, post-thaw thermoresistence and fertility of frozen Murrah buffalo semen under tropical climate. Buffalo J., 1: 35-45.

Dhami, A.J. and K.L. Sahni. 1994. Effects of various cooling from 30oC to 5oC, equilibration and diluents treatments on freezability, post-thaw thermoresistance, enzyme leakage and fertility of bubaline spermatozoa. Buffalo J., 2: 147-159.

Dhami, A.J. and S.B. Kodagali. 1990. Freezability, enzyme leakage and fertility of buffalo spermatozoa in relation to the quality of semen ejaculates and extenders. Theriogenology, 34: 853-863.

Drost, M., W.S. Cripe and A.R. Richther. 1985. Estrus detection in buffaloes Bubalus bubalis: use of an androgenized female. Buffalo J., 12: 159-161.

Du, J., J. Tao, F.W. Kleinhans, A.T. Teter and J.K. Critser. 1994. Determination of boar spermatozoa water volume and osmotic pressure. Theriogenology, 42: 1183-1191.

El-Amrawi, G.A. 1997. Effect of thawing time and post-thaw temperature on fertility of buffalo spermatozoa frozen in straws. In Proceedings of 5th World Buffalo Congress, Caserta, Italy. 1: 850-855.

Ennen, B.D., W.E. Berndtson, R.G. Mortimer and B.W. Pickett. 1976. Effect of processing procedures on motility of bovine spermatozoa frozen in 0.25 ml straws. J. Anim. Sci., 43: 651-656.

Fabbrocini, A., C. Del Sorbo, G. Fasano and G.

Sansone. 2000. Effect of differential addition of glycerol and pyruvate to extender on cryopreservation of Mediterranean buffalo (B.bubalis) spermatozoa. Theriogenology, 54: 193-207.

Fabbrocini, A., C. Del Sorbo, G. Fasano, M.R. Martusciello and G. Sansone. 1995. Effect of cryoprotectant addition mode on freezed-thawed Bubalus bubalis semen. In Proceedings of 30th Symposium “Reproduction and Animal Breeding: Advance and Strategy”, Milano, Italy. 1: 373-374.

Fabbrocini, A., G. Sansone, R. Talevi, R. Gualtieri, M. Palazzo, R. Formato and D. Matassino. 1996. The use of MPA lectin to evaluate the functional integrity of frozen-thawed Bubalus bubalis spermatozoa. In Proceedings of 13th International Congress on Animal Reproduction, Sydney, Australia. 3: 24.

Foote, R.H. 1970. Fertility of bull semen at high extension rates in Tris buffered extenders. J. Dairy Sci., 53: 1475-1477.

Gilbert, G.R. and J.O. Almquist. 1978. Effects of processing procedures on post-thaw acrosomal retention and motility of bovine spermatozoa packaged in 0.3 ml straws at room temperature. J. Anim. Sci., 46: 225-231.

Gilmore, J.A., J. Du, J. Tao, A.T. Peter and J.K. Critser. 1996. Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J. Reprod. Fertil., 107: 87-95.

Graham, E.F., B.G. Crabo and K.I. Brown. 1972. Effect of some zwitterion buffers on the freezing and storage of spermatozoaI bull. J. Dairy Sci., 55: 372-378.


11

Haranath, G.B., T.B. Suryaprakasam, A.V.N. Rao and G. Somasekharam. 1990. Freezability of semen and fertility of frozen semen packaged in mini and medium French straws: a note. In Acharya, R.M., R.R. Lokeshwar and S. Kumar. (eds). Recent Advances in Buffalo Research, 3: 87-88.

Hasan, S., S.M.H. Andrabi, R. Muneer, M. Anzar and N. Ahmad. 2001. Effects of a new antibiotic combination on post-thaw motion characteristics and membrane integrity of buffalo and Sahiwal bull spermatozoa and on the bacteriological quality of their semen. Pak. Vet. J., 21: 6-12.

Holt, W.V. 2000. Basic aspects of frozen storage semen. Anim. Reprod. Sci., 62: 3-22.

Ijaz, A., A. Hussain, M. Aleem, M.S. Yousaf and H. Rehman. 2009. Butylatedhydroxytoluene inclusion in semen extender improves the post-thawed semen quality of Nili-Ravi buffalo (Bubalus bubalis). Theriogenology, 71: 1326-1329.

Kaker, S.S. and S.R. Anand. 1984. Acrosomal damage and enzyme leakage during freeze preservation of buffalo spermatozoa. Indian J. Exp. Biol., 22: 5-10.

Khan, M.I.R. and A. Ijaz. 2008. Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo spermatozoa. Animal, 2: 548-553.

Kolev, S.I. 1997. Effects of Vitamins A, D, E on the motility and acrosomal integrity of cryopreserved buffalo bulls’ spermatozoa. In Proceedings of 5th World Buffalo Congress, Caserta, Italy. 1: 833-835.

Kumar, S., K.L. Sahni and G. Mohan. 1992. Effect of different levels of glycerol and yolk on freezing and storage of buffalo semen in

milk, tris and sodium citrate buffers. Buffalo J., 2: 151-156.

Kumar, S., K.L. Sahni, B.N. Benjamin and G. Mohan. 1993. Effect of various levels of yolk on deep freezing and storage of buffalo semen in different diluters without adding glycerol. Buffalo J., 1: 79-85.

Kumaresan, A., M.R. Ansari, A. Garg and M. Kataria. 2006. Effect of oviductal proteins on sperm functions and lipid peroxidation levels during cryopreservation in buffaloes. Anim. Reprod. Sci., 93: 246-257.

Kundu, C.N., J. Chakraborty, P. Dutta, D. Bhattacharyya, A. Ghosh and G.C. Majumder. 2002. Effects of dextrans on cryopreservation of goat caudaepididymal spermatozoa using a chemically defined medium. Reproduction, 123: 907-913.

Martin, I.C.A. 1965. Effects of cooling to 58C, storage at 58C, glycerol concentration, sodium chloride, fructose and glycine on the revival of deep frozen spermatozoa. J. Agr. Sci., 64: 425-432.

Matharoo, J.S. and M. Singh. 1980. Revivability of buffalo-spermatozoa after deep freezing the semen using various extenders. Zbl. Vet. Med. A., 27: 385-391.

Mazur, P. 1984. Freezing of living cells: Mechanisms and implications. Am. J. Physiol., 247: C125-142.

Mazur, P. 1985. Basic concepts in freezing cells. In Johnson, L.A. and K. Larsson (eds.) Proceedings of 1st International Conference on Deep Freezing of Boar Sperm, Swedish University of Agricultural Sciences, Uppsala, Sweden. 5: 91-111.

Mazur, P. 1977. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology, 14: 251-272.


12

Mazur, P., S.P. Liebo and E.H.Y. Chu. 1972. A two-factor hypothesis of freezing injury. Exp. Cell Res., 71: 345-355.

Mughal, D.H., A. Ijaz, M.S. Yousaf, H. Rehman, M. Aleem, H. Zaneb and F. Wadood. 2013. Assessment of optimal osmotic pressure of citrate egg yolk extender for cryopreservation of buffalo bull (Bubalus bubalis) semen, J. Anim. Plant Sci., 23(4): 964-968.

Nagase, H., T. Niwa, S. Yamashita and S. Irie. 1964. Deep freezing bull semen in concentrated pellet form. II. Protective action of sugars. In Proceedings of 5th International Congress on Animal reproduction and artificial insemination, Trento, Italy. 2: 1111-1113.

Nastri, M.J.F., C. Del Sorbo, A. Fabbrocini, G. Fasano and G. Sansone. 1994. Performances motility in cooled and freeze thawed B. bubalis spermatozoa at different osmotic pressures. In Proceedings of 7th International Symposium on Spermatology, Cairns, Australia. l: 99-100.

Noiles, E.E., P. Mazur, P.F. Watson, F.W. Kleinhans and J.K. Critser. 1993. Determination of water permeability coefficient for human spermatozoa and its activation energy. Biol. Reprod., 48: 99-109.

Oba, E., E.J. Fuck, S.D. Bicudo, F.O. Pap and O.M. Ohashi. 1994. Prelimanary study on different mediums for deep freezing of buffalo semen, p. 579-581. In Vale, W.G., V.H. Barnabe and J.C.A.de. Mattos (eds.) Proceedings of 4th World Buffalo Congress, Sao Paulo, Brazil. International Buffalo Federation, Roma, Italy.

Ohashi, O.M. 1994. Oestrus detection in buffalo cows. Buffalo J., 2: 61-64.

Pace, M.M. and E.F. Graham. 1970. The release

of glutamic oxaloacetic transaminase from bovine spermatozoa as a test method of assessing semen quality and fertility. Biol. Reprod., 3: 140-146.

Parks, J.E. and J.K. Graham. 1992. Effects of cryopreservation procedures on sperm membranes. Theriogenology, 38: 209-222.

Purdy, P.H. 2006. A review on goat sperm cryopreservation. Small Rum. Res., 6: 215-225.

Raizada, B.C., A. Sattar and M.D. Pandey. 1990. A comparative study of freezing buffalo semen intwo dilutors. In Acharya, R.M., R.R. Lokeshwar and A.T. Kumar (eds.). Proceedings of 2nd World Buffalo Congress, New Delhi, India. International Buffalo Federation, Roma, Italy.

Ramakrishnan, P. and M.O. Ariff. 1994. Effect of glycerol level and cooling rate on post-thaw semen quality of Malaysian swamp buffalo. In Proceedings of 4th International Buffalo Congress, Sao Paulo, Brazil. 3: 540-542.

Rasul, Z., N. Ahmed and M. Anzar. 2007.Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm. Theriogenology, 68: 813-819.

Rasul, Z., A. Nasim and M. Anzar. 2001. Changes in motion characteristics, plasma membrane integrity and acrosome morphology during cryopreservation of buffalo spermatozoa. J. Androl., 22: 278-283.

Rasul, Z., M. Anzar, S. Jalali and N. Ahmad. 2000. Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa. Anim. Reprod. Sci., 59: 31-41.

Sahni, K.L. and G. Mohan. 1990. Yolk as a


13

cryoprotectant in deep-freezing of bovine semen. Indian J. Anim. Sci., 60: 828-829.

Sansone, G., M.J.F. Nastri and A. Fabbrocini. 2000. Storage of buffalo (Bubalus bubalis) semen. Anim. Reprod. Sci., 62: 55-76.

Shukla, M.K. and A.K. Misra. 2007. Effect of bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation. Anim. Reprod. Sci., 97: 175-179.

Singh, J., G.R. Pangawkar, R.K. Biswas and N. Kumar. 1991. Studies on buffalo sperm morphology during various stages of freezing in certain extenders. Indian J. Anim. Sci., 12: 126-129.

Singh, P., J.K. Jindal, S. Singh and O.K. Hooda. 2000. Freezability of buffalo bull semen using different extenders. Indian J. Anim. Reprod., 21: 41-42.

Singh, B., D. Chand, P. Singh and N.K. Yadav. 1996. Effect of vitamin C addition in the diluent on the quality of deep frozen Murrah buffalo bull (Bubalus bubalis) semen. Int. J. Anim. Sci., 11: 131-132.

Singh, N.P., R.S. Manik and V.S. Raina, 1989. Effect of cysteine fortification on cryopreservation of buffalo semen in milk whey extenders. Theriogenology, 32: 979-986.

Talevi, R., S. Pelosi, G. Sansone, F. Grasso and D. Matassino. 1994. Effects of different pre-freezing rates on buffalo sperm motility and ultrastructure preservation. In Proceedings of 4th International Buffalo Congress, Sao Paulo, Brazil. 3: 537-539.

Thibier, M. and B. Guerin. 2000. Hygienic aspects of storage and use of semen for artificial insemination. Anim. Reprod. Sci., 62: 233-251.

Thurston, L.M., P.F. Watson, A.J. Mileham and W.V. Holt. 2001. Morphologically distinct sperm

subpopulations defined by Fourier shape descriptors in fresh ejaculate correlate with variation in boar semen quality following cryopreservation. J. Androl., 22: 382-394.

Tuli, R.K., M. Singh and J.S. Matharoo. 1981. Effect of different equilibration times and extenders on deep freezing of buffalo semen. Theriogenology, 16: 99-104.

Vale, W.G. 1997. News on reproductive biotechnology in males. In Proceedings of 5th World Buffalo Congress, Caserta, Italy. 1: 103-123.

Vale, W.G., O.M. Ohashi, H.F.L. Ribeiro and J.S. Sousa. 1991. Semen freezing and artificial insemination in water buffalo in the amazon valley. Buffalo J., 2: 137-144.

Waterhouse, K.E., P.O. Hofmo, A. Tverdal and R.R. Jr. Miller. 2006. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction, 131: 887-894.

Watson, P.F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post thawing function. Reprod. Fertil. Dev., 7: 871-891.

Woelders, H., A. Mathiijs and B. Engel. 1997. Effects of trehalose, and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology, 35: 93-105.

Ziada, M., G. Abdel-Malak and M.S. Abdou. 1992. Effect of glycerol concentration and thawing regimes on the viability of frozen buffulo spermatozoa. In Proceedings of 4th annual congress of Egyptian Society for Animal Reproduction and Fertility, Cairo, Egypt.


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Zicarelli, L., C. De Filippo, M. Francillo, C. Pacelli and E. Villa. 1997. Influence of insemination technique and ovulation time on fertility percentage in synchronized buffaloes. In Proceedings of 5th World Buffalo Congress, Caserta, Italy. 1: 732-737.


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Review Article

INTRODUCTION

Cows and buffaloes sometimes produce milk which has an off-flavor (i.e. bad odor or offensive odor) and a bad taste. Although, there are multiple causes of this problem, the bad odor/bad taste of milk in Pakistan/India seems to be generally associated with certain feeds and presence of some weeds in the fodders. Flavor producing substances present in the feed or weeds are absorbed from the digestive tract into the blood, enter the udder, and appear in milk. The milk carrying abnormal and bad odor is not liked by the consumers and is many cases such milk has to be discarded by the producers. The problem of off-flavor and bad taste in milk is worldwide. Specially trained milk flavor/taste evaluators are used in advanced countries to detect and categorize off-flavors and bad taste of milk. Milk is generally considered to have a flavor defect if it manifest an odor, a foretaste or an aftertaste, or does not leave the mouth in a clean, sweet, pleasant condition following tasting (Alvarez, 2009).

In Pakistan/India, the problem of bad odor in the milk is especially common in spring season and is generally associated with the presence of certain weeds in berseem. The problem is sporadic and affects only some animals in the herd. Not all herds face the problem. Boiling of milk with bad

odor may lead to the presence of bad odor in the kitchen. Tea prepared or curd made from such milk also carries bad odor and a bad taste. In some affected animals, it is a late lactation problem. Problem may persist for months. Sometimes, a bad odor is also detectable from the body and mouth of the affected animals. Addition of wheat straw to fodder generally reduces the severity of the problem. American Dairy Science Association (2005) categorizes the off-flavor of milk into 4 major (A-B-C-D) groupings: Absorbed (‘barny’, ‘cowy’, feed, garlic/onion), Bacterial (acid, bitters fruity/fermented, malty, rancid, unclean [i.e. psychrotrophic]), Chemical (astringent, cooked, lacks freshness, light oxidized, metal oxidized, rancid) and delinquency (flat, foreign, salty, unclean). A ‘cowy’ flavor defect denotes a distinct cow’s breath-like odor and a persistent unpleasant, medicinal or chemical after taste. The difference between ‘cowy’, ‘barny’ and ‘unclean’ off-flavor is that of intensity of bad odor or taste (Alvarez, 2009).

Keywords: buffaloes, Bubalus bubalis, cows, milk, bad taste, off-flavor, Pakistan, India

TROUBLE SHOOTING OFF-FLAVOR (BAD ODOR) AND BAD TASTE IN MILK

Ghulam Muhammad1, Imaad Rashid1,*, Sehrish Firyal2 and Muhammad Saqib1

1Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan, *E-mail: [email protected] Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan


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CAUSES AND TYPES OF MILK OFF-FLAVORS AND BAD TASTE IN MILK

Weeds, odoriferous fodders and silagesMany weeds, certain fodders and strong

smelling silage are common causes of bad odor in milk. Coronopus didymus (= swine cress; known as Pitpapra in Hindi and Jangli Halon in Urdu) is a common weed in berseem incriminated for producing bad odor in milk of cows and buffaloes fed on late cuttings of berseem (Nayyar et al., 2001). This weed contains an odoriferous compound known as benzylthiocianate. Feeding of cabbage, turnips, ragweed, bitter weed, peppergrass, buckhorn and some other weeds, feeding of strong smelling silage (sweet clover, alfalfa or lucerne, corn and soybean), sugar beet tops etc. can also lead to bad odor in milk. The bad flavor of milk associated with some weeds can be controlled if the weeds are withheld from the lactating animals 5 h before milking.

Presence and multiplication of microorganisms in milk

Milk is an excellent growth medium for microorganism. Serious taste and odor defect in milk can result from the multiplication of microorganisms in milk. The growth of microorganisms in milk leads to accumulation of metabolic products of microorganisms. Most of the microorganisms when growing in milk also produce a variety of different lipolytic and lactose destroying enzymes. Sour, bitter, fruity, rancid, malty taste of milk and a variety of bad odors are the consequence of growth of bacteria, yeasts and moulds in milk. Bad taste can be detected when germ concentration in milk exceeds 106 per ml of milk.

Absorption of bad odor from the strong smelling silage or odor from the barn

The natural fat of milk and cream, due to the large surface of fat emulsion and the strongly adsorbing protein-lipid layer on the milk fat globules, is one of the most effective materials known for adsorbing odors (Lasztity, 2014). The presence of strong smelling silage in or near the milking shed should be avoided. The animal sheds should be well ventilated as milk can also absorb bad odor present in poorly ventilated byre.

Digestive disturbances leading to wai badeDisturbances in digestive system

commonly lead to disturbances in the four humors in the body. This condition is colloquially known as wai badee in Punjab and is characterized by slight drooling of saliva, discharge from the eyes, a slight reddish ting of skin and bad odor from the mouth and in the milk.

High concentrate feeding and feeding of roughages with strong smell (e.g. maize or grass silage)

These days, large and modern dairy farms are rapidly increasing in number in Pakistan/India. The management of these farms provides a larger proportion of animal feed in dry form (as hay and concentrate) instead of as pasture grazing, green fodder and silage. In general, dry feeds produce milk with greater susceptibility to oxidized and rancid flavors than do succulent feed (i.e. green fodders). High concentrate feeding to meet the nutritional needs of high producing cows and buffaloes increases the concentration of unsaturated fatty acids in milk fats, with a parallel increase in the chances of oxidation of milk fat leading to off flavor of milk due to rancidity. Sudden change to a new, strong smelling roughages (such as from


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alfalfa hay to maize or grass silage) may also lead to off-flavor in milk. Feeding green maize (corn) fodder can also lead to off-flavor in milk.

Long time interval between milk production and processing

Transportation of milk over long distances usually increases the time between milk production and delivery to milk processing plants. The long storage time of milk even under low temperature may lead to appearance of off-flavor in milk due to oxidation and rancidity of milk fat.

Hydrolytic rancidity, oxidative rancidity (autooxidation) and light activated off-flavor

Hydrolytic milk rancidity occurs when the enzyme lipoprotein lipase hydrolyzes milk lipids resulting in the release of free fatty acids in the milk. This type of milk rancidity is detected by Acid Degree Value (ADV) test that measures the amount of free fatty acids in the milk. When the ADV value is ˃1, the milk will have a noticeable rancid flavor due the presence of short chain fatty acids. Milk rancidity is caused by a weakening of the milk fat globule membrane (MFGM). The MFGM, which is composed of phospholipids and protein, protects the lipids from the activity of lipoprotein lipase. High ADV test values (indicative of hydrolytic rancidity) are primarily associated with milking equipment problems that result in rough handling of milk leading to rupturing of MFGM. Excessive agitation of milk is a common cause of hydrolytic rancidity of milk. Important contributing factors include dietary deficiencies of phosphorus, energy, proteins, high somatic cell count and increase in the percentage of cows or buffaloes in late lactation (Baker, 1990). Rancidity due to oxidization of milk lipids is another type of milk rancidity. The milk affected with oxidized flavor has a tallowy,

metallic or cardboard like off-flavor which may be detected by smell but preferably by taste. The factors generally implicated include contamination of milk with copper, iron, rust (from components of milking equipment), exposure to sun light (which causes demage to riboflavin present in milk) and excessive incorporation of air in the milking system. Milk from some cows becomes oxidized spontaneously. Contamination of milk with iron and copper can lead to the production of unconjugated, unsaturated carbonyl compounds as a result of oxidation of milk lipids (Lasztity, 2014).

Diagnosis of the causes of bad odor/bad taste in milk

Diagnosis of the causes of bad odor/bad taste in milk is often difficult. Generally, if the bad odor/bad taste is strong directly after milking, the cause is probably in the feed, or the accidental addition of detergents, disinfectants, etc., to the milk. If the bad odor/bad taste becomes progressively worse over the 24 h after milking, the cause is probably bacterial or enzymatic and only rarely chemical (e.g., oxidation catalyzed by copper). Further microbiological investigations may help in determining the etiologic nature of bacteria involved (Lasztity, 2014).

TREATMENT AND CONTROL

Withholding suspected feed for 2 to 5 h before milking

The time interval between feeding and milking of cows/buffaloes is an important factor that affects the severity of odors in milk. The bad odor and bad taste in milk is most pronounced when the putative silage is fed 2 to 3 h before milking. The feed suspected for causing bad odor or bad taste in


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milk should not be fed 2 to 5 h before milking. It is recommended that the suspected feed or fodders containing weeds should be fed immediately after milking.

Adding wheat straw to green fodderReducing green fodder and increasing

the amount of wheat straw generally deceases the intensity of bad odor and bad taste in milk.

Vitamin E supplementationGreen succulent fodders containing

vitamin E should be fed when oxidized flavors occur in milk. In many instances, oxidized flavor development may be stopped by feeding cows/buffaloes 1 to 2 grams of alpha-tocopherol acetate (a stable form of vitamin E) per day per animal in the concentrate, (http://www.farminfo.org/dairy/flavor.htm).

Recommendations related to milking machine and milking

Use only high-quality stainless steel, glass, plastic or rubber for all milk contact surfaces. Keep fittings tight and air admission to a minimum. Restrict air admission and prevent equipment leaks. Water treatment/softening may be necessary when water contains abnormally high iron, copper levels. Use iodophor sanitizers preferably. Protect milk from exposure to sunlight or fluorescent lighting; cover glass pipelines. Immediately after milking, cool the milk to at least 40oF (4.4oC) and maintain milk at this temperature (Schroeder, 2012).

Phosphorus supplementationIncreasing dietary P to 0.47% of feed dry

matter often resolves the problem of bad odor and bad taste in milk (Baker, 1990).Feeding cows and buffaloes on ration containing

adequate energy and proteinFeeding of a ration containing adequate

levels of energy and protein was shown to correct the problem of lipolytic rancidity of milk (Baker, 1990).

Weed controlWhen weeds are the suspected cause of

bad taste or bad odor in milk, the use of weedicides e.g. (Atlantis®, Bayer Crop Science, Private, Ltd., Pakistan) should be considered.

Administration of antioxidantsThe cows and buffaloes which are

producing bad flavor or bad tasting milk should be treated with the following by the recipe: Dissolve 15 sachet of Mucolator® (Acetylcysteine; 200 mg per sachet; Abbot Lab. Ltd. Karachi) in 500 ml of distilled or mineral water (e.g. Pure Life®, Nestle). Filter this solution through 2 to 4 layers of muslin cloth. Add this filtered solution to one liter bottle of dextrose 5% solution and inject intravenously. After 30 minutes, administer 20 tablets of vitamin C (e.g. Tab. Cecon®, Abbot Lab.) in the form of a drench. Repeat intravenous administration of Mucolator® and oral administration of Tab. Cecon® for next 2 to 3 days.

Carminative detoxifying mixtureThe following recipe given weekly often

comes very handy in preventing and treating bad odor and bad taste in milk:

Omum (Ajwain in Urdu) = 100 gCumin (Carawy; Zeera in Urdu) = 30 gGinger = 100gSwertia chirata (Chirata in Urdu) = 30 g Aniseed (Saunf in Urdu) = 60 g Green pepper (Haree murch in Urdu) =

http://www.farminfo.org/dairy/flavor.htm

http://www.farminfo.org/dairy/flavor.htm


19

120 g Black pepper (Kali murch in Urdu) = 10 g Citrullus colocynthis (a very bitter

medicinal plant known as Tumma in Urdu) = 60 g Sodium bicarbonate = 100 g Common salt = 100 g Zinc Sulphate = 3 g

Grind and then mix all the above ingredients in one liter of water and give as a drench on weekly basis.

REFERENCES

Alvarez, V.B. 2009. Fluid milk and cream products. In Clark, S., M. Costello and M. A. Drake (eds.) The Sensory Evaluation of Dairy Products. Springer Science-Business Media, New York, USA.

Baker, L.D. 1990. Investigating the cause of chronic milk rancidity in a dairy herd. Veterinary Medicine, 85(8): 901-905.

Dairy Practices Council, Quality Assurance Task Force. 1991. Guidelines for Preventing Off-Flavors in Milk. Dairy Practices Council, Richboro, Pennsylvania, USA.

Lasztity, R. 2014. Milk and milk products. In Food Quality and Standards, 2nd ed. Encyclopedia of Life Support System (EOLSS).

Nayyar, M.M., M. Ashiq and J. Ahmad. 2001. Manual on Punjab Weeds (Part I) Directorate of Agronomy. Ayub Agricultural Research Institute, Faisalabad.

Schroeder, J.W. 2012. Detecting and Correcting Off-flavors in Milk. North Dakota State University, USA, Extension Bulletin AS-1083.


21

Original Article

ABSTRACT

The aim of this study was evaluate the treatment of Bubalus bubalis (dairy buffaloes) affected with clinical mastitis, through the use of Mimosa tenuiflora botanical extract, using as parameters for this study: the semiotic examination and strip cup test, number of colonies count by using Petrifilm plates; isolation and identification of microorganisms found in the animal’s milk before and after treatment, toxicological testing and bactericidal kinetics with strains of Staphylococcus aureus collected from buffalo milk. We observed that the extract of M. tenuiflora exerts bactericidal effect on S. aureus, at the concentration of 107.5 mg/mL as low acute toxicity. In this context, it can be concluded that M. tenuiflora extract presents an antimicrobial activity about S. aureus, with relatively low toxicity. Thus, these outcomes suggested further studies about alternative economically viable for control and prevention of infections in veterinary medicine.

Keywords: phytotherapy, Mimosa tenuiflora, clinical mastitis, dairy buffaloes

INTRODUCTION

Buffalo (Bubalus bubalis) is a rustic animal, resistant to diseases and parasites, however, it presents similar to bovines sanitary problems, such as: mastitis (Carvalho et al., 2007). In mastitis process an intense inflammatory response occurs, causing clinical manifestation, alterations in udder and milk secretion and changes in chemical composition of milk (Deb et al., 2013). Among the microorganisms involved in the mastitis process, Staphylococcus aureus, is the most common, being an inexhaustible source of studies in various countries of the world. S. aureus is a natural inhabitant of human and animal skin and mucosal epithelia (De Los et al., 2014). This microorganism has specific dispersion characteristics among herds, besides the high rate of resistance to drugs used in disease treatment (Troncarelli et al.,

MIMOSA TENUIFLORA EXTRACT REDUCES THE NUMBER OF STAPHYLOCOCCUS AUREUS WITH LOW TOXICITY IN BUBALUS BUBALIS WITH MASTITIS

Andréia Vieira Pereira1,*, Marcelo Biondaro Gois2, Fabiana Nabarro Ferraz3, Jozinete Vieira Pereira4, Sérgio Santos Azevedo5, Ednaldo Queiroga de Lima6,

Onaldo Guedes Rodrigues6, Elizabeth Sampaio de Medeiros7, Rinaldo Aparecido Mota7 and Maria do Socorro Vieira Pereira8

1Department of Experimental Pathology, State University of Londrina, Londrina, Brazil, *E-mail: [email protected] of Morphological Sciences, 3Department of Basic Health Sciences, State University of Maringá, Maringá, Brazil4Department of Dentistry, State University of Paraiba, João Pessoa, Brazil5Department of Veterinary Medicine, 6Department of Biological Sciences, Federal University of Campina Grande, Patos, Brazil7Department of Veterinary Medicine, Federal Rural University of Pernambuco, Recife, Brazil8Department of Biological Sciences, Federal University of Paraiba, João Pessoa, Brazil

mailto:[email protected]


22

2013). Moreover, indiscriminate use of antibiotics promotes the passage of antibiotic residues to milk, in the treatment of lactating females (Freitas et al., 2005). These facts support the search for therapeutic alternatives, i.e., development of new products based on active ingredients from natural origin, in which safety and efficacy are scientifically proven (Troncarelli et al., 2013).

Mimosa tenuiflora is a Mimosoidae subfamily legume, typical of semiarid region of Brazil, and it is widely used by the population because it presents anti-inflammatory and antimicrobial, analgesic, cell regenerating, antipyretic and pectoral astringent activity (Maia 2004). Moreover, M. tenuiflora is rich in tannins and flavonoids, substances responsible for the antimicrobial activity (Meckes- Lozoya et al., 1996). In this context, it is aimed to evaluate the use of M. tenuiflora extract in animals affected with clinical mastitis, aiming to control main microorganisms responsible for bubaline clinical mastitis.

MATERIALS AND METHODS

The study was conducted in department of Chemical and Biological Sciences, Universidade Federal de Campina Grande-UFCG. Parts of the plant used in this study were deposited in the form of voucher specimen in the Herbarium Caririensis Dárdano de Andrade Lima, Universidade Regional Cariri-URCA, Crato-EC, under registration #3275. This research was submitted and approved by CSTR ethics committee (protocol 36-2010).

Collection and preparation of materialTo obtain the M. tenuiflora extract were

used 500 g of stem bark eluted in 1.000 mL ethanol

P.A. for 72 h. The material was concentrated on rota-evaporator and the M. tenuiflora extract concentration was carried out according to Matos methodology (Matos 1997). The concentration of the extract was determined considering their masses, extract concentration and yields, resulted in the following data: 860 mg, 75.43 mg / mL and 1.72%. From which dilutions were made: 430 mg / mL; 215 mg / mL; 107.5 mg / mL; 53.75 mg / mL; 26.87 mg / mL; 13.43 mg / mL; 6.71 mg / mL; 3.35 mg / mL; 1.67 mg / mL.

Microbiological evaluation of milkAcute toxicological assay was made using

forty Swiss mice, males, 6 to 8 weeks old obtained from Universidade Federal de Campina Grande (UFCG). The animals were divided into 4 groups and treated with 0.25 mL of the plant extract, intraperitoneally in a single dose. Each group was treated with the M. tenuiflora extract diluted in proportions 430 mg / mL; 215 mg / ml; 107.5 mg / mL and 53.75 mg / mL. The groups were observed for 24 h, in order to determine the LD50. Moreover, the skin irritation test also was evaluated. Twenty-four hours prior to the test, animals were shaved, in a field of 1.5 and 1.5 cm in mid-dorsal region for application of M. tenuiflora extract. Three mice for each dilution: 107.5 mg / mL and 53.75 mg / mL were used (the choice of these dilutions was determined from the results obtained by MIC and acute toxicological assay) and 0.5 mL of the dilutions was applied. Records were obtained after observation at 3 minutes, 1, 4, 24, 48 and 72 h, registering the skin reactions, according to the index in OECD Guide No. 404 (2002) for sensitivity and irritation test.

Posteriorly, the treatments were performed in 24 mammary quarters of buffalos at different lactation stages, with clinical mastitis detected


23

by strip cup test. For treatment with M. tenuiflora extract (107.5 mg / mL and 53.75 mg / mL concentrations associated with 5% glycerin as fixer (v/v) was utilized 10 mammary quarters and 10 mammary quarters for treatment with antibiotic Cefquinome (Intervet®). For the control group it was stipulated four mammary quarters as non-inoculated controls. In both treatments, it was used an intramammary injection for 5 days (15 mL of the M. tenuiflora extract or Cefquinome 8g) (Costa et al., 1999). Before each application, two daily milkings for total mammary glands depletion and collection of milk samples for further microbiological evaluation, pre and post-dipping were carried out, and the experimental animals were maintained in semi-extensive regime during the whole treatment. Milk samples were collected and analyzed 5 days before treatments, 24 h and 5 days consecutive after administration of treatments.

The physical evaluation of mammary glands and milk udders after the treatments were daily performed through palpation and visual observation, observing color alterations, temperature, edema presence and mammary parenchyma consistency. Black bottom strip cup test for observing milk secretion color, secretion consistency and presence of masses or lumps was conducted in order to determine the occurrence of clinical mastitis (Almeida et al., 2005).

To microbiological evaluation of buffalo milk, approximately 5 mL, were aseptically collected (Bouchot et al., 1985). S. aureus identification was performed by macromorphological characteristics of the colonies (Gram stain) and biochemical tests (Quinn et al., 1994; Santana &and Azeredo, 2005; MacFaddin, 1980). Bacterial curve in face of M. tenuiflora extract was evaluated by Peyret et al. method (Peyret et al., 1990). Three representative S. aureus strains were incubated in nutrient broth

at 37oC / 20 h and subcultured in Muller Hinton-DIFCO /1 h (inoculum of 106 CFU / mL). In 9 mL of bacterial culture, it was added 1 mL of extract and 1 mL of sterile distilled water was added to control tube. The tubes were maintained at 37oC / 24 h, and aliquots were taken after 2, 4, 6, 8, 10 and 24 h and plated on Mueller Hinton / 48 h. Bactericidal effect was defined as the decrease in 3log of CFU / mL or 99.9% cell death over the specified time (May et al., 2000). Moreover, the rapid count of S. aureus was performed by petrifilm® method. The plates were inoculated in duplicate with 1.0 mL of milk samples by the traditional dilutions method 10-1, 10-2 and 10-3 (35 to 37oC / 24 h and further transferred to an incubator at 62±2oC /1 to 4 h).

For statistical analysis of the data, Student’s t test was used for data with normal distribution, and Mann-Whitney U test was used for data with non-normal distribution. Differences were considered statistically significant at P<0.05.

RESULTS AND DISCUSSION

The results of acute toxicological assay conducted with extract of M. tenuiflora presented LD50 of 215 mg / mL. Animals exposed to this dose showed “pre-death” symptoms, as constant urination (urinary incontinence), piloerection, defecation, cyanosis, salivation, corneal opacity, tail relaxation, tachypnea, among others. These symptoms were observed within 24 h after application of the extract. Moreover, no significant presence of edema or erythema was observed in animals in the skin irritation test, obtaining primary irritation index of 0.7 and 0 for dilutions 107.5 mg / mL and 53.75 mg / mL respectively, corresponding to nonirritating classification according OECD guide.


24

Cup test was daily performed in all treated mammary quarters, being possible to observe the disappearance of lumps on the third day after inoculation in both 107.5 mg / mL and 53.75 mg / mL concentrations of the M. tenuiflora extract tested, the same was observed in evaluated mammary quarters treated with antibiotic (Cefquinome). However, in the first inoculation with 107.5 mg

/ mL the mammary quarters showed sensitivity to touch, increased volume and swelling. On the fourth day no sensitivity process was observed.

The bactericidal effect of the hydroalcoholic extract of M. tenuiflora in concentration of 107.5 mg/mL was observed within 4 h after contact for all S. aureus strains isolated from bovine (Table 1). From this result, we selected the concentration of

Table 1. Number of S. aureus colonies in buffalo milk treated with M. tenuiflora extract in 107.5 mg / mL concentration.

Sample Extract Action Time (hours)Bacterial UFC / mL

Time/hours O 2 4 6 8 10 24Sample 1 (C) 2.2 x 106 6.9 x 106 2.2 x 107 8.5 x 107 1.1 x 108 1.4 x 108 1.9 x 108

Sample 1 (T) 2.2 x 106 1.1 x 105 0 0 0 0 0Sample 2 (C) 3.9 x 106 5.2 x 106 4.5 x 107 6.1 x 107 8.7 x 107 1.2 x 108 1.4 x 108

Sample 2 (T) 3.9 x 106 1.4 x 105 0 0 0 0 0Sample 3 (C) 2.1 x 106 7.1 x 106 3.1 x 107 4.3 x 107 1.4 x 108 1.6 x 108 2.3 x 108

Sample 3 (T) 2.1 x 106 1.1 x 106 0 0 0 0 0

(C) Control; (T) Treated with M. tenuiflora extract

0

50

100

150

200

ATB Extract D1 Extract D2 Extract D3

1 2 3 4 5

*

** * * * * * * * * * *

Control

* *

Days

UFC/

mL

Petr

ifilm

pla

te

Figure 1. Count of number of S. aureus colonies (Mean±SD) in buffalo milk. Microbiological evaluation was made by Petrifilm method.*Significant difference (P<0.05) of M. tenuiflora extract 107.5 mg / mL when was compared with antibiotics (ATB) and control.


25

107.5 mg / mL to evaluate the kinetics and bacterial count of the number of microorganisms in petrifilm RSA® system.

In the microbiological evaluations before and after to treatments (extract and antibiotic), the isolated microorganisms were classified as S. aureus. However, a significant decrease in the number of S. aureus colonies was observed for M. tenuiflora extract 107.5 mg / mL when compared with the control in all the dilutions, mainly, from the second day of treatment (Figure 1).

Antimicrobial activity of M. tenuiflora hydroalcoholic extract according to Meckes-Lozoya et al. (1996) can be connected to the presence of tannins and flavonoids. M. tenuiflora extract was the subject of several studies which aimed to verify it’s in vitro effectiveness, testing it on microorganisms such as: Staphylococcus epidermidis, S. aureus, Escherichia coli, Pseudomonas aeruginosa, Micrococcus luteus and Acinetobacter calcoaceticus, as well as fungi such as: Microsporum gypseum, M. canis, Thrichophyton mentagrophytes, T. rubus and Chaetomium indicum (Gonçalves et al., 2005; Lozoya et al., 1989 and Bezerra et al., 2009).

This study contributed to clinical practice innovation, since it suggests an alternative for the treatment of mastitis through clinical use of phytoterapic medicines. In summary, the results suggest the clinical importance of evaluating alternative and economically viable means for infections control in veterinary medicine, showing that M. tenuiflora extract presents an antimicrobial activity with low toxicity in Bubalus bubalis with mastitis.

REFERENCES

Almeida, L.A. do B., M.A.V.P. Brito, J.R.F. Brito, M. de F.A. Pires and N.R. Benites. Almeida LAB, Brito MAVP, Brito J RF, Pires MFA, Benites NR. 2005. Tratamento de mastite clínica experimental por meio de ordenhas múltiplas em vacas leiteiras inoculadas com Staphylococcus aureus. Arq. Inst. Biol., 72(1): 1-6.

Bezerra, D.A.C., Pereira A.V. Pereira, Lôbo K.M.S. Lôbo, Rodrigues O.G. Rodrigues, Athayde A.C.R. Athayde, Mota R.A. Mota, Medeiros E.S. Medeiros and, Rodrigues S.C. Rodrigues. 2009. Atividade biológica da jurema-preta (Mimosa tenuiflora Wild. Poir.) sobre Staphylococcus aureus isolado de casos de mastite bovina. Rev. Bras. Farmacog., 19: 814-817.

Bouchot, M.C., C.J. Catel, C.C. Chirol, G.J.P. Ganiere and M.M.L. Menec. 1985. Diagnostic bactériologique des infections mammaries des bovins. Rec. Med. Vet., 72: 567-577.

Carvalho, L.B., F.R. Amaral, M.A.V.P. Brito, C.C. Lange, J.R.F. Brito and R.C. Leite. 2007. Contagem de células somáticas e isolamento de agentes causadores de mastite em búfalas (Bubalus bubalis). Arq. Bras. Med. Vet. Zootec., 59: 1.

Costa, E.O., R. Sá, H. Ponce, E.T. Watanabe and C.R. Valle. 1999. Avaliação da terapia de mastite clínica: eficácia terapêutica em número de dias em tratamento. Napgama., 2: 10-14.

Deb, R., A. Kumar, S. Chakraborty, A.K. Verma, R. Tiwari, K. Dhama, U. Singh and S.A.K. Kumar. 2013. Trends in diagnosis and control of bovine mastitis: a review. Pak. J.

http://www.ncbi.nlm.nih.gov/pubmed/24506032

http://www.ncbi.nlm.nih.gov/pubmed/24506032


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Biol. Sci., 23: 1653-1661.De Freitas, M.F.L., J.W.P. Júnior, T.L.M. Stamford,

S.S. de A. Rabelo, D.R. Da Silva, V.M. da S. Filho, F.G.B. Santos, M.J. Sena and R.A. Mota. 2005. Perfil da Sensibilidade in vitro de Staphylococcus coagulase positivos isolados de leite de vacas com mastite no Agreste do Estado de Pernambuco. Arq. Inst. Biol., 72: 171-177.

De Los Santos, R., M. Fernández, S. Carro and P. Zunino. 2014. Characterization of Staphylococcus aureus isolated from cases of bovine subclinical mastitis in two Uruguayan dairy farms. Arch. Med. Vet., 46: 315-320.

Gonçalves, A.L., A.A. Filho and H. Menezes. 2005. Estudo comparativo da atividade antimicrobiana de extratos de algumas árvores nativas. Arq. Inst. Biol., 72: 353-358.

Lozoya, X., V. Navarro, J.T. Arnason and E. Kourany. 1989. Experimental evaluation of Mimosa tenuiflora (Willd) Poir. (tepescohuite) I - Screening of the antimicrobial properties of bark extracts. Arch. Invest. Med., 20: 87-93.

Maia, G.N. 2004. Caatinga: Árvores e Arbustos e Suas Utilidades, 1st ed. D and Z. São Paulo, Brasil. 101p.

MacFaddin, J.F. 1980. Biochemical Ttest for Iidentification of Medical Bacteria, 2nd ed. Baltimore: Lippincott Williams and Wilkins. 56p.

May, J., C.H. Chan, A. King, L. Willians and G.L. French. 2000. Time -kill studies of tea tree oils on clinical isolates. J. Antim. Chemother., 45: 639-643.

Matos, F.J.A. 1997. Introdução à Fitoquímica Experimental, 1st ed. Fortaleza, CE, Brasil: edições UFC., 74p.

Meckes-Lozoya, M., X. Lozoya, R. Marles, C. Soucy-Breau and A.J. Avalokitesvarasen. 1990. N, N-Dimethyltryptamine alkaloid in Mimosa tenuiflora bark (Tepescohuite). Arch. Inv. Med., 21: 175-177.

Peyret, M., G. Carret, C. Carre, G. Fardel and J.P. Flandrois. 1990. Etude mathematique de la curve de mortalite d’ Echerichia coli exposes aux polymixines. Path. Biol. , 38: 441-445.

Quinn, P.J., M.E. Carter, B. Markey and G.R. Carter. 1994. Clinical Veterinary Microbiology, 1st ed. London, England: Mosby Wolfe. England. 256p.

Santana, A.S. and D.R.P. Azeredo. 2005. Comparação entre o sistema petrifilm RSA® e a metodologia convencional para enumeração de estafilococos coagulase positiva em alimentos. Ciên. Tecnol. Alimen., 25: 531-535.

Troncarelli, M.Z., H.M. Brandão, J.C. Gern, A.S. Guimarães and H. Langoni. 2013. Mastite bovina sob nanocontrole: a própolis nanoestruturada como nova perspectiva de tratamento para rebanhos leiteiros orgânicos. Vet. e Zootec., 20: 124-136.


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ABSTRACT

The data of buffalo heifers reared in pasture (N=213) and stall feeding system (N=274) were collected to determine the age at puberty and seasonality in breeding behavior through a survey study. The results revealed that the age at puberty and seasonality in breeding behavior was lower in heifer reared in pasture compared to stall feeding system. To determine the effect of mineral supplementation on estrus response, thirty five buffalo heifers were divided into two groups; MIN and CTL. The MIN (N=20) group were fed with mineral mixture (100 grams/day/heifer) for four weeks. Whereas, CTL group (N=15) did not receive any mineral supplementation. Mineral supplementation enhanced estrus response in MIN compared to CTL heifers (65% versus 33%). In conclusion, the age at puberty and seasonality in breeding is less in heifers of pasture system. Furthermore, mineral feeding is helpful to induce estrus in heifers that had achieved average age of puberty.

Keywords: buffalo heifers, management systems, mineral feeding, puberty

INTRODUCTION

The water buffalo is important livestock specie of developing countries in tropical and sub-tropical environments. There are about 29.9 and 33 million heads of buffaloes and cattle present in Pakistan. Although, population of cattle is higher than buffalo, the buffalo milk contributes more than 61% to the total milk (27 million tons) production in the country (GOP 2013). The good feed conversion efficiency and relatively low maintenance requirements are the attributes which make buffaloes ideal in low-input, low-cost production systems. Accordingly, buffaloes have emerged as an increasingly important source of high quality animal protein, milk and meat (Singh et al., 2009). Despite these merits buffalo is blamed for slow reproduction, long calving interval, delayed puberty, poor estrus expression and seasonality in breeding and calving (Naseer et al., 2011).

EFFECT OF TWO MANAGEMENT SYSTEMS AND MINERAL FEEDING ON AGE AT PUBERTY IN NILI-RAVI BUFFALO HEIFERS

Muhammad Tariq Bodla1,*, Muhammad Anwar2, Ejaz Ahmad3, Zahid Naseer4 and Umair Ahsan5

1Artificial Insemination Center, Chowk Azam, Livestock and Dairy Development Department, Punjab, Pakistan, *E-mail: [email protected] Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan3Department of Clinical Sciences, Faculty of Veterinary Sciences, Bahauddin Zakariya University, Multan, Pakistan4Department of Clinical Sciences, Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan5Department of Animal Nutrition and Nutritional Diseases, Adnan Menderes University, Turkey

Original Article


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Moreover, different management systems may affect productive and reproductive performance of the animals such as age at puberty, conception rate, service period, and disease problems (Hansson and Öhlmér, 2008; Di Palo et al., 2009).

Two types of management systems are in practice for buffaloes rearing in Pakistan. In traditional system, buffaloes are grazed on pastures along river banks and merely offered concentrate to milking animals. Breeding bulls are almost invariably present with large grazing herds so the farmers do not have to go for heat detection and artificial breeding. Animals have always access to water for drinking and wallowing that is considered a compulsory exercise in this system. Second type of system is dominant in areas where “cash-crop” production is priority of the farmers and grazing facility is not available. It is also dominant in peri-urban commercial dairying. In this system concentrate and green fodder is provided to buffaloes on the manger where they are kept tied. Usually the bulls are not present in herd, heat detection is relied on farmer’s observation and artificial insemination is practiced for breeding. Due to limited availability of wallowing ponds, animals merely do exercise (personal observation).

Heifer production is most expensive part of the dairy farm operation that requires more input with no visible returns. Attainment of early maturity of heifers results in early returns rather than the more efforts for longer time. Factors involving heat detection system, presence of bull, climate, environment of the area, wallowing facility and exercise during grazing, influence age of maturity in heifers (De Rosa et al., 2009; Gokuldas et al., 2010).

Dietary mineral elements are known to affect the physiological function in general and reproduction in particular (Ullah et al., 2010).

Usually minerals play an intermediate role in the action of reproductive hormones and enzymes at cellular level which ultimately affect the reproductive performance of female (Bearden et al., 2004). Deficiency of mineral elements like phosphorus, copper and zinc are associated with subnormal fertility and anestrous conditions in cows (Campbell et al., 1999). Hence, the balanced feeding including mineral requirements is dire need for the optimal production performance of buffaloes and cows (Bhatti et al., 2007). The present study was designed to determine the effect of pasture or stall feeding system on age of puberty by a survey of buffalo farms and to assess the influence of mineral feeding on estrus induction in Nili-Ravi buffalo heifers.


Area of studyThe study was conducted in Layyah district

(30° 58’ 18” N and 70° 58’ 09” E) Punjab during July to November, 2011. Two types of management system are prevailing for buffaloes rearing in this area; pasture system and stall feeding. In pasture production system, buffaloes kept on grazing on the river bank whereas, in stall feeding system animals are kept tied and fed on the manger.

Survey to assess age of pubertyA survey was carried out to assess and

compare the age of puberty in Nili-Ravi heifers in pasture and stall feeding system. The study was based on retrospective cross-sectional data from dairy farmers in the study area. A total of 83 farmers (53 farmers practiced stall feeding and 30 farmers practiced pasture system) were included who reared their own replacement heifers. Data


29

collection procedure was based on direct question, answer and conversation with farmers. For this purpose a questionnaire was developed to survey the farmers. Very simple question (what was the age of heifer and season at first estrus or at first breeding?) was asked to farmers. The age at first breeding was considered as age of puberty in each individual heifer. Only the data of heifers of age 20 to 50 months were included in this study. Additionally, the questions about feeding (total ration offered/heifer or grazing time allowed/day) and breeding (natural/AI) practices were also asked. It was noticed whether farmers have or have not their own breeding bulls. After collection, the data were organized into different age groups (20 to 26, 27 to 32, 33 to 38 and 39 to 50 months) in both pasture and stall feeding systems separately. Age at puberty and seasonality in breeding behavior were compared between pasture and stall feeding systems.

Effect of feeding mineral mixture on estrus response in heifers This study was conducted during peak breeding season (October to December). A group of thirty five buffalo heifers (42.05±1.18 months of age) from stall feeding system were used that had passed the average age of puberty as assessed by survey. The ovarian status was confirmed twice (11 day apart) for the presence of any functional structure (follicle/corpus luteum) through rectal palpation before the start of trial. All heifers had small inactive ovaries, were divided into two groups randomly: MIN (N=20) and CTL (N=15). MIN heifers were fed with UVAS MMR mineral mixture (Dicalcium Phosphate; 70.81%, Sodium Chloride; 18.91%, Magnesium Sulphate; 8.64%, Ferrous Sulfate; 0.89%, Manganese Sulfate; 0.49%, Zinc Sulfate; 0.22%, Copper Sulfate; 0.03%, Potassium

Iodide; 8.77% and Cobalt Chloride; 0.89%) 100 gram/day/heifer for a period of four consecutive weeks in addition to their normal daily ration. CTL heifers were not fed with any additional mineral supplement and served as control. Both the groups were observed visually for estrus expression daily for a period of three months from the start of the trial. Estrus was confirmed by presence of tone in uterus, vaginal mucus, vulvular swelling and a large follicle observed through rectal palpation.

Statistical analysisThe mean age at puberty of heifers in

pasture and stall feeding system was compared using independent-samples t-test. Whereas, the percentage of buffalo heifers attained puberty in different age groups, seasonality in breeding behavior, farmers having their own breeding bull in pasture or stall feeding system and effect of mineral mixture feeding on estrus response in MIN and CTL were compared using Chi-square test. A probability level of (P<0.05) was considered significant. All data were analyzed using Statistical Package for the Social Sciences (SPSS version17; SPSS Inc., Chicago, IL).

RESULTS

Survey to assess age of pubertyThe number of heifers raised by farmers in

two management systems and their age at puberty is shown in Table 1. The age at puberty in heifers raised in pasture system was lower compared to stall feeding system. Percentage of heifers attained puberty at different ages has been depicted in Figure 1. It is evident that more than 62% heifers in pasture group had attained puberty at the age of 32 months, whereas, in stall feeding system only


30

30.7% heifers reached to puberty at this age. It has been observed that the number of farmers keeping their own breeding bulls was higher in pasture system compared to stall feeding system (Table 1). The seasonality in breeding behavior was lower in heifers in pasture system compared to stall fed system (Table 1).

Effect of feeding mineral mixture on estrus response in heifers The effect of mineral feeding on estrus response has been shown in Table 2. The percentage of heifers which showed estrus signs during the study period remained higher in MIN group (65% versus 33%) compared to that in CTL group.

DISCUSSION

The present study reports that age at puberty in heifers raised in pasture system is lower as compared to the heifers reared in stall feeding system. Furthermore, supplementation of mineral has been found helpful to induce estrus in heifers that had achieved or passed average age of puberty. Our findings are in agreement with previous reports about the improved managmental system for water buffaloes in commercial dairy farming (De Rosa et al., 2009; Neglia et al., 2009). The practice of providing plenty exercise through roaming in ample space, bull presence for breeding and heat detection, grazing on natural grasses and wallowing to combat with heat stress are the advantages of pasture system which minimize the seasonality and pubertal age in buffalo heifers. Oliveira et al. (2009) studied effects of biostimulation (presence of bull in the herd) in Nelore cow heifers kept under extensive management systems in tropical environment. They noted that the exposure of

heifers to a male during the pre-pubertal period decreased age at the first breeding season, resulting in a significant reduction in age of first pregnancy. Bolanos et al. (1997) and Gokuldas et al. (2010) reported similar findings that bull biostimulation effectively enhanced resumption of ovarian activity in cows and buffaloes under intensive system. Bhatti et al. (2007) commented that buffalo heifers attaining their proper weight just before the breeding season were more likely to get bred than those attaining proper weight after this season. However, in case of absence of seasonality of breeding, this possible delay in age at puberty will be ruled out.

In the present study a higher number of heifers were observed in estrus in mineral fed group compared to control. This evidenced that other than the proteins and energy, minerals and vitamins are also vital nutrients which influence the growth of calves to become heifers (Bhatti et al., 2007). Therefore, the balanced feeding to the young stock is essential for the future reproduction. It has been reported that dairy animals reared in the area of having low level of mineral in the forages and soil lead to low production; therefore, the supplementation of required minerals is essential for optimum production (Kumaresan et al., 2010; Khan et al., 2008). Naidu et al. (2009) conducted a similar study in Murrah buffalo heifers and concluded that mineral supplementation could be cheap and convenient source than hormonal therapy to enhance the fertility in delayed pubertal heifers.

CONCLUSIONS

Buffalo heifers reared under pasture system attained puberty earlier than those reared


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Table 1. Age at puberty (months) in buffalo heifers in two management systems in the study area.

Management systemP-ValuePasture (N=213) Stall fed (N=274)

Mean SEM Mean SEMHeifers raised/farmer 7.10 0.45 5.17 0.27 0.000Age at puberty of heifers 32.90 0.43 38.07 0.40 0.000Seasonality in breeding behavior 36.67% - 92.45% - 0.000Farmers having own breeding bull 100.00% - 24.53% - 0.000

Figure 1. Buffalo heifers (%) attained puberty in different age groups reared in pasture or stall feeding system. *P<0.05 in given age group.

Table 2. Estrus response of buffalo heifers in mineral feeding (MIN) and control (CTL) groups.

Group Number of heifers Estrus response (%)MIN 20 65%b

CTL 15 33%a

The values with different superscript within the same column are different (P<0.05).


32

under stall feeding system. Seasonality of breeding in buffaloes was

lower in pasture system due to out-door grazing, ample space per animal, biostimulation and wallowing in river banks.

In addition, the mineral supplementation might be a strategy to induce estrus in heifers that passed average age of puberty onset.

REFERENCES

Bearden, H.J., J.W. Fuquay and S.T. Willard. 2004. Applied Animal Reproduction, 6th ed. Pearson Prentice Hall, New Jersey.

Bhatti, S.A., M. Sarwar, M.S. Khan and S.M.I. Hussain. 2007. Reducing the age at first calving through nutritional manipulations in dairy buffaloes and cows: a review. Pak. Vet. J., 27: 42-47.

Bolanos, T.M., M. Forsberg, H. Kindahl and H. Rodriguez-Martinez. 1997. Biostimulatory effects of estrus cows and bulls on resumption of ovarian activity in postpartum zebu (Bos indicus) cows in the humid tropics. Anim. Reprod. Sci., 48: 180-195.

Campbell, M.H., J.K. Miller and F.N. Schrick. 1999. Effect of additional cobalt, copper, manganese, and zinc on reproduction and milk yield of lactating dairy cows receiving bovine somatotropin. J. Dairy Sci., 82: 1019-1025.

De Rosa, G., F. Grasso, A. Braghieri, A. Bilancione, A. Di Francia and F. Napolitano. 2009. Behavior and milk production of buffalo cows as affected by housing system. J. Dairy Sci., 92: 907-912.

Di Palo, R., B. Ariota, F. Zicarelli, M. De Blasi, G. Zicarelli and B. Gasparrini. 2009. Incidence

of pregnancy failures in buffaloes with different rearing system. Ital. J. Anim. Sci., 8: 619-621.

Gokuldas, P.P., M.C. Yadav, H. Kumar, G. Singh, S. Mahmood and A.K.S. Tomar. 2010. Resumption of ovarian cyclicity and fertility response in bull-exposed postpartum buffaloes. Anim. Reprod. Sci., 121: 236-241.

Government of Pakistan. 2012-13. Pakistan Economic Survey, Economic Advisor’s Wing, Finance Division, Government of Pakistan, Islamabad, Pakistan. http://finance.gov.pk/survey/chapters_13/02-Agriculture.pdf

Hansson, H. and B. Öhlmér. 2008. The effect of operational managerial practices on economic, technical and allocative efficiency at Swedish dairy farms. Livest. Sci., 118: 34-43.

Khan, Z.I., M. Ashraf, K. Ahmad, I. Javed and E.E. Valeem. 2008. A comparative study on mineral status of blood plasma of small ruminants and pastures in Punjab, Pakistan. Pak. J. Bot., 40: 1143-1151.

Kumaresan, A., K.M. Bujarbaruah, K.A. Pathak, Brajendra and T. Ramesh. 2010. Soil-plant-animal continuum in relation to macro and micro mineral status of dairy cattle in subtropical hill agro ecosystem. Trop. Anim. Health Prod., 42: 569-577.

Naidu, G.V., M. Srinivas, N.V.V.H. Krishna and V.D. Prasad. 2009. Management of delayed puberty in graded Murrah heifers under field condition-A practical approach. Buffalo Bull., 28(4): 204-206.

Naseer, Z., E. Ahmad, N. Ahmad and J. Singh. 2011. Fertility following CIDR based synchronization regimens in anestrous Nili-


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Ravi buffaloes. Reprod. Domest. Anim., 46: 814-817.

Neglia, G., M. Rendina, A. Balestrieri, F.L. Grasso, A. Potena, I. Russo and L. Zicarelli. 2009. Influence of a swimming-pool on fertility in buffalo species. Ital. J. Anim. Sci., 8: 637-639.

Oliveira, C.M.G., F.B.D. Oliveira, M.L. Gambarini, M.A.O. Viu, D.T. Lopes and A.P.F. Sousa. 2009. Effects of biostimulation and nutritional supplementation on pubertal age and pregnancy rates of Nelore heifers (Bos indicus) in a tropical environment. Anim. Reprod. Sci., 113: 38-43.

Singh, B., M.S. Chauhan, S.K. Singla, S.K. Gautam, V. Verma, R.S. Manik, A.K Singh, M. Sodhi and M. Mukesh. 2009. Reproductive biotechniques in buffaloes (Bubalus bubalis): status, prospects and challenges. Reprod. Fert. Dev., 21: 499-510.

Ullah, N., M. Anwar, S.M.H. Andrabi, M.S. Ansari, S. Murtaza, Q. Ali and M. Asif. 2010. Effect of mineral supplementation on postpartum ovarian activity in Nili-Ravi buffaloes (Bubalus bubalis). Pak. J. Zool., 43(2): 195-200.


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Original Article

ABSTRACT

Thermal stress resulted in various physio-biochemical alterations in the exposed buffaloes. The rectal temperature and respiration rates increased concurrently with the rising exposure temperature. There was a significant decrease in the electrolyte, triglyceride, thyroid hormone and aldosterone concentration. On the contrary the reactive oxygen radicals and cortisol hormone increased during thermal stress. The study also revealed high methane production at optimum temperatures as compared during heat stress.

Keywords: physio-biochemical, hormone, methane, reactive oxygen radicals, thermal stress

INTRODUCTION

Homeostasis can be altered by environmental fluctuations like very low or high temperatures and the tropical and sub-tropical regions characterized by high summer temperatures can have deleterious impact on animal health and production. These tropical conditions coupled with the global warming scenario can be devastating

to the livestock industry. Several workers have very well documented the negative impact of high temperatures on the livestock (West et al., 2003; Marai and Haeeb, 2010; Baumgard, 2011). Animals suffering from heat stress show compromised heat dissipation mechanisms (conduction, convection, vaporation, radiation) leading to excess heat accumulation and disturbances in metabolism.Although, the buffalo is well adapted to the tropical climate but its heat tolerance capacity is poor when compared with cattle making them more vulnerable to thermal stress. Most of the studies have mainly focused on physiological or biochemical aspect during hyperthermia. With this perspective the study was planned to observe the buffaloes’ integrated physiological, metabolic, endocrinal acclimation and methane emission patterns so that proper ameliorative measures could be taken during heat stress.


Four adult dry indigenous Murrah buffaloes (< 3 y, live weight -492.14±9.58 kg) were housed in environmental chamber at Physiology and Climatology Division, Indian Veterinary Research

BIOCHEMICAL PROFILE AND METHANE EMISSION DURING CONTROLLED THERMAL STRESS IN BUFFALOES (BUBALUS BUBALIS)

Alok K. Wankar1,*, Gyanendra Singh2 and Brijesh Yadav3

1Department of Veterinary Physiology, College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Mathura, India, * E-mail: [email protected] and Climatology Division, Nuclear Research Laboratory, Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India3College of Veterinary and Animal Husbandry, Mathura, India


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Institute. After exposing the animals at 25oC (optimum conditions) they were moved to 30oC (mild stress), 35oC (acute stress) and 40oC (severe stress) respectively, 21 days at each treatment. Daily exposure period was fixed from 1000 to 1500 h. Basal diet of wheat straw ad libitum along with required amount of concentrate mixture was offered during entire trial.

The rectal temperature (RT) and respiration rates (RR) were measured daily immediately before and after exposure by standard procedures. Blood collection was done at 5 days interval (before/after exposure) by jugular veni-puncture in sterile glass test tubes. In all serum samples sodium, potassium, chloride, calcium, aspartate aminptransferase (AST), alanine aminptransferase (ALT), triglyceride concentrations were measured on double beam UV-VIS spectrophotometer. Serum superoxide dismutase (SOD) was measured using the method as described by Madesh and Balasubramanian (1998) and reactive oxygen radicals (ROS) were estimated by procedure suggested by Brambilla et al. (2001). Thyroxine (T4), triiodothyronine (T3), cortisol and aldosterone were determined by radioimmunoassay technique on SR-3000 Stratec Counter (Stratec biomed systems, Germany).Methane was collected by indirect open circuit calorimetry method as standardized by Mc lean and Tobbin (1987) and Derno et al. (2009) during last 2 days at each treatment (day 20 to 21). Gas collection lasted for total 12 h and analysis was done on infrared gas analyzer (2RJF4C25, Fuji Electronic Systems, Japan). Methane concentration in the expired air was calculated by using following formula,

CH4 (L/Day) = VSTP x X/1000000, VSTP=V x 273 x P-Vp 273+T 760Where,

V=Total volume of air, T=Dry bulb temperature, P=Borometric pressure in mm Hg, Vp=Partial pressure of water vapor, X=Methane % in ppm (CH4 out-CH4 in)All the data were analyzed by using SPSS 16.0 software package.


Rectal temperature increased significantly at all the treatments except at 35oC (Figure 1). At 25oC it might be due to thermogenic activity of the animals but the subsequent rise in RT reflected heat load on the animals. Respiration rates also paralleled with the RT, except at 25oC and 35oC (Figure 2). This significant increment in RT and RR with thermal stress clearly indicate heat load on the animals and compromised heat loss mechanisms, culminating into panting as only means to lose excess heat quickly. Present results of increased RT and RR are in accordance with those reported by Banerjee and Ashutosh (2011a, b) and Pereira et al. (2008) in heat stressed cattle. Methane production was highest at optimum conditions and at 30oC, while it decreased at acute and chronic thermal stress (Figure 3). Total methane produced depends on many factors like dry matter intake, diet digestibility, digesta passage rates, fibre proportion in feed, ruminal pH and temperature and fermentation patterns. Christopherson and Kennedy (1983) attributed low thyroid hormone levels for decreased motility and longer retention of digesta in the gut. Low thyroid activity was also confirmed during thermal stress, possibly increasing digestibility and reducing methane production in present trial. Similarly, Dmytruk et al. (1995) and Mc Ginn et al. (2008) observed low methane emission during heat stress which supports


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our findings.The essential electrolytes sodium and potassium decreased significantly during heat stress conditions which might be due to overall negative mineral balance and increased excretion through urine and skin (Table 1). Similarly, Aboul-Naga (1983) and Banerjee and Ashutosh (2011b) also reported depletion in sodium and potassium concentrations in cattle exposed to high environmental temperatures which support present findings. On the contrary no variation was recorded for serum calcium and chloride indicating optimum metabolism for these minerals even during stressful conditions (Table 1). No variation was seen for free radical during acute thermal stress but a significant rise was observed at chronic conditions (Table 1). This increment in the ROS reflected faster production than that could be possibly neutralized by different antioxidant systems, thus increasing oxidative stress and cellular injury (Zhao et al., 2006). Bernabucci et al. (2002) also reported increased ROS levels in heat stressed dairy cows

(TBARS; 8.8±0.4 vs 7.6±0.4 nmoL/mL). The superoxide dismutase did not varied significantly at any of the treatment (Table 1), possibly due to synergistic action of other antioxidant systems (catalase, glutathione peroxidase, vitamins A, E and C, ubiquinone and flavonoids) in neutralizing free radicals. Similarly, Calamari and co workers found no significant variation for SOD in heat exposed dairy cattle which corroborate present observations (Calamari et al., 2011).

Serum triglyceride decreased significantly during acute and chronic heat stress but no variation was seen at optimum or mild stress conditions (Table 1). Cortisol is a known lipolytic agent; mobilizing fatty acids (FA) and triglycerides thus mediating the essential stress adaptations for providing extra energy (Cunningham and Klein, 2007). High levels of cortisol were also noted in present trial during heat stress which possibly increased lipolysis and FA catabolism, resulting into low triglyceride levels. Abeni et al. (2007) also

Figure 1. Pooled means±SEM for rectal temperatures (oC) of animals exposed at 25oC, 30oC, 35oC, 40oC. *Superscript a, b, differ significantly at P<0.01. aThe post exposure means are cited from Wankar et al. (2014).


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Figure 2. Pooled means±SEM for respiration rates (Breaths/Min) of animals exposed at 25oC, 30oC, 35oC, 40oC. *Superscript a, b, differ significantly at P<0.01. aThe post exposure means are cited from Wankar et al. (2014)

Figure 3. Average methane emission for buffaloes at different treatments.


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Table 1. Pooled means±SEM of various biochemical and endocrinal variants at different exposure temperatures. SEM=Standard error of means, ROS=Reactive oxygen radicals, SOD=Superoxide dismutase, AST=Aspartate aminotransferase, ALT=Alanine aminotransferase, T3=Tri-iodothyronine, T4=Thyroxine.

Parameter Period 25oC 30oC 35oC 40oC SEM Sodium

(mmoL/L)Pre-exposure 137.28 148.05a 143.42a 128.69

1.248Post-exposure 131.17 120.78b 127.94b 139.50

Potassium(mmoL/L)

Pre-exposure 4.60 5.02 5.45 5.66a

0.072Post-exposure 4.97 4.63 5.08 4.81b

Chloride(mmoL/L)

Pre-exposure 86.72 99.67 91.49 87.031.691

Post-exposure 99.81 101.65 102.84 98.52Calcium(mmo/L)

Pre-exposure 2.27 2.24 2.21 2.260.039

Post-exposure 2.42 2.53 2.42 2.34ROS

(mg H2O2/dL)Pre-exposure 0.05 0.04 0.05 0.05b

0.001Post-exposure 0.04 0.04 0.05 0.06a

SOD (U/mL)

Pre-exposure 229.34 218.31 154.59 207.966.024Post-exposure 226.67 211.98 209.52 192.77

Post-exposure 24.09 27.42 31.59 32.53Triglyceride(mmoL/L)

Pre-exposure 0.93 0.94 0.93a 0.85a

0.017Post-exposure 0.79 0.78 0.69b 0.64b

AST (IU/L)

Pre-exposure 162.1a 169.31a 195.24a 166.222.892

Post-exposure 131.57b 137.21b 158.47b 139.46ALT

(IU/L)Pre-exposure 72.20 99.34 120.91 105.91

3.769Post-exposure 61.52 74.44 105.14 105.21

T3 (nmoL/L)

Pre-exposure 1.56 1.50a 1.70a 1.62a

0.026Post-exposure 1.50 1.23b 1.18b 1.12b

T4 (nmoL/L)

Pre-exposure 38.18 34.64 35.40 30.900.694

Post-exposure 43.80 36.16 38.18 32.20Cortisol

(nmoL/L)Pre-exposure 1.32 1.13 2.50b 1.73

0.769Post-exposure 0.71 1.96 4.56a 3.51

Aldosterone(nmoL/L)

Pre-exposure 0.02a 0.01 0.02a 0.02a

0.000Post-exposure 0.01b 0.01 0.01b 0.01b

*Superscript a, b, within a column differ significantly at P<0.01 for a respective parameter. *The post exposure means except that for triglyceride are adopted from Wankar et al. (2014).


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reported decrease in triglycerides levels during hyperthermia in Friesian cows, due to higher energy demand which confirm present findings. Low Aspartate aminotransferase (AST) activity at optimum condition is quiet surprising but could be due to thermogenic activity, further decrease was probably due increased gluconeogenesis and muscle catabolism during thermal stress (Table 1). Similarly, a significant decrease (P<0.01) was also noted in Merino (84.67 to 30.67 IU/l) and Omani (145.00 to 85.83 IU/l) sheep during heat stress, respectively (Srikandakumar et al., 2003). However, no variation was seen for alanine aminotransferase (ALT) which might be due to absence of any hepatocellular injury or optimum hepatic metabolism. Our results are in accordance with Yokus and Cakir (2006) observing similar non significant variation for ALT in sheep during summer (119.3±291.97 U/l) than winter (29.9±36.05 U/l) months.

Tri-iodothyronine (T3) declined with the heat increment in the present trial (Table 1), probably to reduce the metabolic heat generation as is concerned with thermogenesis in domestic animals (West et al., 2003). Other reason for this decline could be low thyroid gland activity or low levels of thyroid stimulating hormone or higher glucocorticoid activity during heat stress. Similar, decrease in T3 was also confirmed by many researchers in heat stressed animals which validate present findings (Pereira et al., 2008 and Saber et al., 2009). However, thyroxine (T4) did not vary statistically at any treatment which is in accordance to observations made in sheep during hot and cold seasons (Yokus et al., 2006). Cortisol levels were significantly higher at acute thermal stress possibly due to the activation of hypothalamo-pituitary-adrenal axis, facilitating physio-metabolic adaptation (Table 1). Present observations of

increased glucocorticoid agree with previous reports in heat stressed ruminants (Meghad et al., 2008 and Sejian et al., 2010). No notable difference for cortisol at 40oC reflected gradual adaptation and confirm pertinent role of cortisol in both short and long term stress adaptation. Aldosterone decreased after exposure at all the treatments except at 30oC (Table 1). Similarly, Beatty et al. (2006) also reported low plasma aldosterone in Bos taurus and Bos indicus cattle during heat stress. This decrease in aldosterone during acute and chronic heat stress was probably to reduce the potassium losses and to maintain extracellular fluid (ECF) volume. However, the decrease in aldosterone at 25oC is quiet surprising and there are no previous reports in ruminants for confirmation.

Physiological and behavioral responses are activated earliest followed by endocrinal changes, working in unison thus enabling the necessary acclimatization during heat stress. We observed a positive correlation between heat stress and reduction in methane emission which indicate major changes in fermentation and energy metabloism. It can be suggested from the study that heat stress is deleterious for the well being of animals and proper ameliorative measures should be taken to reduce the effect.

REFERENCES

Abeni, F., L. Calamari and L. Stefanini. 2007. Metabolic conditions of lactating Friesian cows during the hot season in the Po valley. 1. Blood indicators of heat stress. Int. J. Biometeorol., 52(2): 87-96.

Aboul-Naga, A.I. 1983. The role of heat induced physiological changes of minerals metabolism in the heat stress syndrome in


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cattle. M.Sc. Thesis. Faculty of Agriculture, Mansoura University, Egypt.

Banerjee, D. and Ashutosh. 2011a. Effect of thermal exposure on diurnal rhythms of physiological parameters and feed, water intake in Tharparkar and Karan Fries heifers. Biol. Rhythm Res., 42(1): 39-51.

Banerjee, D. and Ashutosh. 2011b. Circadian changes in physiological responses and blood ionized sodium and potassium concentrations under thermal exposure in Tharparkar and Karan Fries heifers. Biol. Rhythm Res., 42(2): 131-139.

Baumgard, L.H. and R.P. Rhoads. 2012. Ruminant nutrition symposium: Ruminant Production and Metabolic. J. Anim. Sci., 90: 1855-1865.

Beatty, D.T., A. Barnes, E. Taylor, D. Pethick, M. McCarthy and S.K. Maloney. 2006. Physiological responses of Bos taurus and Bos indicus cattle to prolonged, continuous heat and humidity. J. Anim. Sci., 84: 972-985.

Bernabucci, U., B. Ronchi, N. Lacetera and A. Nardone. 2002. Markers of oxidative status in plasma and erythrocytes of transition dairy cows during hot season. J. Dairy Sci., 85: 2173-2179.

Brambilla, G., M. Fiori and L.I. Archetti. 2001. Evaluation of the oxidative stress in growing pigs by microplate assays. J. Vet. Med. A., 48(A): 33-38.

Calamari, L., F. Petrera, F. Abeni and G. Bertin. 2011. Metabolic and hematological profiles in heat stressed lactating dairy cows fed diets supplemented with different selenium sources and doses. Livest. Sci., 142: 128-137.

Christopherson, R.J. and P.M. Kennedy. 1983. Effect of thermal environment on digestion

in ruminants. Can. J. Anim. Sci., 63: 447-496.

Cunningham, J.G. and B.G. Klein. 2007. Veterinary Physiology, 4th ed. Saunders Elsevier, Missouri, USA.

Derno, M., H.G. Elsner, E.A. Paetow, H. Scholze and M. Schweigel. 2009. Technical note: A new facility for continuous respiration measurements in lactating cows. J. Dairy Sci., 92: 2804-2808.

Dmytruk, O., G.W. Mathison, T.A. McAllister and K.J. Cheng. 1995. Effect of environmental temperature and level of feeding on methane production in steers. Proceedings Western Section American Society of Animal Science, 46: 487-490.

Madesh, M. and K.A. Balasubramanian. 1998. Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J. Biochem. Biophys., 35(3): 184-188.

Marai, I.F.M. and A.A.M. Haeeb. 2010. Buffalo’s biological functions as affected by heat stress-A review. Livest. Sci., 127: 89-109.

Mc Ginn, S.M., D. Chen, Z. Loh, J. Hill, K.A. Beauchemin and O.T. Denmead. 2008. Methane emissions from feedlot cattle in Australia and Canada. Aust. J. Exp. Agr., 48(2): 183-185.

Mclean, J.A. and G. Tobin. 1987. Animal and Human Calorimetry, Cambridge University Press: Cambridge, UK.

Megahed, G.A., M.M. Anwar, S.I.M. Wasfy and E. Hammadeh. 2008. Influence of heat stress on the cortisol and oxidant-antioxidant balance during estrous phase in buffalo-cows (Bubalus bubalis): Thermo-protective role of antioxidant treatment. Reprod. Dom. Anim. 43: 672-677.


42

Pereira, A.M.F., F. Baccari, E.A.L. Titto and J.A. Afonso. 2008. Effect of thermal stress on physiological parameters, feed intake and plasma thyroid hormones concentration in Alentejana, Mertolenga, Frisian and Limousine cattle breeds. Almeida. Int. J. Biometeorol., 52: 199-208.

Saber, A.P.R., M.T. Jalali, D. Mohjeri, A.A. Akhoole, H.Z.N. Teymuourluei, M. Nouri and S. Garachorlo. 2009. The effect of ambient temperature o thyroid hormones concentration and histopthological changes of thyroid gland in cattle in Tabriz, Iran. Asian J. Anim. Vet. Adv., 4(1): 28-33.

Sejian, V., V.P. Maurya, M.K. Sayeed and Naqvi. 2010. Adaptive capability as indicated by endocrine and biochemical responses of Malpura ewes subjected to combined stresses (thermal and nutritional) in a semi-arid tropical environment. Int. J. Biometeorol., 54: 653-661.

Srikandakumar, A., E.H. Johnson and O. Mahgoub. 2003. Effect of heat stress on respiratory rate, rectal temperature and blood chemistry in Omani and Australian Merino sheep. Small Ruminant Res., 49(2): 193-198.

Wankar, A.K., G. Singh and B. Yadav. 2014. Thermoregulatory and adaptive responses of adult buffaloes (Bubalus bubalis) during hyperthermia: Physiological, behavioral, and metabolic approach. Vet. World, 7: 825-830.

West, J.W. 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci., 86: 2131-2144.

Yokus, B., D.U. Cakir, Z. Kanay, T. Gulten and E. Uysal. 2006. Effects of Seasonal and Physiological Variations on the Serum Chemistry, vitamins and thyroid hormone

concentrations in sheep. J. Vet. Med. A., 53(6): 271-276.

Yokus, B. and U.D. Cakir. 2006. Seasonal and physiological variations in serum chemistry and mineral concentrations in cattle. Biol. Trace Elem. Res., 109: 255-266.

Zhao, Q.L., Y. Fujiwara and T. Kondo. 2006. Mechanism of cell death induction by nitroxide and hyperthermia. Free Radical Bio. Med., 40(7): 1131-1143.


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ABSTRACT

In present study, it is investigated the toxins in drinking water samples due to microbe’s activities, very harmful health effect on humans and animals and especially their liver functions can disturb badly. Liver and kidneys problems in cows and buffalos have a major economic impact on the beef and milk processing business for the reason that liver disease problem can shrunk body size and animal performance. For this purpose draw the blood samples of both cows and buffaloes for LFTs (liver function tests) and RFTs (renal function tests) medical laboratory test.This study had the documentary proof the bad health of large animals (cows and buffaloes) that was due to microcystins toxins effects, these findings in case of cows and buffaloes with hepatic problems, collected the 116 samples of buffaloes and 116 of cows, but 82.2% and 87.93% seen as positive in range for microcystins, respectively. In details, animal samples was 116 collected including 50 (43.1% of total sample) cows and 66 (56.8% of total sample) buffaloes but find 47 out of 50 (94%) of cows and 63 out of 66 (95.45%) buffaloes suffered from liver diseases were investigated. Liver swellings were confirmed by performing the LFTs biochemical tests profile

of liver and disease sign and symptoms. Water sources treatment is only solution of these problems; in this case we use the coagulation process with ferric chloride solution of different doses. In this study, it has proved that microcystins are removed using the concentration of ferric chloride dose is 16 mg/l. Liver function tests were also played very important role to know the actual working positions of liver function, so values of Rfts indicated the abnormalities of liver in cows and buffaloes due to continues taking the microcystin toxins from water and food sources.

Keywords: liver function, proteins, blood testing, serum albumin

INTRODUCTION

The cyanobacteria are named as blue-green algae and its name due to the existence of photosynthetic pigments inside cells of cyanobacteria. A cyanobacterium is a main group of bacteria that take place all over the world. Cyanobacteria of freshwater may accumulate on the surface of water supplies in form of blooms,

EFFECTS OF MICROCYSTINS TOXINS CONTAMINATED DRINKING WATER ON HEPATIC PROBLEMS IN ANIMALS (COWS AND BUFFALOS) AND

TOXINS REMOVAL CHEMICAL METHOD

M. Badar1,*, Fatima Batool2, Safder Shah Khan1, Irshad Khokhar1, M.K. Qamar3 and Ch. Yasir1

1Department of Environmental Management, National College of Business Administration and Economics, Lahore, Pakistan, *E-mail: [email protected] Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan3Government of the Punjab, Planning and Development Department, Lahore, Pakistan

Original Article


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it may high concentrate on the surface in form of blue-green is known as scums. Cyanobacteria some species produce toxins, these toxins are categorised according to their type of action with other such as neurotoxins (e.g. anatoxins), hepatotoxins (e.g. microcystins), skin toxin produce irritants as feeling on skins and many other types of toxins producing from various other sources. Hepatotoxins and neurotoxins both are produced by cyanobacteria as normally and it is found on surface of water and they are related to water supplies as drinking source (Abenavoli et al., 2011).

The toxin of Microcystins is the most commonly known as hepatotoxin, and it is the common dose variant with an LD50, have value 50.0 µg/kg in mice observed by method of intra peritoneal injection. Microcystins is 200 times more toxic and poison than cyanide metal. This toxin has structural variants include amino acid substitutions and alterations such as methylation and demethylation. Drinking water supplies contaminated with Cyanobacteria toxins is a main cause of a health hazard for human beings, domestic animals both large and small, and wildlife animals (Badar et al., 2016). Cyanobacteria can be produced both toxins microcystins and nodularin which are known as the hepatotoxins, and they are powerful tumour promoters and can bind to serine and threonine protein phosphatase enzymes and slow the protein activity by reaction mechanism. The results of this hepatotoxicity from the entering ability of microcystins and nodularin to hepatocytes process where they make strong cause hyperphosphorylation of liver proteins and destruction of liver cells (Ayers et al., 2008).

Basically, Liver is important one with the largest part of animal’s body and works as

a central title role in the process of metabolic chemical reactions which essential for the life and survival. It controls several important functions in the body such as proteins, carbohydrates and fat absorption in animals’s body and also excrete the substances formed in the duration of body growth (Bakoyiannis et al., 2013).

Liver function medical tests are very important for the identification of hepatic illnesses in both animals and animals such as, estimation of serum albumin, globulin, ALT, bilirubin, AST and GGT levels along with several specific enzymes as well. But, there is lack of information in literature regarding work on these tests for hepatic insufficiency in buffaloes and cattle. Keeping this in view the present study has pictured with the objective of assessment of some liver function tests for the diagnosis of hepatic insufficiency in clinical cases of infectious and non-infectious diseases in buffaloes and cows under over sampling regime (Crump et al., 2003).

Toxins accumulating within the blood were responsible for other end-organs being damaged. Some of these factors may be due to changes in the cellular component of blood, but many of these deleterious effects are due to changes in the humoral component of circulating blood. These toxins may arise either as a consequence of a failure of normal hepatic functions, or elsewhere in the body as an importance of simple liver disease (da Hora VP et al., 2011).

These toxic factors in the blood affect the function of many organ systems, such as the systemic and portal vasculature and the brain, as well as the liver itself. The exact nature of these toxins is unknown and may be different and multiple for each organ system damaged. Ammonia, aromatic amino-acids, tryptophan, indoles, mercaptans and endogenous benzodiazepines are implicated in the


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development of hepatic encephalopathy (de la cruz et al., 2011).

Whereas, prostanoids, inflammatory cytokines, nitric oxide and oxidative stress, are all considered to be important factors in the development of the haemodynamic and renal changes seen in liver failure. It is, however, the substances that are directly hepatotoxic that are particularly important in terms of recovery, as they may perpetuate liver injury invoking a downward spiral with further reduction in functional liver mass and increased toxin load. It is notable that many of the suggested toxins are insoluble in water and exist in the circulation bound to albumin (Eckburg et al., 2005).

The most commonly used chemicals in this process include aluminium or ferric chloride. More recently some synthetic organic polymers gained some approval. It is very effective for removing the cyanobacteria cells and it is possible to removal soluble microcystins by strong chemical coagulants such as polyaluminium chloride, alum and ferric sulphate. The effectiveness depends on coagulation doses but high dose can produce fungus or algae (Tehrani et al., 2012).

In present study, we are investigating contaminated drinking water toxic effect that microcystins toxins on animal’s basic health and find a possible a method for moving from drinking water.


Collection of drinking water samples Three types of drinking water samples are collected as randomly given in list below, 1. Ground water

2. Canal water 3. Upper water storage tanks And the temperature of the day when collect the samples was 27oC. All sample collect in sterilized PVC bottles as the container and water sample container were filled 100% of the volume capacity. Water sampling is followed the standards methods of water sampling.

Collection of blood samples Blood sampling has been taking day time at 11:00 am after breakfast at their normal body temperature and blood pressure. In animals blood samples collection after surveys and interviews we selected animals (60 cows and 40 Buffaloes) from houses of diseases infected persons. Selected animals blood samples are for toxins analysis and to estimate the live functions strength due to bioaccumulation of toxins. All the samples shifted to Microbiological Division of Chemical Biotech Lab for Biochemical and Microbiological analysis and detail of temperature and humidity given in Figure 1.

Animal blood sampling Collect the random animal blood sampling from different places (animals use for meat and milk) was with the frequency of samples (n=116). All samples were collected by syringe in sterilized blood vessel used as container and blood sample 5 ml collected by volume and actual capacity of container was 5 ml. The temperature of the day when collect the samples was 16oC.

Microcystins toxin testing method ELISA is the most reliable method for rapid screening of samples for detection of microcystins because its sensitivity and specificity


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very clear with ease operation in devices. ELISA assay provide the information about the total toxin concentration in the sample.If the water samples very clear or filtered, then the testing were started with following assay protocol with better sensitivity, where the analysis of the three microcystin-LR calibrators are 0.16, 1.5 and 2.5 ppb performed, were diluted as 1:3 by adding 100 μL of each to 200 μL of kit water and then give the concentrations of calibrators as 0.05, 0.2 and 0.83 ppb respectively (Ethelberg et al., 2004).

Liver function tests Biochemical Testing methods are available for liver function Tests of animals (Buffaloes and Cows) to know how liver damage due to excess inhaling the toxins (Falconer et al., 2005). Blood samples is used for complete count with regard to the liver enzymes, was investigated in a whole blood by help of clinical method (Scott, 2013). Serums of samples were collected after mechanical centrifugation of the samples blood, and start the analysis of clinical chemistries of blood samples. It was used the Chemical reagent (in form kits) to determine concentrations of following parameters in the serum as,

1. ProteinAspartate aminotransferase (AST), Alanine amino transferase (ALT), G-glutamyl transferase (GGT), Alkaline phosphatase (ALP) Total Bilirubin Direct bilirubin

Preparation of coagulants solutionsFerric chloride (FC) In the experiments, it was used the coagulant Ferric chloride as chemical formula (FeCl3. 6H2O). Different composition of solutions

was prepared using calculated amount of Ferric chloride salt dissolved in deionised water. Solution of different concentration used in this study as given (3, 6, 9, 11, 16).

Coagulation experiments At room temperature (27oC), Coagulation experiments were performed using two jar test equipment. In this process, it was taken the untreated water from different three drinking water samples from different sources mentioned previously. The in each jar, sample volume of untreated water was taken 2L, but actual capacity of jar was 5 litter. To calculated the performances efficiency of Ferric Chloride in different concentration (mentioned as 3, 6, 9, 11, 16) as coagulation process for removing the toxins from drinking samples of water (Ho et al., 2011).

RESULTS

Drinking water supplies contaminated with Cyanobacteria toxins is a main cause of a health hazard for animals, domestic animals both large and small. Cyanobacteria can be produced both toxins microcystins and nodularin which are known as the hepatotoxins, and they are powerful tumour promoters and can bind to serine and threonine protein phosphatase enzymes and slow the protein activity by reaction mechanism. In Figure 2, it is clearly observed the cyanbectria population in different samples of drinking water as shown, respectively.

Toxins analysis in blood 1 show that different values of micocystins in blood samples of cows and buffaloes which is not good sign for good health and have bad


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effects on their milk and meats production. Excess concentration of toxins find in animals blood samples are meant that food chain including animals drinking water source is the cyanobacterial contaminated. Cows have more immunity of detoxification of toxins as compare to buffaloes and that is why buffaloes s blood samples with more toxins values as observed in Table 1. 116 blood samples were taken from buffaloes and cows, but 82.2% and 87.93% seen as positive in range for microcystins toxins, respectively. In details, animal samples was 116 including 50 (43.1% of total sample) cattle and 66 (56.8% of total sample) buffaloes. But find 47 out of 50 (94%) of cattle and 63 out of 66 (95.45) buffaloes suffered from liver abscesses were investigated. Liver swellings were confirmed by performing the LFTs tests profile of liver and disease sign and symptoms.

Effect of toxins accumulation in body on liver functions performance According to literature it is confirmed that liver function, performance is disturbed due to accumulation of toxins in the body because the work function of the liver is to detoxify the toxins and other harmful substances but if the toxin load increased liver function values changed and disrobed. Especially ALT liver function tests depend on liver hotness and blood purifications, its value increase showed that blood has high level toxin concentrations. Animals were also infected because their SGPT values in the range of 78 to 82 which is no good sign for milk producing animals, similarly SGOT was high, which was showed, that liver status was not good and these effects may appear in their meat and milk production. Identification of toxins in blood samples showed that toxins present in food chain and this was a serious threat to all

living things. The results of complete LFTs blood tests revealed that all animals had the liver enzymes, there were mild elevation of ALT (80.50±3.90; reference range 5 to 40 units/L) and GGT (24.80±4.30, reference range 12 to 64 units/L) in buffalo animals given in Table 4.3 and, while AST and ALP were also not within reference range in all examined animals (AST: 84±4.1, reference range 5 to 42 units/L, ALP: 416±10, reference range 410 to 420 units/L, all the units values given from kit bio-check company. Similarly in Table 2, it was clearly showed those cattle’s liver enzymes in abnormal form as given values in ascending order because castles had to used canal water for drinking purposes due to some economic reasons under our surveys. We had already discussed the quality results of canal water as previous given in Figure 1. Liver damage situation is a very serious health issue across world due to drinking of bad quality water. Liver function tests are usually represent and recognized as the reliable indicator of liver performance of detoxification function. Inside the liver, enzymatic activity have been raised up, this is may be due to synthesis of enzymes, their low levels indicate that the enzymatic inhibition due to liver injury without specific regeneration. Among liver enzymes, amylase GOT, GPT and ALT were elevated in the samples of animals blood, it was showing acute liver damage (hepatitis), while in samples of animal’s blood, all these enzymes were inhibited showing hypocondition or dysenzymia. The raised values of LFTs showed the enzymatic activity in the blood which attributed to liver damage, while their low values of LFTs showed in Table 2, the regenerative power of liver to minimize and cell membrane may injury. The increased enzymatic activities in the


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Table 1. (Mean±S.D.) values blood samples analysis of animals.

Type of animal blood samples

Microcystin (Toxin)Mean±S.D. Range

Cows (mg/l) 5.7±0.5 1-7.9Buffalos (mg/l) 9.7±1.4 7-11

Table 2. Live function clinical test of animals (buffaloes and cows).

Blood Parameters Unit

Buffaloes blood samples Cows blood samples

Mean±SD Range Reference range Mean±SD Range Reference

rangeSGPT (ALT) U/L 80±3.9 78-82 5.0-40 50±2.9 47-53 5.0-40SGOT (AST) U/L 84±4.1 80-87 5.0-42 72±3.1 68-74 5.0-42Alkaline Phosphatase (ALP) U/L 416±10 410-420 98-279 299±7.8 290-310 98-279

GGT U/L 16.7±1.6 15-18 6.0-8.5 4.2±1 15-18 6.0-8.5Total proteins g/dl 20±2.2 18-22 12-64 7±1.2 12-64 3.5-5.0Globulin g/dl 11±0.9 10-12 1.2-3.2 7±0.7 6-12 1.2-3.2Bilirubin total mg/dl 10±0.7 8-12 0.2-1.2 3±0.4 1-5 0.2-1.2

Table 3. Renal function clinical test of animals.

Parameters UnitsBuffaloes Cows Reference

rangeMean±SD Range Mean±SD Range Urea mg/dl 4.1±1.1 4-5 3.2±0.6 2.5-4 0.9-1.1Creatinine mg/dl 4.7±0.5 4-5.3 3.7±0.4 3-4.3 1.2-1.9Ammonia µg/dl 127±10 120-140 116±10.4 110-127 13.0-108.0Glucose mg/dl 47±3 45-52 60.7±4 55-63 75-115Calcium mg/dl 9.3±0.9 7-11 7±0.9 6-10 1.3-4.6Potassium mg/dl 9±3 10-11 7.2±0.5 6-9 1.5-5.4Sodium mg/dl 1.4±0.3 0.9-2 0.97±0.3 0.7-1 0.9-3


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Figure 1. Condition of temperature and humidity during samples testing in laboratory.

Table 4. Different coagulant doses (Ferric Chloride) for removing toxins from canal drinking water source samples.

Coagulant Dose (mg/l)

Ferric Chloride

Microcystin (mg/l)(Actual value)

Microcystin(mg/l) (Value after treatment)

Microcystin (%)Values after treatment

Mean Range Mean Range Mean Range 3 25 17-27 17 16-19 68 63-74 6 25 16-23 12 10-14 48 45-53 9 22 16-23 9 7-10 40.90 38-4311 22 16-23 5 4-7 22.72 20-2716 22 16-23 2 1.5-3 9.09 7-11


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Figure 2. Detection c in different water samples analysis.

Figure 3. Different doses of ferric chloride used for toxins removing from canal drinking water source samples used for animals.


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liver might be due to increased enzyme synthesis to the damage the liver in the collected samples of humans and animals. The decreased activities of enzymes in liver may be attributed to decreased enzyme synthesis and it may also be due to changes in absorptivity of hepatic cells. Results clear about amylase activity in the present investigation increased in both of samples of cows. Amylase is secreted by the exocrine region of pancreas in mammalians system by help of liver function. The increased activity may be due to pancreatitis or due to the damage of the amylase secretary cells inside body. There is also the possibility that greater amounts of amylase were secreted into the intestine, which causes the consequently enhanced starch digestion, and transferred itself to the degradation products into portal blood, and then into liver and hepatic cells through assimilation, which may also be caused for hyperglycemic response in animals.

Effects of toxins on renal function of kidneysLiver and kidneys are worked together

because if one of them does not give properly routine function its means one of organ in process of damaging. According to the literature, it is sure abnormal values of liver enzyme due to microcystins toxicity may cause of kidneys problems such as metabolic protein convert into ammonia and then urea by liver break down reaction but only kidneys cannot excrete without breaking down. If kidneys work as it is, they must damage because such big molecule can damage the sensitive filtering tissues of kidneys.

Previous studies had shown that liver problems affect the kidneys functions that may lead different bundle of diseases as similar observed in this research. Both human and animals liver and kidneys functions are distributed as shown

in Tables 2 and 3 for liver and renal or kidneys functions tests. A change in the amount of urine produced or blood in the urine may indicate kidney toxicity. Sodium, potassium and calcium are the minerals that show the kidneys performance.

Effect of coagulant ferric chloride on removing of toxins dissolved in drinking water

FeCl3 is an ionic molecules that have a great charge density due to attachment of ferrous metals known as heavy metal. When FeCl3 is hydrolysed in water it decomposes into positive and negative ions. Fe metal is electropositive and very soon attached with organic molecules as waste product in waste water and cause of toxicity. Reaction mechanism of FeCl3 coagulation is explained in literature with good manners as can applicable on this research. If we are compared toxins removing values after FeCl3 coagulation treatment it is clearly proved working efficiency of FeCl3 much times better than alum coagulation as shown in Table 4. Toxins basically have weak electrical change when it comes with high polar atom then it attracts automatically and this reason is enough for removing the toxins from drinking water samples. One is most important use FeCl3 coagulation process, its chemical reaction is going to very sudden because FeCl3 molecule has maximum change density and able to donate the electrons in this chemical reactions. Low concentration of FeCl3 is used as compare to Aluminium salt in this research, this situation is very favourable toward where residues of FeCl3 is less in quantity, this is stopped for further microbes growths as shown in Figure 3.


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DISCUSSION

Dull demeanor, altered appetite and weight loss were recorded in some examined animals. It was measured 55 to 100 beats/minute as heart rate and 15 to 50 breaths/ minute as respiratory rate occurred. Grunting, arched back and other tucked up abdomen were observed in both cows and buffaloes. Thirty Two cows and fifty two buffaloes had scant hard feces, while one cow and three buffaloes had diarrhea. The results of complete blood count revealed that all animals had the liver enzymes, there were mild elevation of ALT (60.50±11.90, reference range 11 to 40 units/L) and GGT (24.80±4.30, reference range 6 to 40 units/L) in four animals (3 cattle, one buffalo), while AST and ALP were within reference range in all examined animals (AST: 80.64±20.20, reference range 78 to 132 units/L, ALP: 140±5.50, reference range 0 to 500 units/L. Liver is highly susceptible for parenchymal, vascular and biliary system lesions. Bacterial, chemical, viral, toxic or immune-mediated insults may cause focal or diffuse hepatic abnormalities or lesions. These signs are not specific signs and considered as general signs for many diseases. These results are partially similar to those described previously in cattle. Other report stated that cattle with liver abscesses seldom exhibit any clinical signs and abscesses can be detected only at the time of slaughter (Hoeger et al., 2002). Moreover, Hypoalbuminaemia with hyperglobulinaemia were reported in all diseased animals under investigation. These results are in accordance with those obtained previously in cattle and in camel. With regard to the liver enzymes (AST, ALT, ALP and GGT) and renal function parameters, blood urea and creatinine

were within normal range in our study, only mild elevation in ALT and GGT were detected in cows and buffaloes. Generally, hematology and liver function tests are reliable indicators of liver abscesses. The usefulness of the technique for diagnosis of various liver abnormalities including abscesses in animals has been well documented (Khan et al., 2001) (Bakoyiannis et al., 2013). In USA, the death rate due to typhoid illness are twenty or more people per hundred thousand of population was measured common, the ratio was 58.7 in Minneapolis, in London the rate was only 3.3 per hundred thousand (AWWA, 1996). However, despite advances in water treatment many people have no reach to adequately treated potable water. The World Health Organisation assessments on 1 billion people internationally no access to safe drinking water sources and 2.6 billion people suffering from poor sanitation. Diseases causes due to unsafe water drinking, poor sanitation and hygiene are as results of expected 1.7 million deaths annually (Lahti et al., 2001).

In developed countries water treatment and sanitation has removed the problem of diseases such as typhoid and cholera. These diseases, however, among other water related issues, remain a serious problem in developing countries. Modern water treatment processes control the spread of water related disease; remove numerous contaminants, such as organic chemicals and heavy metals, producing safe water. However the presence of pharmaceutical residues, disinfection by-products, and the possibility of disease causing agents as Cryptosporidium, which are unaffected by common water purification processes and so, need of new treatment technologies for this purposes (Frank et al., 2008). The single largest consumer issue affecting potable water under developing states is off-flavour. Off-flavour is caused by compounds in


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water that are known for their undesirable taste and odour characteristics. A survey conducted of more than 800 water usages in the America and Canada found the 16% of utilities experience the serious taste and odour problems, spending approximately 4.5% of their total budget for taste and odour control (Lehman et al., 2007).

Nitrogen based biological compound inside samples of canal water can be detached by aluminium sulphate setting if the matter is based on organic compound a minor in quantity, Pietsch et al. (2001) initiate that the removal of nitrogenous matter is problematic to attain with simple coagulation in some cases and the nitrogen based compound are detached by microbial degradation and zonation processes (Lequin et al., 2005).

Moreover, Vilge-Ritter et al. (1999) reported that bio-organic based compounds resemble to if a minor in percentage in the any kind of samples of water; their elimination is so much poor because Aluminium and ferric salts not able to coagulate them. Removal of cyanobacteria and algae in coagulation and clarification process is dependent on optimization of chemical doses of coagulation with Aluminium coagulants (McKaigeny et al., 2013).

Specific dose of Coagulant is essential to removal cyanobacteria and algal cell which is relative to the cell number of logarithm (Perelle et al., 2005). Minimizing turbidity in jar test is not sufficient to remove algae and cyanobacteria toxin. Cyanobacteria will not be removed on insufficient coagulant dose of aluminium sulphate (Radostits et al., 2007). Aluminium sulphate dosed at 20 mg/l without polymer addition removed about 80% of the toxicity from neurotoxic bloom of microcystins, Coagulation had an ability to eliminate the toxins in water samples in several studies (Cobbold et

al., 2004). These studies tested the coagulant as Aluminium sulphate in different concentrations. Coagulation and clarification studies have had mixed results on cell lysis and the subsequent release of cyanobacterial algal toxins (Schlosser et al., 2001).Results of this study shows that microcystin toxins are produced by cyanobacteria that is commonly called blue-green algae although it is really an algae and algal toxins are released from cyanobacteria as a bloom nears the end of its lifecycle, or the cells are lysed (split apart) and the toxins are released.

As previous studies on treatment of potable water using with ferric chloride had shown the results showed that different optimum dose of coagulant can be effected on disinfect the microbes in different types of samples effectively (Schmidt et al., 2002).

Some studies showed a 5 mg/l was ineffective for destroying algal toxin extracts but it is demonstrated that joint flocculation management procedures which involved chlorination 0.7 mg/l dose also successful for killing of microbes (Scott MC et al., 2002).

CONCLUSION

Microcystins are very toxic compounds and their presence may cause of serious acute or chronic toxicity in any level of cattle. Microcystins toxicity can damage the cattle liver easily that can be observed in kidneys functions tests. Kidneys functions tests are also important tools for observing on the filtration process of animals kidneys. High values of Rfts are indicated that kidneys s filtrations process not be normal along losing of animal’s weight and healthy habits.

Large volume of drinking water for animals


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is not easily practice and it is made possible by using the coagulation process by adding the calculating amount of ferric chloride. This process is showed that more than 98% microcystin are removed and finally achieve the desire WHO drinking water limits. So, we can say that WHO drinking water standard for microcystin is free from any toxicity causes in human and animals.

In present study, it is investigate the toxins in drinking water samples from microbe’s activities, very harmful health effect on humans and animals and especially their liver functions disturb badly, for this purpose draw the blood samples of both humans and animals for LFTs medical lab test. Liver abscesses in cattle and buffalos have a major economic impact on the beef and milk processing business for the reason that of liver problem can shrunk body size and animal performance.

REFERENCES

Abenavoli, L., G. Aviello, R. Capasso, N. Milic and F. Capasso. 2011. Milk thistle for treatment of nonalcoholic fatty liver disease. Hepat. Mon., 11: 173-177.

Ayers, T. and I. Williams. 2008. Outbreak net team: electronic foodborne reporting system (eFORS) and national outbreak reporting system (NORS). Presented for the CDC Enteric Diseases Epidemiology Branch Program Plans. Atlanta, GA.

Bakoyiannis, A., S. Delis, C. Triantopoulou and C. Dervenis. 2013. Rare cystic liver lesions: A diagnostic and managing challenge. World J. Gastroentero., 19: 7603-7619.

Badar, M., Irshad Khokhar, Fatima Batool and Ch. Yasir. 2016. Managing the chlorine dose for disinfection of drinking water. Sci. Int., 5:

563-4568.Crump, J., C. Braden, M. Dey, M.

Hoekstra, J. Rickelman-Apisa, D. Baldwin, S. De Fijter, S. Nowicki, E. Koch, T. Bannerman, F. Smith, J. Sarisky, N. Hochberg and P. Mead. 2003. Role of toxins in animal’s liver. Epic. Infect. Disea., 131: 1055-62.

Da Hora, V.P., F.R. Conceição, O.A. Dellagostin and D.L. Doolan. 2011. Non-toxic derivatives of LT as potent adjuvants. Vaccine, 29: 1538-1544.De la cruz, A. 2011. Can we effectively degrade microcystins? implications on human health. Anti-Cancer Agent. Me., 11: 19-37.

Eckburg, P.B., E.M. Bik, C.N. Bernstein, E. Purdom, L. Dethlefsen, M. Sargent, S.R. Gill, K.E. Nelson and D.A. Relman. 2005. Diversity of the human intestinal microbial flora. Science, 308(5728): 1635-1638.

Ethelberg, S., K. Olsen, F. Scheutz, C. Jensen, P. Schiellerup, J. Engberg, A.M. Petersen, B. Olesen, P. Gerner-Smidt and K. Molbak. 2004 Virulence factors for hemolytic uremic syndrome, Denmark. Emerg. Infec. Dis., 10: 410-416.

Falconer, I.R. 2005. Cyanobacterial toxins of drinking water supplies. Cylindrospermopsins and Microcystins, CRC Press, Boca Raton, FL.

Frank, C., S. Kapfhammer, D. Werber, K. Stark and L. Held. 2008. Cattle density and shiga toxin-producing escherichia coli Infection in Germany: Increased risk for most but not all serogroups. Vector-Borne Zoonot., 8: 635-642.

Ho, L., P. Lambling, H. Bustamante, P. Duker and G. Newcombe. 2011. Application of powdered activated carbon for the adsorption of


55

cylindrospermopsin and microcystin toxins from drinking water supplies. Water Res., 45: 2954-2964.

Hoeger, S.J., D.R. Dietrich and B.C. Hitzfeld. 2002. Effect of ozonation on the removal of cyanobacterial toxins during drinking water treatment. Environ. Health Persp., 110: 1127-1132.

Khan, A.S., D.L. Swerdlow and D.D. Juranek. 2001. Precautions against biological and chemical terrorism directed at food and water supplies. Public Health Rep., 116: 3-14.

Lahti, K., J. Rapala, A.L. Kivimaki, J. Kukkonen, M. Niemel and K. Sivonen. 2001. Occurrence of microcystins in raw water sources and treated drinking water of finnish waterworks. Water Sci. Technol., 43: 225-228.

Lehman, E.M. 2007. Seasonal occurrence and toxicity of Microcystis in impoundments of the Huron River, Michigan, USA. Water Res., 41: 795-802.

Lequin, R.M. 2005. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin. Chem., 51(12): 2415-2418.

McKaigeny, C. 2013. Hepatic abscess: Case repot and review. West. J. Emerg. Med., 14: 154-157.

Radostits, O.M., C.C. Gay, D.C. Blood and K.W. Hinchcliff. 2007. A Textbook of the Diseases of Cattle, Sheep, Pigs, Goats and Horses. W.B. Saunders, London. 393-395.

Schlosser, O., C. Robert, C. Bourderioux, M. Rey and M.R. de Roubin. 2001. Bacterial removal from inexpensive portable water treatment systems for travelers. J. Travel Med., 8: 12-18.

Schmidt, W., H. Willmitzer, K. Bornmann and J.

Pietsch. 2002. Production of drinking water from raw water containing cyanobacteria-pilot plant studies for assessing the risk of microcystin breakthrough. Environ. Toxicol., 17: 375-385.

Scott, M.C., G.S. Helfman, M.E. Mctammany, E.F. Benfield and P.V. Bolstad. 2002. Multiscale influences on physical and chemical stream conditions across blue ridge landscapes. J. Am. Water Resour. As., 38: 1372-1392.

Scott, P. 2013. Diagnosis and treatment of liver abscesses in cattle. Livest. Sci., 18: 20-23.

Tehrani, A., J. Javanbakht, M. Hassan, M. Zamani, M. Rajabian, H. Akbari and R. Shafe. 2012. Histopathological and bacteriological study on hepatic abscesses of herrik sheep. Medical Microbiology and Diagnosis, 1: 115-119.

Westrick, J., D. Szlag, B. Southwell and J. Sinclair. 2010 A review of cyanobacteria and cyanotoxins removal/inactivation in drinking water treatment. Anal. Bioanal. Chem., 397: 1705-1714.

Wright, J.M., J. Schwartz and D.W. Dockery. 2004. The effect of disinfection by-products and mutagenic activity on birth weight and gestation duration. Environ. Health. Pers., 112: 920-925.

Wu, W.W., M.M. Benjamin and G.V. Korshin. 2001. Effects of thermal treatment on halogenated disinfection by-products in drinking water. Water Res., 35: 3545-3550.


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ABSTRACT

Xylazine is very popular sedative, analgesic and muscle relaxant. Ketamine is widely used dissociative anaesthetic. The prsesent comminucation gives an insight into the effect of xylazine, ketamine alone and their combination on haemato-biochemical parameters after lumbar epidural administration in buffalo calves.

Keywords: buffalo calves, ketamine, spinal epidural anaesthesia, xylazine

INTRODUCTION

Xylazine was introduced for veterinary use as a sedative, analgesic and muscle relaxant in late 1960‘s (Clarke and Hall, 1969; Kerr et al., 1972). Epidural xylazine produces the local analgesic effect by inhibiting impulse conduction at adrenoreceptors in the CNS and spinal cord. Xylazine is the most popular alpha-2 adrenoreceptor agonist. Xylazine has been used for intraspinal (epidural or subarachnoidal/intrathecal) administration for quite some time now but other alpha-2 agonists have been used for the purpose only in few experimental studies in the recent years. Reports regarding epidural use of xylazine and ketamine alone and its

combination in buffaloes are scanty. Ketamine hydrochloride is a congener of phencyclidine and chemically designated as (2-{0-chlorophenyl}-2 methylaminocyclohexanone). The commercial product is a 50:50 racemic mixture (Ryder et al., 1978). The positive isomer is more potent than negative isomer for analgesic properties (White et al., 1980). Ketamine rapidly crosses the blood brain barrier, quickly enters the brain and the brain/plasma concentration ratio becomes constant in less than one minute. The analgesic properties of ketamine may be mediated via blockade of high affinity monoaminergic uptake sites and inhibition of reuptake of neurotransmitters (Smith et al., 1981). Therefore the present study was undertaken to study the effect of xylazine, ketamine alone and it’s in combination after lumbar epidural administration in buffalo calves.


Non-descript, healthy male buffalo calves (5) ranging from 6 to 8 months of age and weighing from 55 to 75 kg were used in this study. The animals were stall fed, provided clean drinking water and kept under uniform managemental conditions throughout the period of observation. Each animal was kept off feed for 24 h and water was withheld for 12 h prior to start of experiment.

HAEMATO-BIOCHEMICAL RESPONSE TO LUMBAR EPIDURAL ANAESTHESIA USING XYLAZINE, KETAMINE ALONE AND ITS COMBINATION IN BUFFALO CALVES

P.K. Pandey, S.K. Tiwari, Deepak Kumar Kashyap*, Govina Dewangan and Devesh Kumar Giri

Department of Veterinary Surgery and Radiology, College of Veterinary Sciences and Animal Husbandry, Anjora, Durg (C.G.), India, *E-mail: [email protected]

Original Article



58

The animals were restrained in standing position and the lumbar region (between 1st and 2nd) space was prepared for aseptic injection of drug. The animals were divided in 3 treatment groups A, B and C comprising 5 animals in each group. In group A, xyalzine 0.1 mg/kg, group B ketamine 2.50 mg/kg body weight and in group C xylazine 0.1 mg/kg+ketamine 2.50 mg/kg body weight was injected epidurally in lumbar region. The total volume of the drug injected was made 4 ml in all the treatment groups after reconstituting them with distilled water for lumbar epidural injection.

Haematological parameters Venous blood (1 ml) from external jugular vein was collected before and at 30, 60, 120, 240 minutes and 24 h interval after administration of drug in clean dry glass vials containing 2 mg of ethylene diamine tetra acetic acid. The various haematological parameters included estimation of haemoglobin, packed cell volume, total erythrocyte count and differential leucocyte count (Jain, 1986).

Biochemical parameters Venous blood (8 to 10 ml) was collected in dry test tube before and at 60, 120, 240 minutes and 24 h interval following administration of drugs. Serum was separated for estimation of serum glucose, serum total protein, serum urea nitrogen, creatinine and aspartate aminotransferase levels using Computerized Semi-Automated Analyzer (ROBONIK-ASP 300). The data obtained by using different combination of anaesthetic drugs were analysed as per Snedecor and Cochran (1967).


Haematological parametersA non-significant decrease in haemoglobin

concentration in all the groups was observed (Table 1) between half an hour to 2 h of analgesia. The values ranged from 7.82±0.05 gm/dl to 8.62±0.05 gm/dl in different groups of animals at various intervals, which might have resulted from pooling of circulating blood cells in the spleen secondary to decreased sympathetic activity which was also observed in buffaloes (Sharda et al., 2008). The decrease in haemoglobin during the period of anaesthesia might also be due to shifting of fluids from extravascular to intravascular compartment in order to maintain normal cardiac output in the animals (Wagner et al., 1991).

Similarly non-significant decrease in packed cell volume (Table 1) between half an hour to 2 h was observed in all the treatment groups. The mean values ranged from 23.47±0.07 percent to 26.29±0.09 percent in different groups at various intervals. The decrease in packed-cell volume and total erythrocyte count during the period of anaesthesia might also be due to shifting of blood from extravascular compartment to intravascular compartment (Wagner et al., 1991) to maintain normal cardiac output in animals. Non significant decrease was recorded in neutrophil count in all the groups (Table 1) between half and 2 h and the values had reached to near or above the base line within 24 h in all the groups. The mean values ranged from 32.20±0.37 to 34.80±0.09 percent in all the animals of three groups at various intervals. Pooling of the circulating blood cells in the spleen or other reservoirs secondary to decreased sympathetic activity could be the reason for decrease in Hb, PCV and TLC recorded in this study as also reported with other tranquillizers in dog (Soliman


59

Tabl

e 1.

Effe

ct o

n ha

emat

olog

ical

par

amet

ers

afte

r lum

bar e

pidu

ral a

dmin

istra

tion

of X

ylaz

ine,

Ket

amin

e al

one

and

thei

r com

bina

tion

in b

uffa

lo

cal

ves. Pa

ram

eter

sG

roup

sTi

me

Inte

rval

(h)

O½

12

24

Hb

(gm

/dl)

A B C

8.57

±0.0

78.

62±0

.05

8.21

±0.0

4

7.82

±0.0

58.

11±0

.02

7.99

±0.0

5

8.21

±0.0

38.

34±0

.01

8.06

±0.0

6

8.34

±0.0

68.

41±0

.09

8.10

±0.0

5

8.38

±0.0

28.

58±0

.04

8.15

±0.0

5

PCV

(%

)

A B C

24.6

6±0.

1626

.29±

0.09

24.3

9±0.

04

23.7

5±0.

1725

.40±

0.12

24.0

0±0.

02

23.4

7±0.

0726

.04±

0.15

24.1

2±0.

06

24.1

4±0.

0426

.14±

0.06

24.2

0±0.

06

24.5

0±0.

0826

.28±

0.16

24.3

1±0.

02

TLC

(x

103

cum

m-1)

A B C

4.88

±0.0

14.

72±0

.02

4.34

±0.0

1

4.29

±0.0

34.

30±0

.02

3.97

±0.0

1

4.15

±0.0

24.

11±0

.02

3.87

±0.0

1

4.09

±0.0

34.

38±0

.04

3.99

±0.0

1

4.77

±0.0

34.

65±0

.02

4.27

±0.0

3

D L C (%)

I. N

eutro

phils

A B C

33.0

0±0.

7032

.20±

0.37

32.4

0±0.

06

34.0

0±0.

7033

.60±

0.67

33.4

0±0.

06

34.6

0±0.

5034

.40±

0.58

34.4

0±0.

06

34.4

0±0.

3934

.20±

0.37

34.6

0±0.

05

33.6

0±0.

4834

.40±

0.58

34.8

0±0.

39

II. L

ymph

ocyt

esA B C

56.4

0±0.

6757

.40±

0.67

55.4

0±0.

50

55.2

0±0.

6657

.00±

0.54

54.6

0±0.

50

54.8

0±0.

6656

.00±

0.83

53.8

0±0.

37

55.0

0±0.

4456

.60±

0.97

53.6

0±0.

24

55.4

0±0.

4456

.20±

0.81

53.8

0±0.

37

III.

Mon

ocyt

esA B C

5.40

±0.5

86.

00±0

.31

5.00

±0.3

1

4.20

±0.3

74.

80±0

.37

5.20

±0.4

8

3.80

±0.3

94.

40±0

.24

5.40

±0.3

7

4.00

±0.0

04.

20±0

.31

5.00

±0.0

0

4.10

±0.2

44.

20±0

.19

5.20

±0.1

9

IV. E

osin

ophi

lsA B C

5.20

±1.2

44.

40±0

.50

7.20

±0.7

3

6.60

±1.1

64.

60±0

.97

7.00

±0.8

9

6.80

±0.9

45.

20±1

.28

6.40

±0.3

1

7.00

±0.7

25.

00±0

.86

6.80

±0.3

7

6.90

±0.7

95.

20±1

.07

6.20

±0.2

4


60

Tabl

e 2.

Effe

ct o

n bi

oche

mic

al p

aram

eter

s af

ter

lum

bar

epid

ural

adm

inis

tratio

n of

Xyl

azin

e, K

etam

ine

alon

e an

d th

eir

com

bina

tion

in b

uffa

lo

ca

lves

.

Para

met

ers

Gro

ups

Tim

e In

terv

al (h

)O

12

24

Glu

cose

(m

g/dl

)

A B C

60.4

0±0.

9261

.20±

0.66

61.8

0±0.

66

73.2

0±1.

19**

a

69.2

0±0.

70**

b

76.2

0±0.

66**

ab

68.4

0±0.

50**

a

65.0

0±0.

54**

b

82.2

0±0.

86**

ab

62.2

0±0.

7961

.60±

0.37

62.4

0±0.

70

Tota

l Pro

tein

(m

g/dl

)

A B C

6.44

±0.0

96.

61±0

.01

6.65

±0.0

2

6.38

±0.0

96.

46±0

.01

6.57

±0.0

2

6.40

±0.0

96.

52±0

.01

6.59

±0.0

1

6.43

±0.0

96.

56±0

.01

6.61

±0.0

2

BU

N

(mg/

dl)

A B C

6.03

±0.0

66.

04±0

.04

6.17

±0.0

3

6.59

±0.0

2**a

6.19

±0.0

3*b

7.15

±0.0

3**ab

6.41

±0.0

4*a

6.22

±0.0

4**b

7.19

± 0.

03**

b

6.12

±0.0

66.

09±0

.04

6.31

±0.0

4

Cre

atin

ine

(mg/

dl)

A B C

1.09

±0.0

41.

08±0

.01

1.19

±0.0

1

1.89

±0.0

2**a

1.29

±0.0

1*a

1.64

±0.0

1** b

1.91

±0.0

2**a

1.17

±0.0

1a

1.40

±0.0

1**b

1.16

±0.0

11.

13±0

.01

1.20

±0.0

2

AST

(U

/L)

A B C

21.6

0±0.

3924

.60±

0.50

24.2

0±0.

58

23.6

0±0.

39**

a

25.6

0±0.

50b

26.2

0±0.

58**

ab

23.6

0±0.

39a

24.6

0±0.

50a

26.8

0±0.

73**

ab

22.0

0±0.

5424

.20±

0.39

25.4

0±0.

50

Mea

ns b

earin

g di

ffere

nt su

pers

crip

ts d

iffer

Sig

nific

antly

at c

orre

spon

ding

inte

rval

s (P<

0.05

)*P

<0.0

5 =

Sign

ifica

nt a

t 5%

leve

l**

P<0.

01 =

Sig

nific

ant a

t 1%

leve

l


61

et al., 1965). A transient and fluctuating count of monocyte, eosinophil was recorded in all the three treatment groups which varied non significantly.

Biochemical parametersA significant (P<0.01) increase in serum

glucose (Table 2) concentration from 60 to 120 minutes interval was seen following administration of various drug combination epidurally in animals of all the 3 groups. However, the values of glucose increased significantly (P<0.01) from 30 minutes interval in group A and C as compared to group B (ketamine) animals. Thereafter, the glucose level returned gradually to near normally. Hyperglycaemia might have resulted due to increased concentration of adreno-cortical hormone in blood or increased sympathetic activity and suppression of microsomal enzymes (Thurmon et al., 1978) or increased glucose production in liver (Tranquilli et al., 2007). These findings simulates with the observations reported by Raidurg et al. (1995) in cow calves. The decrease in plasma total protein between 1 to 2 h periods following epidural injection of various drugs was non-significant in animals of all the groups. This decrease could be attributed to the increased levels of glucocorticoids, adrenal activity and increased protein turn over resulting in decreased plasma protein and albumin. It is also mentioned that decreased insulin level may modify general metabolism and impair protein synthesis and adrenal steroids also reduce the rate of protein synthesis by antagonizing the effect of insulin (Turner and Bagnara, 1976).

A significant (P<0.01) increase in the values of serum urea nitrogen and creatinine between 60 to 120 minutes post epidural injection was recorded in all the groups (Table 2). Serum urea nitrogen showed a significant (P<0.05)

increase between 1 to 2 h in all the groups during post-injection period. This might be attributed to the temporary inhibitory effect of drugs on renal blood flow, which in turn might have caused a rise in serum urea nitrogen (Kinjavdekar, 1998). A significant (P<0.01) increase in Aspartate aminotransferase (AST) in groups A and C at 1 h and between 1 to 2 h of observation but in group B, a non significant increase was observed. This might be due to the hypoxia produced due to respiratory centre depression in group A and C due to systemic absorption of xylazine. Alpha-2 agonists including xylazine are potent CNS depression agents. Some alternations might also take place in cell membrane permeability which may permit these enzymes to leak from the cells with intact membrane. As the values returned to pre-administration level by 24 h of observation and the values were which the normal physiological range, possibility of pathological changes in liver could therefore, be ruled out. It corroborates with the findings of Koichev et al. (1988) after detomidine administration in cattle and sheep. Non significant changes in group B suggested that changes in AST in groups A and C could be attributed to the effect of xylazine alone.

These observations on various haemato-biochemical parameters suggested that the alterations recorded at various time intervals following epidural injection of xylazine, ketamine along with its combination were not of great magnitude. The changes were transient and more or less same in animals of all the groups and returned to base levels within 24 h. Thus, xylazine, ketamine alone or along with its combination can be safely used for epidural anaesthesia in bovines.


62

SUMMARY

Haemato-biochemical response to lumbar epidural anaesthesia using xylazine, ketamine alone and its combination in buffalo calves has been reported.

REFERENCES

Clarke, K.W. and L.W. Hall. 1969. Xylazine”-a new sedative for horses and cattle. Vet. Rec., 85: 512-517.

Jain, N.C. 1986. Schalm’s Veterinary Haematology, 4th ed. Lea and Febinger, Philadelphia. 1221p.

Kerr, D.C., E.W. Jones, D. Holbert and K. Huggins. 1972. Comparison of the effects of xylazine and acetylpromazine maleate in the horse. Am. J. Vet. Res., 33: 777-784.

Kinjavdekar, P. 1998. Spinal analgesia with alpha-2 agonists and their Combinations with ketamine and lignocaine in goats. Ph.D. Thesis, Indian Veterinary Research Institute Deemed University, Izatnager (Uttar Pradesh), India.

Koichev, K., D. Golemanov, H. Houbenov and B. Aminokov. 1988. Experimental study on the effect of Â“DomosedanÂ” in sheep and cattle. Journal Association of Veterinary Anaesthetist, 15: 114-116.

Raidurg, R., B.N. Ranganath, S.M. Jayadevappa and C.L. Srinivas. 1995. Study of xylazine as an epidural. Indian Vet. J., 72: 894-895.

Ryder, S., W.L. Way and A.J. Trevor. 1978. Comparative pharmacology of the optical isomers of ketamine in mice. International Journal of Pharmacology, 49: 15-23.

Sharda, R., G.K. Dutta, S.K. Tiwari and N. Sharda.

2008. Effect of xylazine, Ketamine and their combination ass epidural anaesthesia in buffalo calves. Indian Vet. J., 85: 608-610.

Smith, D.J., A.J. Azzaro, S.B. Zaldivar, S. Palmer and H.S. Lee. 1981. The properties of the optical isomers and metabolites of ketamine on the high affinity transport and catabolism of monoamines. Neuropharmacology, 20: 391-396.

Snedecor, G.W. and W.G. Cochran. 1994. Statistical Methods, 8th ed. East West Press, New Delhi. 254-268.

Soliman, M.K., S.E. Amrousi and M.Y. Khamis. 1965. The influence of tranquilizers and barbiturate anaesthesia on the blood picture and electrolytes of dogs. Vet. Rec., 77: 1256.

Thurmon, J.C., D.R. Nelson, S.M. Harsfield and C.A. Rumore. 1978. Effects of xylazine hydrochloride on urine in cattle. Aust. Vet. J., 54(4): 178-180.

Tranquilli, W.J., J.C. Thurmon and K.A. Grim. 2007. Lumb and Jones, Veterinary Anaesthesia and Analgesia, 4th ed. Blackwell Publishing, USA. 45-57.

Turner, C.D. and J.T. Bagnara. 1976. General Endocrinology, 6th ed. W.B. Saunders Company, Philadelphia, London.

Wagner, A.E., W.W. Miur and K.W. Hinchoff. 1991. Cardiopul-monary effects of xylazine and detomidine in horses. Am. J. Vet. Res., 52(5): 651-657.

White, P.F., J. Ham, W.L. Way and A.J. Trevor. 1980. Pharmacology of ketamine isomers in surgical patients. Anesthesiology, 52(3): 231-239.


63

ABSTRACT

The present study was to assess the effect of concentrate supplementation during pre-partum and post-partum on the performance of buffaloes. Twenty eight pre-partum buffaloes (10 to 12 weeks before calving) were randomly divided in two groups control (n=14) and treatment (n=14) to ascertain the effect of concentrate supplementation during pre-partum and post-partum and its influence on feed intake, body condition, lactation and reproductive performance in buffaloes. In control group, pregnant buffaloes were fed as per farmer’s practices (0.5 to 1.0 kg concentrate), while in treatment group, pregnant buffaloes were given specially formulated concentrate mixture (CP 22%) 2.0 to 2.5 kg per day. After parturition the lactating buffaloes of treatment and control group were further divided into two sub groups (7 each) to receive either concentrate supplementation or as per farmers’ practices. The basal diet of wheat straw was offered ad libitum. Concentrate supplementation during pre-partum significantly (P<0.05) improved total dry matter intake and body condition score of pregnant buffaloes as compared to control. Concentrate supplementation during postpartum period also appreciably improved feed intake, body condition, lactation and reproductive

performance of buffaloes.

Keywords: supplementation, body condition, lactation, performance, buffaloes

INTRODUCTION

Most of the animals in developing countries including India are fed on agriculture by-products and low quality crop residues, which have got inherent low nutritive value and digestibility. Feeding on such poor quality roughages with imbalanced supplementation is the major reason for poor reproductive and production performance of buffaloes (Qureshi et al., 2002). Under the conventional farming system, diets are not formulated according to the requirements of individual animals, resulting in decreased production and poor health and reproduction (Qureshi, 1995). The lactating buffaloes are fed green fodders plus concentrate feeds but dry and pregnant buffaloes are considered uneconomical and are mostly fed only low quality green fodders. Consequently, animals which receive adequate nutrients have higher body condition scores, which enable them to produce higher quantities of milk and they also are bred earlier.

EFFECT OF PRE AND POST-PARTUM SUPPLEMENTATION TO BUFFALOES ON BODY CONDITION, LACTATION AND REPRODUCTIVE PERFORMANCE

Original Article

B.K. Ojha1,2, Narayan Dutta1, S.K. Singh1, A.K. Pattanaik1 and Amit Narang1

1Centre of Advanced Faculty Training in Animal Nutrition, Indian Veterinary Research Institute, Izatnagar, India2Present Address: College of Veterinary Science and Animal Husbandry, Rewa, India, *E-mail: [email protected]


64

High producing buffaloes in early lactation do not consume sufficient dry matter to support maximal production of milk (Goff and Horst, 1997). Demand for energy is very high during early stage of lactation but supply is not commensurate with demand due physiological stage or limited intake may affects production potential of animal in the whole lactation length (Sirohi et al., 2010). Hence, during early lactation, dairy animals are often forced to draw on body reserves to satisfy energy requirements (negative energy balance); this leads to substantial loss in body weight which adversely affects production, resulting in lower yield (Kim et al., 1993). So, proper pre-partum nutrition is required to maintain post-partum body condition and milk production in buffalo. Little information is available regarding the effect of pre-partum concentrate supplementation in field conditions which has practical applicability. Therefore an attempt was made to assess the effects of concentrate supplementation in the dry period and early lactation on body condition, lactation and reproductive performance in buffaloes.


Experimental design and feeding A set of 20 farm families, collectively owning 28 pre-partum (10 to 12 weeks) buffaloes in their 2nd to 4th parity, were selected for the on-farm trial and randomly divided into two equal groups control and treatment to ascertain the effect of pre-partum supplementation on feed intake, body condition, milk yield and composition and reproductive performance of buffaloes. All the buffaloes were maintained at the livestock owner’s farms under field condition in Bareilly district of UP. In control group, pregnant buffaloes were fed

as per farmer’s practices (0.5 to 1.0 kg concentrate); while in treatment group, pregnant buffaloes were given specially formulated concentrate mixture (CP 22%) 2.0 to 2.5 kg/d. After parturition the lactating buffaloes of control group were further divided into two (7 each) sub groups, one fed as per farmers’ practices (CC) while another strategically supplemented (CT). Similarly, lactating buffaloes of treatment group were divided into two (7 each) sub groups, one group fed as per farmers’ practices (TC) and another group continued to receive the strategic supplementation (TT) during post-partum upto 120 days. The amount of farmers formulated or treatment group concentrate mixture was given individually to each animal 1 kg/2 litres milk production and adjusted fortnightly as per change in the milk yield in consultation with the farmers. The basal diet of wheat straw was offered ad libitum. A small amount of green berseem was also provided to animals to take care their vitamin A requirement. Dry matter (DM) content of feeds offered and leftovers was determined to calculate the daily DM intake of buffaloes.

Body weight and condition score Body weight of the buffaloes was calculated from their heart girth and body length measurements by Shaffer’s formula (Sastry et al., 1982). Body condition score of the buffaloes were performed fortnightly by using a procedure 5 points’ scale (Edmonson et al., 1989).

Sample collection The milk samples were drawn from individual buffaloes during both the times of milking in a day at fortnight intervals. After thorough mixing, a sample of 50 to 100 ml was taken by means of a dipper and transferred to a sample bottle with rounded corners (to avoid


65

lodging of the milk solids) up to 3/4th level, and then bottle was corked tightly by a rubber stopper. The sample bottles were labeled properly and dispatched to laboratory in an ice box. Immediately after estimation of fat content of milk, the samples (50 ml each) were stored at 4oC after adding 2 to 3 drops of potassium dichromate as a preservative, until further analysis. Milk samples were warmed in water bath at 38oC and mixed well for homogenous solution.

Chemical analysis Representative samples of feed offered and residue left were processed and analyzed for proximate principles AOAC (2005). Neutral detergent fibre (NDF) and Acid detergent fibre (ADF) content of feeds and residues were estimated by the method of Van Soest et al. (1991). Milk samples were analyzed separately from each buffalo to determine the percentage of fat, protein, total solids and solids-not-fat (SNF) content. Fat percent of milk was determined by Babcock method as per procedure described by Agarwala and Sharma (1961). Protein was determined by using Kjeldhal Method as described in AOAC (2005). Oven drying method was used for determining Total Solids content of milk as described by Eckles et al. (1951).

Reproduction parameters The signs of oestrus were detected from different signs such as bellowing, mounting over other animals, less interest in feed and vaginal discharge. Animals detected in estrus in morning were inseminated in the evening on the same day and those detected in heat in the evening were served next day in the morning. After 60 days of insemination, pregnancy diagnosis was carried out by rectal palpation of the uterus.

Statistical analysis The data were analyzed statistically using standard methods (Snedecor and Cochran, 2004) for one way analysis of variance (ANOVA) using general linear model of SPSS version 17 and Duncan’s multiple range tests was applied to test the significance. Significance was declared when P value is less than 0.05 unless otherwise stated.


Pre-partum performance The chemical composition (% DM basis) of concentrate mixture, wheat straw and berseem offered to buffaloes during experimental period is given in the Table 1. Pre-partum average DM intake expressed as (kg/d) or live weight (% LW) by buffaloes was significantly (P<0.05) higher in treatment group as compared to control group. Similarly, concentrate and crude protein intake (kg/d) was significantly (P<0.05) higher in treatment group. The dietary treatments had no influence on the body weight changes over the prepartum period (Table 2). The body condition score of pre-partum buffaloes was significantly (P<0.05) higher in treatment group as compared to control. Increasing the concentrate proportion of the diet is one method used to improve energy supply during the pre-partum period of the transition phase. Present results are in agreement with the findings of Kokkonen et al. (2004), who reported that supplying additional concentrate to cattle during pre-partum increases pre-partum DM intake. Similarly, Rabelo et al. (2003) reported that cows fed with high energy diet pre-partum consumed 19% more DM than the cows fed the low energy diet. Contrary to this Reynolds et al. (2004) does not support the conclusion that increasing


66

Table 1. Chemical composition of feeds offered (% DM basis).

Parameter Concentrate mixture

Wheat straw Berseem ControlI TreatmentII

Dry matter 90.94±0.38 90.57±0.29 90.25±0.45 18.40±0.33Organic matter 91.27±0.06 93.25±0.07 94.53±0.22 87.15±0.15Crude Protein 16.48±0.11 22.18±0.10 3.25±0.10 18.90±0.57Ether extract 4.17±0.10 2.85±0.06 1.43±0.13 3.27±0.10Total ash 9.73±0.06 6.75±0.07 5.47±0.22 12.85±0.15NDF 37.89±0.36 25.06±0.29 78.25±0.91 37.54±0.92ADF 13.28±0.14 10.78±0.08 50.89±0.71 24.99±0.89

IComposed of Wheat bran (62%), Rice polish (20%), Mustard cake (15%), Mineral mixture (2%), Salt (1%). IIComposed of Wheat bran (50%), Rice polish (20%), Deoiled Soyabean meal (27%), Mineral mixture (2%), Salt (1%).

Table 2. Mean daily feed intake (kg DM basis) and body weight changes of experimental buffaloes during the pre-partum period.

Attributes Control Treatment SEM P ValueFeed (DM) intakeConcentrate, kg/d 0.66a 2.01b 0.14 <0.01Roughage, kg/d 10.97 11.52 0.30 0.37Total, kg/d 11.63 a 13.53b 0.376 0.01DMI, %BW 2.37 a 2.72 b 0.04 <0.01CPI, g/d 543.79a 962.05 b 40.27 <0.01Body weight changes (kg)Initial (60 d prepartum) 491.72 496.88 13.06 0.84Final (before parturition) 521.24 542.63 13.21 0.43Net gain during prepartum 29.51a 45.75b 1.74 <0.01BCS (60 d prepartum) 2.59 2.69 0.03 0.15BCS at calving 3.34 a 3.51 b 0.04 0.03

abMean values with different superscripts with in a row differ significantly (P<0.05).


67

Table 3. Effect of strategic supplementation on post-partum body condition and voluntary feed intake.

Attributes CC CT TC TT SEM P-valueAverage weight (kg) just after calving

469.66 494.21 464.36 511.59 13.17 0.58

Average weight (kg) at the end of lactation

442.90 490.67 453.60 512.02 13.90 0.26

BCS at one month of lactation 2.65a 2.70 a 2.85ab 2.97b 0.04 0.03BCS at two month of lactation 2.17a 2.23a 2.37b 2.38b 0.03 0.04BCS at three month of lactation 2.12a 2.13a 2.28ab 2.38b 0.03 <0.01BCS at the end of trial (120d) 2.28a 2.35a 2.45ab 2.55b 0.03 <0.01Concentrate DMI, kg/d 1.63a 2.34b 2.52b 3.57c 0.16 <0.01Roughage DMI, kg/d 10.10 10.42 9.88 10.22 0.21 0.96Total DMI, kg/d 11.73a 12.76ab 12.40ab 13.78b 0.27 0.04Total DMI g/kgW0.75 122.60 125.01 127.84 130.41 1.66 0.39Total DMI,% BW 2.62 2.73 2.77a 2.82 0.05 0.85CPI, g/d 792.54a 1134.87b 1117.67b 1399.79c 48.15 <0.01

abcMean values with different superscripts with in a row differ significantly (P<0.05).

Table 4. Effect of strategic supplementation on milk yield and composition in buffaloes.

Attributes CC CT TC TT SEM P-valueMilk yield Whole milk (kg/d) 3.62a 5.10b 5.58b 7.88c 0.35 <0.014 % FCM (kg/d) 5.45a 7.40b 8.15b 10.90c 0.33 <0.01ECM (kg/d) 5.58a 7.67b 8.41b 11.50c 0.37 <0.01Fat yield (g/d) 266.5a 356.7b 394.4b 517.0c 20.94 <0.01Protein yield (g/d) 132.10a 193.58b 206.83b 310.43c 14.29 <0.01Total milk yield (kg) 431.25a 610.00b 666.25b 943.75c 41.78 <0.01Peak yield (kg) 4.08 5.92 6.42 8.83 0.39 <0.01Milk composition (%)Fat 7.34c 7.02b 7.13b 6.60a 0.07 <0.01Protein 3.67a 3.81ab 3.73a 3.95b 0.02 <0.01Lactose 4.81 4.87 4.72 4.61 0.04 0.15Ash 0.77 0.77 0.74 0.76 0.01 0.25SNF 9.27 9.46 9.18 9.33 0.04 0.12Total Solid 16.62 16.48 16.32 15.91 0.08 0.21

abcMean values with different superscripts with in a row differ significantly (P<0.01).


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Figure 1. Effect of strategic supplementation on lactation performance of buffaloes.

Table 5. Effect of strategic supplementation on reproductive performance of buffaloes.

Attributes CC CT TC TT SEM P-valueNo. of Buffaloes 7 7 7 7 - -First postpartum oestrus (days) 115b 110b 99.6a 92a 2.35 <0.01No. of Buffaloes showing oestrus 3 5 5 6 - -No. of inseminations per conception 1.33 1.20 1.20 1.17 0.10 0.96Conception rate (%) 42.85 71.42 71.42 85.71 - -Service period (days) 122b 122.6b 112.20b 96.4a 3.37 <0.01

abMean values with different superscripts with in a row differ significantly (P<0.01).


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the concentrate proportion of the pre-partum diet improves DMI during the prepartum portion of the transition period. The increased BCS in treatment group was due to the higher nutrients (energy and protein etc.) availability to pre-partum buffaloes. Similar findings were also observed by Khan et al. (2004), who reported that BCS for both low and high energy group increased during the pregnancy period but the increment was more for the cows that were kept on high level of energy. Similarly, Mee et al. (2000) reported that cows fed silage and concentrates prepartum had significantly (P<0.05) higher BCS at calving than cows fed silage and straw.

Post-partum performanceFeed intake The overall total DM consumption (kg/d) by lactating buffaloes was 11.73, 12.76, 12.40 and 13.78 in CC, CT, TC and TT groups, respectively, which was significantly (P<0.05) higher in TT

group than CC group, while CT and TC had an intermediate position between TT and CC groups. Similarly DMI when expressed as g/kgW0.75 or % BW did not differ significantly (P>0.05) among the treatment groups (Table 3). The concentrate intake (kg/d) was significantly (P<0.01) higher in TT group followed by comparable intake between CT, TC and CC, respectively, however, roughage moiety did not differ significantly (P>0.05) among the treatment groups. The intake of CP was significantly (P<0.01) higher in TT group followed by comparable intake between CT, TC and CC groups, respectively. The concentrate supplementation of the buffaloes was based on the milk production and there was significant (P<0.01) differences in milk yield of the buffaloes under the four groups hence the DMI through concentrate was significantly (P<0.01) higher in TT group

followed by comparable intake between CT, TC and CC, respectively. The results of present study are in consistency with the findings of Rabelo et al. (2003), who reported that cows fed high energy density diet (1.63 Mcal NEL/kg, 25% NDF and 47% NFC) had higher DMI and energy intake for the first 20 d of lactation as compared to cows fed low energy density diet (1.57 Mcal NEL/kg, 30% NDF and 41% NFC). They further reported that increasing concentrate level of the diet immediately postpartum instead of delaying the increase until d 21 postpartum is related with a higher rate of increase in milk yield and DMI. Similarly, some earlier reports (Dann et al., 1999; Mashek and Beede, 2000; Doepel et al., 2002) also indicated that increased DMI or energy intake during the close-up period improves post-partum intake and performance of cows.

Body weight changes and condition score The post-partum initial and final body weights of dairy buffaloes did not differ significantly (P>0.05) among treatment groups (Table 3). The mean BCS during 3rd to 4th month of lactation was significantly (P<0.01) higher in TT group as compared to CT, CC, however, TC group had an intermediate position. Similar to present results, Singh et al. (2003) reported that average live weights of crossbred cows up to 3rd fortnight decreased at different plane of nutrition, indicating that the stress of pregnancy followed by initiation of milk secretion lead to body weight losses during early lactation. As the stress reduced, animals started gaining weight and the animal fed at higher plane of nutrition (20% above NRC during pre-partum or both during pre-partum and post-partum) gained more weight as compared to those fed as per NRC during 4 to 8th fortnight. Kokkonen et al. (2004) reported that supplying additional


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concentrate pre-partum improves postpartum BCS of the animal. Body condition is a reflection of the body fat reserves carried by the animal. These reserves can be used by the buffalo in periods when she is unable to eat enough to satisfy her energy needs.

Milk yield and composition The daily milk yield, 4% FCM, ECM (kg/d), fat and protein yield (g/d) and total milk yield (kg) were significantly (P<0.01) higher in lactating buffaloes of TT group followed by comparable yield between buffaloes of CT, TC and CC groups, respectively (Table 4). Fortnightly changes in milk yield are depicted in Figure 1, which showed that daily milk yield and peak yield were higher in TT group as compared to other three groups throughout the study period. Present results are in agreement with the findings of McNamara et al. (2003). They fed three pre-partum diets containing grass silage and straw, grass silage alone and grass silage with 3 kg/d of additional concentrate, respectively. Cows fed the pre-partum diet containing silage and straw had lower milk yield than cows fed the silage or silage and concentrate treatments. It appears that additional concentrate pre-partum improves lactation performance when dietary energy is limiting. Similarly, Narang et al. (2012) reported considerable improvement in productivity in terms of total and daily milk yield (kg), and 4% FCM (kg) through strategic supplementation in pre-partum buffaloes. Sihag et al. (2009) reported that by increasing 10% CP in the prepartum diet, milk yield and 4% FCM yield increased significantly (P<0.05). The milk composition is presented in Table 4. Milk fat % was significantly (P<0.01) higher in CC group followed by comparable fat level between CT, TC and TT groups, respectively. Crude protein % of milk was significantly (P<0.01)

higher in TT as compared to CC, TC groups, however, CT group had an intermediate position. The percent total solids, SNF, lactose and ash in milk did not differ significantly (P>0.05) among treatment groups. Similar to present results MacRae et al. (1988) reported that additional protein feeding to lactating cows increases milk and milk protein output. Kokkonen et al. (2004) reported that rapid increase of concentrate tended to increase milk and milk component yields during the first 5 weeks of lactation but at the expense of decreased efficiency of energy utilization. In addition, milk fat content decreased with increased proportion of concentrate in the prepartum diet. Similarly, Murphy (1999) observed that feeding cows more than normal before calving produces milk with slightly high protein content. Singh et al. (2003) reported that the higher level of feeding during pre-partum period alone or both during pre- partum and post-partum period had improved the quality of milk significantly.

Reproductive performance Overall reproductive performance of buffaloes was better in supplemented group during pre-partum and post-partum (TT) than that of three other groups. Time required (days) from calving to first oestrus was significantly (P<0.01) lower for the buffaloes of TT and TC groups than that of CT and CC groups. The days required for calving to conception (service period) was significantly (P<0.01) lower for the buffaloes of TT as compared to comparable days among TC, CT and CC groups. Number of service per conception did not differ significantly (P>0.05) among different groups. Conception rate after insemination was 85.71% in TT group followed by 71.42% in TC and CT and 50% in control (CC) group, respectively. Present results are in agreement with the earlier report of


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Singh et al. (2003), they reported that first post-partum estrus was observed much earlier (P<0.05) in crossbred cows fed at higher plane of nutrition during pre-partum or during pre as well as post-partum periods than the animals fed as per NRC standard. It might be attributed to body weight loss, which might have affected the initiation of ovarian activity after calving. Negative energy balance and rate of mobilization of body reserve appeared to be directly related to the post-partum interval to first ovulation and lower conception rate (Butler and Smith, 1989). Hence, the poor reproductive performances of buffaloes of CC group could be associated to a low BCS. Spitzer et al. (1995) reported that increase in pre-partum BCS was associated with reduced post-partum interval to first estrus (PPI) in beef heifers. This association is explained by the positive relationship that exists between pre-partum BCS and post-partum LH. Luteinizing hormone is responsible for final follicular maturation and ovulation, and ovulation failure is the primary cause of prolonged PPI (Wright et al., 1992). Hassein and Abdel-Raheem (2013) reported that reduction in DMI below NRC recommendations (50%) impairs reproductive performance in terms of reduced folliculogenesis, prolongs the period for attaining puberty, and reduce the pregnancy rate in buffalo heifers. Wongsrikeao and Taesakul (1984) also observed that improved nutrition reduced the post-partum service period in Swamp buffaloes and increased the growth rate in their calves as observed in the present study. From the present results, it can be concluded that considerable improvement in nutrient intake and body condition could be achieved through strategic concentrate supplementation in pre-partum buffaloes. Concentrate supplementation during post-partum period also appreciably improved

overall performance of lactating buffaloes.

ACKNOWLEDGEMENTS

This study was financially supported by funds provided by the Indian Council of Agriculture Research, New Delhi, India.

REFERENCES

Agarwala, A.C. and R.M. Sharma. 1961. A Laboratory Manual of Milk Inspection, 4th ed. Asian Publishing House, Bombay.

AOAC. 2005. Official Methods of Analysis of the Association of Official Analytical Chemist, 18th ed., Horwitz William Publication, Washington DC, USA.

Butler, W.R. and R.D. Smith. 1989. Interrelationships between energy balance and postpartum reproductive function in dairy cattle. J. Dairy Sci., 72(3): 767-783.

Dann, H.M., G.A. Varga and D.E. Putnam. 1999. Improving energy supply to late gestation and early postpartum dairy cows. J. Dairy Sci., 82: 1765-1778.

Doepel, L., H. Lapierre and J.J. Kennelly. 2002. Peripartum performance and metabolism of dairy cows in response to prepartum energy and protein intake. J. Dairy Sci., 85(9): 2315-2334.

Eckles, C.H., W.B. Combs and H. Macy. 1951. Milk and Milk Products, 4th ed. McGrow Hill Book Company, New York-Toranto-London.

Edmonson, A.J., I.J. Lean, L.D. Weaver, T. Farver and G. Webster. 1989. A body condition scoring chart for Holstein dairy cows. J.


72

Dairy Sci., 72: 68-78.Goff, J.P. and R.L. Horst. 1997. Effects of the

addition of potassium or sodium, but not calcium, to prepartum rations on milk fever in dairy cows. J. Dairy Sci., 80: 176-186.

Hassan, H.A. and S.M. Abdel-Raheem. 2013. Effect of feed intake restriction on reproductive performance and pregnancy rate in Egyptian buffalo heifers. Trop. Anim. Health Prod., 45: 1001-1006.

Kim, Y.K., D.J. Schingoethe, D.P. Casper and F.C. Ludens. 1993. Supplemental dietary fat from extruded soybeans and calcium soaps of fatty acids for lactating dairy cows. J. Dairy Sci., 76: 197-204.

Khan, M.A.A., M.N. Islam, M.A.S. Khan and M.A. Akbar. 2004. Effects of feeding high and low energy levels during late pregnancy on performance of crossbred dairy cows and their calves. Asian-Aust. J. Anim. Sci., 17: 947-953.

Kokkonen, T., A.M. Tuori and L. Syrjälä-Qvist. 2004. Concentrate feeding strategy of dairy cows during the transition period. Livest. Prod. Sci., 86: 239-251.

MacRae, J.C., P.J. Buttery and D.E. Beever. 1988. Nutrition interaction in the dairy cow, 55p. In P. Garnsworthy (ed.) Nutrition and Lactation in the Dairy Cow. Butterworths, London, England.

Mashek, D.G. and D.K. Beede. 2001. Peripartum responses of dairy cows fed energy dense diets for 3 or 6 weeks prepartum. J. Dairy Sci., 84: 115-125.

McNamara, S., F.P. O’Mara, M. Rath and J.J. Murphy. 2003. Effects of different transition diets on dry matter intake, milk production and milk composition in dairy cows. J. Dairy Sci., 86: 2397-2408.

Mee, J.F., S.E.M. Snijders and P. Dillon. 2000. Effect of Genetic Merit for Milk Production, Dairy Cow Breed and Pre-calving Feeding on Reproductive Physiology and Performance. Fermoy, Co. Cork: Teagasc.

Murphy, J.J. 1999. Effect of dry protein feeding on post-partum milk production and composition. Livest. Pro. Sci., 57(2): 169-179.

Narang, A., N. Dutta, A.K. Pattanaik, S.K. Singh and K. Sharma. 2012. Effect of pre-partum strategic supplementation on the performance of buffaloes. In Proceeding 8th Biennial ANA Conference, Bikaner, India.

Qureshi, M.S. 1995. Conventional buffalo farming system in north-west Frontier province of Pakistan. Buffalo Bull., 34: 38-41.

Qureshi, M.S., G. Habib, H.A. Samad, M.M. Siddiqui, N. Ahmad and M. Syed. 2002. Reproduction-Nutrition Relationship in Dairy Buffaloes. I. Effect of Intake of Protein, Energy and Blood Metabolites Levels. Asian-Aust. J. Anim. Sci., 15: 330-339.

Rabelo, E., R.L. Rezende, S.J. Bertics and R.R. Grummer. 2003. Effects of pre-and postfresh transition diets varying in dietary energy density on metabolic status of periparturient dairy cows. J. Dairy Sci., 88: 4375-4383.

Reynolds, C.K., B. Dürst, B.D. Lupoli, J. Humphries and D.E. Beever. 2004. Visceral tissue mass and rumen volume in dairy cows during the transition period from late gestation to early lactation. J. Dairy Sci., 87: 961-971.

Sastry, N.S.R, C.K. Thomas and R.A. Singh. 1982. Farm Animal Management and Poultry Production, 2nd ed. Vikas Publishing House Pvt. Ltd., New Delhi, India.

Sihag, S., S.Z. Sihag and D.S. Dahiya. 2009.


73

Effect of higher plane of nutrition on the performance of crossbred pregnant heifers. Indian J. Anim. Nutr., 26(1): 34-39.

Singh, J., B. Singh, M. Wadhwa and M.P.S. Bakshi. 2003. Effect of level of feeding on the performance of crossbred cows during pre- and post-partum periods. Asian-Aust. J. Anim. Sci., 16: 1749-1754.

Sirohi, S.K., T.K. Walli and R.K. Mohanta. 2010. Supplementation effect of bypass fat on production performance of lactating crossbred cows. Indian J. Anim. Sci., 80: 733-736.

Snedecor, G.W. and W.G. Cochran. 2004. Statistical Methods, 9th ed. The Iowa State University Press, Ames, Iowa, USA.

Spitzer, J.C., D.G. Morrison, R.P. Wettemann and L.C. Faulkner. 1995. Reproductive responses and calf birth and weaning weights as affected by body condition at parturition and postpartum weight gain in primiparous beef cows. J. Anim. Sci., 73: 1251-1257.

Van Soest, P.J., J.B. Robertson and B.A. Lewis. 1991. Method of dietary fibre, neutral detergent fibre and non-starch polysaccharide in relation to animal nutrition. J. Dairy Sci., 74: 3583-3587.

Wongsrikeao, W. and S. Taesakul. 1984. Effect of feeding of urea ensiled wheat straw during pre-and postpartum on reproductive performance of buffaloes, 1: 486p. In Proceedings of 3rd Asian-Australasian Association of Animal Production Societies, Seoul, Korea.


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ABSTRACT

An extensive survey was made to collect the information on buffalo husbandry practices in the Panchmahals district of central Gujarat through specially designed questionnaires on buffalo husbandry practices. Buffaloes were reared under intensive and semi intensive system of management. They were allowed for grazing on forage and foliage along with the road side, community land, forest land and fallow field for 4 to 6 h daily in rainy season in the year when green grasses are available. Only 27.08% and 15.42% of the farmers regularly fed common salt and mineral mixture respectively. Majority (72.08%) of the respondent fed concentrate to lactating buffaloes after the milking and 81.25% respondent fed concentrate mixture as a special ration to advance pregnant buffaloes.It was also observed that 95.41% of the respondent resorted to Artificial Insemination and 82.08% inseminate their buffaloes at mid heat stage. Majority (85%) farmer’s believed in quick treatment for anestrous/repeater animals and 69.58% buffalo’s rearers followed pregnancy diagnosis. It was also observed that 73.74% of the respondent got treated their sick animal by live stock inspector /veterinary doctor. Regarding vaccination against foot-and

mouth disease and hemorrhagic septicemia disease 76.25% of the respondents got vaccination their animals. Majority 63.75% of the buffalo’s keeper isolated their sick animals from healthy animals.

Keywords: breeding, buffalo keepers, feeding, health care, management practices

INTRODUCTION

Livestock sector plays a critical role in the welfare of India’s rural population. It contributes 9% of GDP and employs 8% of the labour force. This sector has emerged as an important growth leverage of Indian economy (Kurup, 2000). The role of buffalo as a main milk producing species is well known especially because buffalo is the main source of marketable surplus milk in India. There is no dichotomy about the view that if the buffalo is properly looked after, it can emerge as a more suitable animal for milk production than the imported Holstein (Kurian, 1988). India ranks first in the world with a total of 108.70 million buffalo population (GOI, 2012). Feeding management plays a very significant role in exploiting real potential of dairy animals (Sinha et al., 2009).

Breeding and Health care management like

STUDY OF BUFFALO HUSBANDRY PRACTICES IN RURAL AREA OF CENTRAL GUJARAT IN INDIA

B.S. Khadda1,*, Kanak Lata1, Brijesh Singh2 and Raj Kumar1

1Krishi Vigyan Kendra-Panchmahals, (CIAH-ICAR) Vejalpur, Godhra, Panchmahal, Gujarat, India, *E-mail: [email protected] of Livestock Production Management, College of Veterinary and Animal Sciences, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, India

Original Article


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preventive measures, vaccination, deworming and timely treatments ensure proper health of animals that promotes their productivity. In India 73% households have their own livestock. Tending, grazing, feeding and milking cows and buffaloes is one the largest sources of productive employment in rural India. In Gujarat state, especially in rural areas, the majorities of buffalo keepers are agriculture farmers and have not yet developed a commercial attitude towards dairy farming. Understanding the livestock management practices followed by farmers is necessary to identify the strengths and weaknesses of the rearing systems and to formulate suitable intervention policies. Keeping in view, above a comprehensive study was conducted to find out the various husbandry practices followed by the buffalo keepers in rural area of central Gujarat in the aspects of feeding, breeding and health care management.


The present study was conducted in the tribal dominated area of Panchmahals district of central Gujarat during the year 2012 to 2014. The area of study is characterized as hot semi-arid climate and rainfed farming and livestock husbandry are the way of life of the rural masses. The mean summer temperature is 34.9oC while the mean winter temperature is 21.3oC indicating that the area falls under hyperthermic soil regime. The annual water needed or potential evapotranspiration of the area ranges between 1500 to 1600 mm, whereas actual mean usual precipitation is about 831 mm thus causing an annual water deficit of nearly 769 mm, Rain is confined to three months (July to September) with average rainy days about 31. The mean monthly maximum temperature

ranges from 26 and 40oC, while the minimum monthly temperature varies between 9oC and 26oC. The percentage of buffalo’s population of district is 4.45% of Gujarat state and the breed viz Surti, Mehsana, and Banni are being reared. For data collection, four tehsil i.e. Godhra, Kalol, Goghamba and Jambughoda were selected from the district. Six villages from the each selected tehsil and ten buffalo rearing families from each village were selected randomly. Thus the data for study were collected from a total of 240 household by adopting the Proportionate Random Sampling Method. The data were collected by personal interview techniques through an interview schedule by administrating a developed questionnaire and also by direct observation in the farmer’s flocks. The existing management practices relating to feeding, breeding and health care management were separately enlisted. The collected data were subjected to basic statistical analysis as per Snedecor and Cochran (1989).


Feeding management practicesThe data related to existing feeding

management practices followed by buffalo keepers are presented in Table 1. The results of the present study revealed that the majority of buffalo keepers follow semi stall feeding system (66.25%) followed by stall feeding (33.75%). 65.42% of the respondents are allowed for grazing on forage and foliage along with the road side, community land and forest land and about one third (34.58%) of the respondents grazed their animals on their own pasture land with harvested and fallow field for 4 to 6 h daily in rainy season in the year when green grasses are available. Majority of (87.92%) farmers


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Table 1. Feeding management practices.

Particulars Frequency PercentFeeding system of animals

Stall feeding 81 33.75Semi stall feeding 159 66.25Grazing only 00 00.00

Grazing siteCommon pasture land 157 65.42Harvested/fallow field 83 34.58

Method of feeding for milch animalsGroup feeding 29 12.08Individual feeding 211 87.92

Type of dry fodderPaddy straw 32 13.33Maize, Bajra and Jowar stover 168 70.00Paddy straw+Maize, Bajra stover+Wheat straw 40 16.67

Method of dry fodder feedingAs such 219 91.25Chaffed 21 08.75

Green fodder feeding Yes 240 100.00No 00 00.00

If yes, thenRound the year 91 37.92Seasonal 149 62.08

Method of green fodder feedingAs such 233 97.08Chaffed 07 2.92

Green fodder productionRound the year 91 37.92Seasonal 149 62.08

Type of concentrate mixtureHome prepared 14 5.83Readymade 158 65.84Mixture of home prepared and Readymade 68 28.33

Method of concentrate feedingAs such 00 00.00Soaking 189 78.75Soaking and boiling 51 21.25


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Table 1. Feeding management practices. (Cont.)

Particulars Frequency PercentMode of concentrate feeding to lactating buffaloes

Before milking time 18 7.50At milking time 49 20.42After milking 173 72.08

Concentrates feeding to advanced pregnant buffaloesNo special feeding 00 00.00For last one month 36 15.00For last two month 195 81.25Confirmed to pregnancy 09 03.75

Special feeding after calvingYes 240 100.00No 00 00.00

Concentrate feeding to young calf Yes 240 100.00No 00 00.00

Concentrate feeding to heiferYes 197 82.08No 43 17.92

Quantity of concentrate fed to the lactating buffaloes per day1–2 kg concentrate 19 7.922–3 kg concentrate 189 78.753–5 kg concentrate 32 13.33

Feeding of common saltRegularly 65 27.08Occasionally 78 32.50Not feeding 97 40.42

Feeding of mineral mixtureRegularly 37 15.42Occasionally 104 43.33Not feeding 99 41.25

Frequency of Watering2 times 49 20.423 times 174 72.50Free assess of water 17 7.08


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adopted individual feeding system to their milch animals as well as others to maintain the uniform plane of nutrition for milk production. Adoption of this practice shows full awareness of farmers in this behalf. This finding was in conformity with that of Modi (2003); Patel et al. (2005); Chowdhry et al. (2006) and Sabapara et al. (2010). The study also indicate that the 70% farmers fed their animals Maize, Bajra and Jowar stover as dry fodder followed by Paddy straw and Maize, Bajra, Jowar stover and Wheat straw (16.67%) and rest fed only paddy straw (13.33%). In addition to Maize, Bajra and Jowar stover and Paddy straw all the respondents provided some quantity of dry grasses collected during crop weeding to their animals as dry fodder.

The similar findings were observed by Deoras et al. (2004); Rathore et al. (2010) and Sabapara et al. (2010) in their studies in various regions of India. Majority of farmers (91.25%) practiced to fed dry fodder as such only 8.75% of the farmers offered chaffed dry fodders and all the farmers fed green fodder as such to their animals. It was observed that majority of farmers were unaware about the importance of using chaffed fodders. It might be due to inadequate knowledge of efficient utilization of feed and fodders. All the farmers practiced to feed green fodder to their animals as shown. Cultivation of green fodder Jowar, Maize, hybrid Napier grass and Lucerne is done round the year only by the farmers who had irrigation facilities (37.92%). The majority (65.84%) of the respondent fed readymade concentrate mixture to their animals followed by mixture of home prepared and readymade (28.33%) and home prepared (5.83%).Contrasting to these finding Chowdhry et al. (2006) and Sabapara et al. (2010) reported that majority of the respondents fed home prepared concentrate mixture to their animals. The main reason for fed

readymade concentrate mixture to their animals is to provide cheap and nutritious concentrate mixture for all the dairy farmers on subsidized rate from the Panchmahal District Cooperative Milk Producer Union, Godhra. Regarding pre treatment of concentrate mixture 78.75% of the respondents soaked concentrate mixture before feeding and 21.25% soaked and boiled concentrate mixture before feeding.

These findings are almost similar as observed by Malik et al. (2005); Kumar et al. (2006) and Rathore et al. (2010).Concentrate mixture was offered to the buffaloes twice in a day. Further it was observed that 72.08%, 20.42% and 7.50% of the farmers practiced to fed concentrates after milking, during milking and before milking, respectively. Practice of feeding concentrates mixture after milking was done with the idea to inculcate in them the habit of let down milk without concentrate being offered during milking. The present findings are in conformity with Sabapara et al. (2010). Contrary to report by this Rathore et al. (2010) reported that majority of animals were fed concentrates during milking. The data related to concentrate feeding to advance pregnant buffaloes were encouraging, because majority of buffalo keepers (81.25%) practiced to feed concentrates to their dairy animals during last 2 months of pregnancy. This is a good practice adopted by buffalo keepers because maximum development of fetus occurs during last 6 to 7 weeks of pregnancy. Present finding is an indication of successful communication by the technical agencies working in this area resulted in proper adoption by the farmers.

This finding was in agreement with findings of Sabapara et al. (2010). Modi (2003) and Chowdhry et al. (2006) also reported that the concentrates feeding during last 2 to 4 weeks


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of pregnancy in 70% and 74% of dairy animals, respectively. Their findings are also in agreement with the present findings. All the respondents followed to feed special feeding after calving. They fed energy rich guar, wheat, barley, coconut etc. feed mixed with ajuvayan, Asaliya, Suva, Methi, etc. to prevent stress and to provide sufficient energy for freshening and increasing milk production. Similar findings were reported by (Patel et al., 2005; Sabapara et al., 2010). All the respondents followed to feed concentrate feeding for young calves and majority (82.08%) to feed concentrate feeding to their heifers. Farmers were feeding concentrate to their animals on the basis of their milk production. The majority of (78.75%) farmers fed 2 to 3 kg concentrate to the lactating buffalo per day. Only 27.08% respondents regularly provided extra salt to their milch animals whereas 32.50% of farmers occasionally follow this practice.

Very low percent of followers to feeding extra salt may be due to the practice of feeding compound cattle feed by about 94.17% of the farmers in the present study. Compound cattle feed contains nearly 2% to 3% of salt. Similar findings were reported by Singh et al. (2007a); Rathore et al. (2010) and Sabapara et al. (2010). In contrast to present findings Sohane et al. (2004) and Malik et al. (2005) observed supplementation of common salt followed by 60.74% and 88% respondents, respectively, in their surveys. Mineral mixture supplements were provided regularly by only 15.42% farmers to their milch animals where as 43.33% of farmers follow this practice occasionally. It might be due to the dairy farmers not aware about the benefits of mineral mixture feeding and unwillingness in use due to additional cost of mineral mixture they have to incur for feeding. More or less similar findings were reported by Modi (2003); Sohane et al. (2004); Patel et al.

(2005); Chowdhry et al. (2006); Rathore et al. (2010) and Sabapara et al. (2010). Contrasting to these finding Madke et al. (2006) reported very low (6.67%) of farmers fed mineral mixture to their animals. Almost all farmers provided water to their milch animals ad lib. in quantity but restricted in frequencies in which two and three times 20.42% and 72.50% respondents, respectively in a day were common in summer. Whereas, 7.08% respondents allowed buffaloes to free access for watering as water troughs were attached with manger. These findings are in line with Chowdhry et al. (2006) reported that the 72% of the respondents provide water 3 times a day but as much as the animals can drink.

Breeding management practicesThe results regarding various breeding

practices followed by the buffalo keepers are presented in Table 2. The results of the study revealed that all the respondents followed heat detection practice regularly based upon behavioral signs of estrus only. Among the various behavioral signs of estrus, majority (80.00%) of farmers believed on mucus discharge and bellowing as the symptoms of heat, whereas others trusted only on mucus discharge, frequent urination, mounting and continuously let down of milk as sole symptom of heat. It was also found that estrus symptoms were mostly pronounced in morning or cool hours of day. Similar findings were reported by Patel et al. (2005); Chowdhry et al. (2006) and Sabapara et al. (2010) in North Gujarat. The majority of buffaloes come in heat during the month of October to December. As regards to the stage of heat at which buffaloes were allowed for insemination 13.33%, 82.08% and 4.58% of the respondents followed the practice in early heat, mid heat and later heat, respectively. Majority of farmers (95.41%) used


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Table 2. Breeding management practices.

Particulars Frequency PercentHeat detection

Yes 240 100.00No 00 00.00

Methods of heat detectionSymptoms 240 100.00Teaser 00 00.00

Symptoms of heat detectionMucus discharge 27 11.25Mucus discharge + bellowing 192 80.00

Frequent urination 07 2.91Mounting 11 4.58Any other 03 1.25

Stage of heat at which buffaloes were allowed for insemination/serviceEarly heat 32 13.33Mid heat 197 82.08Later heat 11 4.58

Method of breedingNatural service 06 2.5Artificial insemination 229 95.41Both 05 2.08

Quality of breeding bull if natural service is followPure-bred 11 100.00Nondescript 00 00.00

Pregnancy diagnosis (PD)Yes 167 69.58No 73 30.42

If yes, thenOwn judgments 23 9.59Qualified veterinarian 29 12.08LI or AI worker 188 78.33

Treatment of Anoestrous/repeatersYes 204 85.00No 36 15.00

If yes, thenBy veterinary doctor/stockman 169 70.42

By quacks 71 29.58


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scientific method of artificial insemination (AI) for breeding their dairy animals. Higher proportion for use of artificial insemination may be due to the availability of good infrastructure facilities, for the preservation and timely AI services with satisfactory results provided by AI workers in villages. Similarly, Chowdhry et al. (2006) and Sabapara et al. (2010) observed that majority of farmers adopted AI in dairy animals in North Gujarat. Regarding quality of breeding bull all the respondents used purebred bull. Regarding practice of pregnancy diagnosis was followed by 69.58% of the buffalo keepers, whereas remaining 30.42% of the respondents did not follow pregnancy diagnosis practice for their buffaloes. Among pregnancy diagnosis practice adopted, 78.33% pregnancy diagnosis were done by either livestock inspectors or AI workers followed by qualified veterinarian (12.08%) and own judgments (9.59%) at about 3 months of pregnancy. This finding is in accordance with findings of Sabapara et al. (2010). 85.00% respondents reported that they treated to their buffaloes for anoestrous and repeat breeding. Regarding the treatment of anoestrus and repeat breeding problem, majority 70.42% of the respondents properly treated their problematic buffaloes with the help of veterinary doctor and

stockman.These findings are almost similar to Malik

et al. (2005). Contrasting to this finding Rathore et al. (2010) reported that only 18.00% of the respondents properly treated their problematic cows by veterinary doctor and stockman. The majority (54.58 %) of the farmers breeding their buffaloes 3-5 months after calving followed by 2 to 3 months (38.75%) and after 5 months (6.67%). Calving interval was 10.83%, 82.50% and 6.66% of buffaloes had 12 to 15 months, 16 to 18 months and more than 18 months, respectively. These observations are similar to that of Patel et al. (2005); Chowdhry et al. (2006) and Sabapara et al. (2010) for crossbreed cattle and buffaloes. The results of the present studies are indicative of very high level of awareness regarding this most important economic trait of dairy animal. Thus, it quite evident from the emerging results of various breeding practices followed by the buffalo keepers in the study area that majority of the respondents were adopting the recommended breeding practices.

Health management practices The data related to health management

practices followed by buffalo keepers are

Table 2. Breeding management practices. (Cont.)

Particulars Frequency PercentBreeding after calving

2–3 months 93 38.753–5 months 131 54.58After 5 months 16 6.67

Calving interval12–15 months 26 10.8316–18 months 198 82.5More than 18 months 16 6.66


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Table 3. Health care management practices.

Particulars Frequency PercentVaccination against FMD and HS

Yes 183 76.25No 57 23.75

Deworming of milch animalRegular 64 26.67Occasional 152 63.33Not practiced 24 10

Deworming of calvesRegular 87 36.25Occasional 136 56.67

Not practiced 17 7.08Navel disinfection of calf after birth followed

Yes 31 12.92No 209 87.08

Practices to control ecto- parasitesFollowed 240 100Not followed 00 00

If yes, thenManual 165 68.75Dusting of insecticides 75 31.25

Sanitary condition of shed / shelter / standing placeGood 33 13.75Satisfactory 62 25.83Poor 145 60.42

Treatment of sick animal byVeterinary doctor 52 21.66Livestock Inspector 125 52.08Quacks 63 26.25

Isolate the sick animals from healthy animalsYes 153 63.75No 79 36.25

Availability of veterinary facilitiesGood 63 26.25Satisfactory 140 58.33Poor 37 15.42


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Figure 1. Housing and feeding system at progressive farmer’s flock.

Figure 2. Feeding system at tribal poor farmer’s flock.


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presented in Table 3. The results of the present study revealed that the vaccination was adopted by 76.25% respondents for their animals against foot-and-mouth disease and hemorrhagic septicemia disease, while, 23.75% farmers did not follow vaccination practice against these diseases. No regular vaccination for BQ and anthrax was done in this area. The present findings are encouraging than finding of (Singh et al., 2007 and Sabapara et al., 2010). This practice was widely accepted by farmers which might be due to high level of awareness regarding protecting the animals from diseases by vaccination. Regular deworming in milch animals were followed by only 26.67% respondents whereas 63.33% respondents followed occasionally and remaining 10.00% did not give any medication to control the endo-parasites. This finding is well comparable with finding of Pawar et al. (2006) and Sabapara et al. (2010). It is also observed that very few (36.25%) respondents practiced deworming to their calves at regular interval while 56.67% respondents practiced deworming for calves in occasionally. Majority (87.08%) of respondents did not follow any practice to navel disinfection of calf after birth. However, Pawar et al. (2006) and Rathore et al. (2010) reported cutting and disinfection of navel cord in 31% to 37% cases. All the respondents practice to control ecto-parasites.

The majority (68.75%) of the buffalo keepers follow manual method of picking followed by (31.25%) dusting of insecticides to control to control ecto-parasites. However, Sabapara et al. (2010) reported that the majority of respondents (78.50%) did not follow any practice to control ecto-parasites in south Gujarat. Regarding to Sanitary condition of shed it was found that 13.75% animals sheds cleaned and good condition followed by satisfactory (25.83%). While 60.42% farmers did

not give more attention towards sanitary condition of shed. This might be due to that the farmers not aware about sanitary and hygienic condition in animal shed and insufficient space, inadequate drainage facility in shed thus ultimately leads to dampness and insanitary condition. The majority of (52.08%) respondents got treated their sick animals by livestock inspector followed by 26.25% of the respondents got treated their sick animals by quacks first and if sick animals were not recovered, then they contacted to veterinary doctor or stockman for treatment but that time the condition of sick animal become very serious. Only 21.66% of the buffaloes keepers got treated their sick animals properly by veterinary doctor. Our study revealed that 63.75% of the buffalo keepers isolated their sick animals from healthy ones. This finding is well comparable with finding of Rathore et al. (2010) but lower than reported by Kumar et al. (2006). The percentage of respondents regarding veterinary facilities as good, satisfactory and poor was 26.25%, 58.33% and 15.42% respectively.It is observed that majority of buffalo keepers are not aware about scientific rearing of buffalo’s particularly balanced feeding, vaccination, deworming and health care. Based on the observations collected it may be concluded that enhanced productive and reproductive performance of buffaloes and also a good amount of income can be generated by providing scientific knowledge to the buffalo keepers about buffalo rearing, which will not only be remunerative as source of income for livelihood but also contribute to the nutritional security.

ACKNOWLEDGEMENTS

The authors are thankful to the Director, CIAH Bikaner for encouragement and providing


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facilities and are also thankful to Dr. J.L. Choudhary, Director, Planning and Monitoring, MPAU&T-Udaipur for valuable suggestion and comments in this article.

REFERENCES

Chowdhary, N.R., J.B. Patel and M. Bhakat. 2006. An overview of feeding, breeding and housing practices of dairy animals under milk co-operative system in Banaskantha district of North Gujarat region. Dairy Planner, 5(12): 8-10.

Deoras, R., R.K. Nema, S.P. Tiwari and M. Singh. 2004. Feeding and housing management practices of dairy animals in Rajnandgaon of Chhatisgarh plain. Indian J. Anim. Sci., 74(3): 303-306.

Government of India, (G.O.I. ). 2012. 19th Livestock Census, 2012. Department of Animal Husbandry and Dairying, New Delhi, India.

Kumar, U., R.K. Mehla, R. Chandra and B. Roy. 2006. Studies on managemental practices followed by the traditional owners of Sahiwal cows in Punjab. Indian J. Dairy Sci., 58(2): 123-128.

Kurian, V. 1988. Experience in dairy development in India. In Proceedings of 2nd World Buffalo Congress, New Delhi, India.

Madke, P.K., J.S. Murkute, S.V. Upadhye and C.P. Vedpathak. 2006. Adoption of scientific feeding practices by dairy farmers. Indian J. Anim. Res., 40(2): 155-57.

Malik, B.S., B.S. Meena and S.V.N. Rao. 2005. Study of existing dairy farming practices in Uttar Pradesh. Journal of Dairying, Foods and Home Sciences, 24(2): 91-95.

Modi, R.J. 2003. Study of dairy animal management

practices in Sabarkantha district of North Gujarat. M.V.Sc. Thesis, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar.

Patel, J.B., N.B. Patel, K.B. Prajapti and K.G. Brahmxatri. 2005. Animal husbandry practices for dairy animals in semi-arid region of Patan district, 253p. In Proceeding of National Seminar on Recent Advances in Conservation of Biodiversity and Augmentation of Reproduction and Production in Farm Animals, Sardar Krushinagar Dantiwada Agricultural University, Sardar Krushinagar.

Pawar, B.K., T.H. Nalawade and D.Z. Jagtap. 2006. Adoption of bovine heeding practices and constraints faced by tribal farmers of Pune district. J. Maharashtra Agri. Univ., 31(3): 329-330.

Rathore, R.S., R. Singh, R.N. Kachwaha and R. Kumar. 2010. Existing management practices followed by the cattle keepers in Churu district of Rajasthan. Indian J. Anim. Sci., 80(8): 798-805.

Sabapara, G.P., P.M. Desai, V.B. Kharadi, L.H. Saiyed and R.R. Singh. 2010. Housing and feeding management practices of dairy animals in the tribal area of South Gujarat. Indian J. Anim. Sci., 80(10): 1022-1027.

Singh, M., A. Chauhan, S. Chand and M.K. Garg. 2007. Studies on housing and health care management practices followed by the dairy owners. Indian J. Anim. Res., 41(2): 79-86.

Sinha, R.R.K., T. Dutt, R.R. Singh, B. Bhushan, M. Singh and S. Kumar. 2009. Feeding and housing management practices of dairy animals in Uttar Pradesh. Indian J. Anim. Sci., 79(8): 829-833.

Snedecor, G.W. and W.G. Cochran. 1989. Statistical


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Method, 8th ed. Iowa state University Press, Ames, Iowa, USA.

Sohane, R.K., P.B. Jha and A. Kumari. 2004. Land utilization, feed resources, feeding practices, milk production and disposal pattern in some districts of North Bihar. Rajasthan Agricultural University of Journal Research, 14(1): 157-160.


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Original Article

ABSTRACT

This study aimed to determine utility of a short-term controlled internal drug release (CIDR)-based protocol for hormonal therapy to large ovarian cysts and inactive ovaries in postpartum anestrous Mehsana buffaloes under field conditions in a highland area of Thailand. Anestrous buffaloes were diagnosed by transrectal ultrasonography as having ovarian cysts (n=5) or inactive ovaries (n=14). These 19 buffaloes were submitted to a CIDR-prostaglandin F2α (PGF2α)-pregnant mare serum gonadotropin (PMSG)-based protocol with human chorionic gonadotropin (hCG) injections. Cows that exhibited estrus were bred with bulls. Pregnancy was diagnosed by transrectal ultrasonography at 35 days after mating; the non-pregnant cows were continuously resynchronized by the same protocol. In the initial synchronization and resynchronization phases, the estrous and pregnancy rates did not differ between buffaloes diagnosed with large ovarian cysts or inactive ovaries. Although the pregnancy outcome did not differ between the two groups, 57.9% of the anestrous Mehsana buffaloes became pregnant after therapy with the short-term CIDR-based protocol. Thus, these data demonstrated that a CIDR-PGF2α-PMSG-based protocol with hCG injection effectively treated large cystic and inactive ovaries in infertile buffaloes, allowing

for subsequent pregnancy; this hormonal therapy is recommended for anestrous Mehsana buffaloes under field conditions in the highland areas of Thailand.

Keywords: Mehsana buffaloes, anestrus, highlands, inactive ovary, ovarian cyst

INTRODUCTION

Mehsana buffaloes (Bubalus bubalis) are distributed in Mehsana, Banaskantha and Sabarkantha districts of North Gujarat, India (Pundir et al., 2000; Muhaghegh and Goswami, 2006). The buffaloes are used for milk production. In 1996, the National Dairy Development Board of India provided Mehsana buffalo to the Royal Project Foundation of Thailand; these buffalo are reared in the highlands of northern Thailand. However, a majority of all cases of reproductive failures in postpartum Mehsana buffaloes result from infertility. In buffaloes, the delay in resumption of ovarian cyclic activity has been attributed to the long calving interval (Malik et al., 2011). In buffaloes, postpartum anestrus (inactive ovary) was reported to be as high as 31% to 42% for more than five months after calving (El-Wishy, 2007). Moreover, cystic ovaries have been recognised clinically as a cause of reproductive failures, and are an important

RETURN TO FERTILITY IN POSTPARTUM MEHSANA BUFFALOES AFTER THERAPEUTIC APPROACH FOR LARGE OVARIAN CYSTS AND INACTIVE OVARIES

USING A SHORT-TERM PROGESTIN-BASED REGIME UNDER HIGLAND FIELD CONDITIONS IN THAILAND

T. Moonmanee*, S. Tangtaweewipat, J. Jitjumnong and P. Yama

Department of Animal and Aquatic Sciences, Chiang Mai University, Chiang Mai, Thailand, *E-mail: [email protected]


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ovarian dysfunction in buffaloes. The incidence of cystic ovarian disease has been reported to be 0.5% to 1.4% in buffaloes (Purohit, 2014). Due to these incidences, hormonal therapies are rarely utilised in buffalo reproductive management.

Until now, few studies have focused on a short-term progestin-based therapy for cystic and inactive ovaries in postpartum Mehsana buffaloes under field conditions in a highland area. We hypothesized that a therapeutic approach to treat large ovarian cysts and inactive ovaries with a short-term progestin-based protocol would influence the return to fertility and subsequent pregnancy outcome in postpartum Mehsana buffalo. Therefore, the objective of this present study was to determine utility of a short-term controlled internal drug release (CIDR)-based regime for hormonal therapy to large ovarian cysts and inactive ovaries in anestrous Mehsana buffaloes under field conditions in a highland area of Thailand.


Experimental location, animal and feedingThis experiment was conducted at a

buffalo dairy farm, the Mae Tha Nuea Royal Project Development Center, Thailand, situated at longitude 99o18′06.2″E, latitude 18o41′48.0″N and an altitude of 520 to 1,250 m above sea level (defined here as a highland area). The climate was tropical, with distinct differences between the dry (October to April) and wet (May to September) seasons. Nineteen postpartum anestrous Mehsana buffaloes (>90 days after calving) were treated for cystic and inactive ovaries. All buffaloes received chopped corn stover as roughage ad libitum and concentrate supplementation. Water was available

ad libitum.

Ovarian diagnostic ultrasonographyCysts and inactive ovaries were diagnosed

by transrectal ultrasonographic genital monitoring with a 7.5 MHz rectal transducer (HS-1600 V, Honda Electronics, Japan). The buffalo cows were diagnosed with large ovarian cysts, if they had anovulatory follicle-like conformations (fluid-filled structures) larger than 25 mm in diameter on their ovaries (Noseir and Sosa, 2015) that persisted for more than 10 days without a corpus luteum (CL) (Purohit, 2014). The diagnostic criterion for inactive ovaries was the absence of luteal structures and follicles more than 10 mm in diameter (Yotov et al., 2012; Ingawale and Bakshi, 2016). Based on the transrectal ultrasounds of the ovarian structures, postpartum anestrous buffaloes were diagnosed with either cystic ovaries (n=5) or inactive ovaries (n=14).

Short-term CIDR-based protocol and pregnancy diagnosis

Nineteen Mehsana buffaloes, categorised into cystic and inactive ovarian groups, were submitted to a short-term CIDR-based protocol. Hormonal synchronization was adapted from previously described procedures (Yotov et al., 2012). Briefly, buffalo cows were treated with an intravaginal CIDR insert (Eazi-Breed CIDR, Zoetis Limited, Thailand) containing 1.9 g of progesterone (P4) for 7 days. On the day of the CIDR withdrawal, 25 mg of prostaglandin F2α (PGF2α; Dinoprost, Lutalise, Zoetis Limited, Thailand) and 500 IU of pregnant mare serum gonadotropin (PMSG; Folligon, MSD-Animal Health, New Zealand) were injected intramuscularly. Two days after CIDR removal, buffalo cows received 500 IU of human chorionic


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gonadotropin (hCG; Chorulon, MSD-Animal Health, New Zealand). After hCG administration, all cows were observed for estrus with a teaser bull. Commencement of estrus was defined as the time when the cows displayed mounting behavior followed by vaginal mucus discharge, as well as enlarged vulva and vagina (Singh, 2003). Then, the cows that exhibited estrous behavior were bred with a Mehsana bull. Pregnancy was diagnosed by transrectal ultrasonography of uterine contents and fetal structures at 35 and 60 days after mating. Buffaloes diagnosed as not pregnant at day 35 were continuously resynchronized for a second round of hormonal therapy by a short-term CIDR-based protocol.

Statistical analysisThe proportion of buffalo cows in estrus

and pregnant were analyzed using chi-square analysis (Steel et al., 1997) by procedure of SAS (SAS Institute Inc, Cary, NC, USA). Differences with P<0.05 were considered significant.


The incidence of infertility was observed, with the majority of the buffaloes having inactive ovaries (73.7%, Figure 1a) and some of them having large ovarian cysts (26.3%, Figure 1b). The present study provides the first description of the incidence of reproductive failures from inactive ovaries and large ovarian cysts in postpartum Mehsana buffaloes under field conditions in a highland area. This observation is inconsistent with Modi et al. (2011), who reported that the incidences of true anestrus and follicular cysts in dairy buffaloes in the Mehsana milk-shed area of Gujarat (India) were 20.84% and 0.07%, respectively.

In the initial synchronization and resynchronization phases, the estrous and pregnancy rates of buffaloes diagnosed with large ovarian cysts or inactive ovaries did not differ (Table 1). Overall, the pregnancy outcome after synchronized treatment did not differ significantly between inactive and cystic ovarian buffaloes (Table 1). Although the incidences of smooth inactive ovaries and large ovarian cysts in anestrous Mehsana buffaloes did not demonstrate a difference in the pregnancy outcome after therapy with the short-term CIDR-based protocol, more than 20.0% and 33.3% of the non-pregnant buffaloes in the two groups were diagnosed with now active ovaries, respectively (Table 1, Figures 1c and 1d). The estrous and pregnancy rates of the anestrous buffaloes did not differ significantly between the initial synchronization and resynchronization phases (Table 2).

These results demonstrated that treating postpartum anestrus Mehsana buffaloes with a hormonal protocol did not significantly different between those with cystic ovaries or inactive ovaries. The results of this study support the hypothesis that hormonal therapy for cystic ovarian and inactive ovarian buffaloes benefited the pregnancy outcome. More than 64.3% and 40.0% of the anestrous Mehsana buffaloes with inactive ovaries and large ovarian cysts, respectively, in the present study returned to pregnancy after initial synchronization and resynchronization using a short-term CIDR-based protocol. This was consistent with Yotov et al. (2012), who demonstrated that the pregnancy rate of buffalo diagnosed with inactive ovaries was 55.6% in the Murrah breed after therapy with a progesterone-releasing intravaginal device (PRID)-based protocol. Regardless of the cause of anestrous (cystic ovaries or inactive ovaries) in Mehsana


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Figure 1. Sonogram images illustrate smooth inactive ovary (a: small arrowhead) and large ovarian cyst (b: arrow) of anestrous Mehsana buffaloes. After therapy with a short-term CIDR-based protocol, sonogram images illustrate non-pregnant buffaloes with active ovaries (c and d), as indicated by the appearance of a CL (large arrowhead) and/or follicles more than 10 mm in diameter (dotted arrows). The black areas in the sonogram images correspond to the follicle areas.


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Table 1. Estrous and pregnancy responses of anestrous Mehsana buffaloes after therapy for large ovarian cysts and inactive ovaries with a short-term, CIDR-based regime.

ItemBuffalo group

Cystic ovary Inactive ovaryInitial synchronization

Buffalo cows (no.) 5 14Estrous rate (%) 40.0 (2/5) 50.0 (7/14)Pregnancy rate (%) 50.0 (1/2) 42.9 (3/7)

ResynchronizationBuffalo cows (no.) 4 11Estrous rate (%) 50.0 (2/4) 72.7 (8/11)Pregnancy rate (%) 50.0 (1/2) 75.0 (6/8)

Pregnancy outcome (%) 40.0 (2/5) 64.3 (9/14)Non-pregnant buffaloes with active ovaries (%)a 33.3 (1/3) 20.0 (1/5)

aThe percentage of non-pregnant buffaloes with active ovaries was calculated per total buffalo cows for return to active ovaries, as indicated by the appearance of a CL and/or follicles more than 10 mm in diameter.

Table 2. Estrous and pregnancy responses of anestrous Mehsana buffaloes after initial synchronization and resynchronization with a short-term CIDR-based regime.

ItemSynchronization phase

Initial synchronization ResynchronizationBuffalo cows (no.) 19 15Estrous rate (%) 47.4 (9/19) 66.7 (10/15)Pregnancy rate (%) 44.4 (4/9) 70.0 (7/10)Pregnancy outcome (%) 21.1 (4/19) 46.7 (7/15)


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buffaloes, a short-term CIDR-PGF2α-PMSG-based program with hCG injection influenced estrous activity and increased the pregnancy outcome by 57.9%. It has been suggested that a P4-PGF2α-PMSG-based protocol for treating cystic ovaries and stimulating ovarian activity in buffalo cows (Yotov et al., 2012) activates the maturation of dominant follicles, either directly through the action of PMSG or indirectly by P4 device synchronizing a surge of luteinizing hormone (LH) (Naseer et al., 2013). In fact, the successful therapy of ovarian cysts was improved 65% to 80% by stimulating the LH concentration with hCG. The hCG was used because of its greater LH activity (Noseir and Sosa, 2015). Moreover, P4 device disintegrates cysts by resuming the responsiveness of the hypothalamus to the positive feedback mechanism of estradiol, which influences atresia of cysts, and subsequently retunes estrus and ovulation after P4 device withdrawal (Jeengar et al., 2014).

Based on our results, 1) stimulating the endocrine milieu that enhanced the follicular growth by using PMSG, 2) inducing the atresia of cysts by using a CIDR device, and 3) influencing the ovulatory activity by using hCG improved the traditional therapeutic treatment approach for large ovarian cysts and inactive ovaries in anestrous buffaloes. Thus, a CIDR-PGF2α-PMSG-based protocol, in combination with hCG, effectively treated large cystic and inactive ovaries in infertile Mehsana buffaloes, returning the cows to fertility; this hormonal therapy is recommended for anestrous Mehsana buffaloes under field conditions in the highland areas of Thailand.

ACKNOWLEDGEMENTS

This work was supported by the Royal

Project Foundation of Thailand under grant number 3045-A067. We would like to acknowledge the Research Administration Center, Office of the University, Chiang Mai University, Thailand for improving the English in this paper.

REFERENCES

El-Wishy, A.B. 2007. The postpartum buffalo. II. Acyclicity and anestrus. Anim. Reprod. Sci., 97: 216-236.

Ingawale, M.V. and S.A. Bakshi. 2016. Effect of early post-partum GnRH and PGF2 alpha administration on follicular activities in Murrah buffaloes. Buffalo Bull., 35: 317-324.

Jeengar, K., V. Chaudhary, A. Kumar, S. Raiya, M. Gaur and G.N. Purohit. 2014. Ovarian cysts in dairy cows: old and new concepts for definition, diagnosis and therapy. Anim. Reprod., 11: 63-73.

Malik, R.K., P. Singh, I.J. Singh, R.K. Sharma, S.K. Phulia, R.K. Tuli and R.K. Chadolia. 2011. Ovarian response and fertility of Ovsynch-treated postpartum anestrus Murrah buffaloes. Buffalo Bull., 30: 272-276.

Modi, L.C., P.A. Patel, S.P. Patel, A.H. Joshi and D.N. Suthar. 2011. Prevalence of reproductive problems in buffalo in Mehsana milk-shed area of Gujarat. Int. J. Agro. Vet. Med. Sci., 5: 424-428.

Muhaghegh, M.D. and S.L. Goswami. 2006. Single strand conformation polymorphism (SSCP) in 3’ region of growth hormone gene in five breeds of Indian buffalo. Anim. Sci. Pap. Rep., 24: 159-162.

Naseer, Z., E. Ahmad, N. Ullah, M. Yaqoob and Z. Akbar. 2013. Treatment of anestrous


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Nili-Ravi buffaloes using eCG and CIDR protocols. Asian Pac. J. Reprod., 2: 215-217.

Noseir, W.M.B. and G.A.M. Sosa. 2015. Treatment of ovarian cysts in buffaloes with emphasis to echotexture analysis. J. Dairy Vet. Anim. Res., 2: 1-7.

Pundir, R.K., G. Sahana, N.K. Navani, P.K. Jain, D.V. Singh, S. Kumar and A.S. Dave. 2000. Characterization of Mehsana buffaloes in India. Anim. Gen. Res. Inform., 28: 53-62.

Purohit, G.N. 2014. Ovarian and oviductal pathologies in the buffalo: Occurrence, diagnostic and therapeutic approaches. Asian Pac. J. Reprod., 3: 156-168.

Singh, C. 2003. Response of anestrus rural buffaloes (Bubalus bubalis) to intravaginal progesterone implant and PGF2α injection in summer. J. Vet. Sci., 4: 137-141.

Steel, R.G.D., J.H. Torrie and D.A. Dickey. 1997. Principles and Procedures of Statistics: a Biometrical Approach, 3rd ed. McGraw-Hill Press, New York, USA, 256p.

Yotov, S., A. Atanasov and Y. Ilieva. 2012. Therapy of ovarian inactivity in postpartum Bulgarian Murrah buffaloes by PRID and Ovsynch estrus synchronization protocols. Asian Pac. J. Reprod., 1: 293-299.


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ABSTRACT

The aim of the present study was to evaluate the response of progesterone administration on resumption of cyclicity on post-partum anestrus buffaloes located in and around Danapur, Patna, Bihar area. Only those buffaloes were selected which did not show sign of estrus for one year after parturition and were maintained in small unorganized herd. The investigation was conducted in hot summer months (viz. March to June) season. The enumerated buffaloes were examined per rectally and a total of 38 apparently healthy buffaloes were selected with normal genitalia without having palpable corpus luteum on ovaries and pathological lesion. Selected animals were subjected to deworming prior to this study with broad-spectrum anthelmintic Fenbendazole (3 grams) once. The animals were first treated with PGF2α at the dose rate of (25 mg) intramuscularly on day ‘0’. Out of 38 animals 2 buffaloes that had shown signs of estrus have been dropped and they were discarded from further investigation. Finally total of 24 buffaloes were selected randomly and divided into 4 groups (6 animals in each group) to observe the effect of progesterone (Duraprogen, 250 mg) at different dosages on resumption of cyclicity. Animals of

Group control were treated with normal saline (2 ml) on day 39, 43, 47 and 51 while those under Group T1,, T2 and T3 were treated with Duraprogen intramuscular on day 39. The animals of Group T1 were further treated with Duraprogen on day 41, 43, 45, 47 and 49 (i.e. on alternate day). The animals of Group T2 were further treated with Duraprogen on day 43, 47 and 51 (on three day interval) while the animals of group T3 were further treated on day 46 (on six day interval). Results showed that in group T1, animals treated with progesterone (Duraprogen

TM250 mg,1ml) I/M on alternate day (i.e. day 39, 41, 43, 45, 47 and 49), five out of 6 animals showed signs of estrus. Two animals showed signs of estrus on day 44 while other three showed the estrus sign on day 45, 46 and 49. The intensity of estrus was moderate in 2 animals and strong in 3 animals. In group T2, where animals were treated with progesterone (Duraprogen TM250 mg, 1 ml) I/M on every 4th day (on day 39, 43, 47, 51), three out of 6 animals showed signs of estrus. Two animals showed sign of estrus on day 46 and one on day 50. The intensity of estrus was moderate in 2 animals and strong in 1 animal. In group T3, where animals were treated with progesterone (Duraprogen TM250 mg) I/M on every 7th day after administration of 1st injection (on day 39 and 46), one out of 6 animals showed sign of estrus on day 43 and the intensity

STUDY THE RESPONSES OF PROGESTERONE ADMINISTRATION ON RESUMPTION OF CYCLICITY ON POST-PARTUM ANESTRUS BUFFALOES

Deepak Suvarn1, C. Singh1 and M.M. Ansari2,*

1Department of Physiology, Bihar Veterinary College, Patna, Bihar, India2Division of Veterinary Surgery and Radiology, Faculty of Veterinary Sciences and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shuhama, Alesteng, Jammu and Kashmir, India, *E-mail: [email protected]

Original Article


98

was moderate. Moreover, resumption of cyclicity was highly significant in group T1 in which repeated dosages of progesterone (Duraprogen TM250 mg, 1 ml) i.e. on alternate days, were administered. The effect of progesterone on cyclicity decreases with decrease in frequency of dose i.e. on three day interval and on six day interval.

Keywords: postpartum anestrus, cyclicity, buffalo, progesterone

INTRODUCTION

Indian economy is predominantly agrarian and livestock constitutes an integral part of the agrarian economy. The overall output from livestock sector is about 25% of the total output from agriculture sector in which milk alone constitutes around 63% to the total output from the livestock. Dairying provides millions of small marginal farmers and landless labours means for their subsistence. India has the largest holding of bovine population, about one third of the world’s population and about half of the Asian population. India is now the largest milk producer in the world producing about 84.6 million tones of milk per annum (India, 2004). Buffalo alone accounts for 53% of total milk production in India. So its importance cannot be ignored in dairy industry. Full genetic potential in terms of milk production from buffalo can be explored only when the reproduction is normal.

In India delayed puberty, acyclicity after attaining puberty, and post-partum anestrus which lead to prolong inter calving period are major causes of poor reproductive efficiency in cattle and buffaloes. Real anestrus with inactive, smooth, small and round and flat ovarian condition

is a major limiting factor in greater utilization of artificial insemination for rapid improvement of livestock productivity. This also results in loss of production and increased cost of maintenance. The problem of anestrus has been recognized as having moderate to high incidence affecting the fertility of the animal vis-à-vis economy of the farmer (Kurien and Madhavan, 1985; Kumar et al., 1986 and Sinha et al., 1987).

Anestrus is one of the most commonly occurring reproductive problems in cattle and buffalo in India, affecting livestock productivity and enterprise to a great extent. Buffalo has higher incidences of functional anestrus than cattle as the post-partum estrus interval is longer and especially during summer. The problem is more severe in sub urban and rural areas of the country. It is a functional disorder of the reproductive cycle which is characterized by absence of overt signs of estrus manifested either due to lack of expression of estrus or failure of its detection. Anestrus is observed in post pubertal heifers, during pregnancy, lactation and in early postpartum period in adult animals. The condition may be associated with uterine pathology such as pyometra, fetal resorption, maceration and mummification. Expression of estrus is also influenced by seasonal changes, stress and aging. In heifers, it poses a herd problem possibly due to low plane of nutrition, stress of seasonal transition or extremes of climatic conditions. Expression of overt signs of estrus is greatly affected by heat stress in buffaloes. Besides breed and climate, management and nutrition also play vital role in determining the reproductive disorder in cattle and buffalo. Reproductive failures such as anestrum, repeat breeding and pathological condition of the genital tract suggest the nutritional deficiencies, hormonal imbalance and deranged enzymatic activity affect the normal reproductive behavior


99

of the animal, causing serious morphological and physiological alterations (Roberts, 1971). Nutritional deficiencies and excesses may cause infertility. They may act via the hypothalamus and anterior pituitary thus influencing the production of gonadotropins or directly on the ovaries, thus influencing oogenesis and endocrine function. Considering above mentioned point in view, it is proposed to investigate the effect of progesterone (Duraprogen TM250 mg, 1 ml) administration on resumption of cyclicity on post-partum anestrus buffaloes and as a managemental aid for better reproductive efficiency located in and around Danapur (Patna) area.


The present study was done in and around the rural area of Danapur situated on western proximity of Patna, Bihar, India. The experiment was based upon small and unorganized dairy units popularly known as ‘Khatals’. Danapur is located 25º36’ North (latitude) and 85º06’ East (longitude) at an altitude of about 60 meters from mean sea level. The total annual rainfall ranges from 100 to 120 cm and the maximum temperature goes above 38º C during May to June.

At first, the private dairy units distributed in the study area consisting of non-descript buffaloes were enumerated through a “door to door” survey method. Only those buffaloes, which did not show estrus up to one year after parturition were selected for this investigation. The selected buffaloes were examined per-rectally for their reproductive status. The animal showing infectious conditions like pyometra, metritis etc. was not selected. Their rectal temperature, respiration rate, pulse rate and ruminal motality rate were also recorded and those within

the normal range were selected. Selected buffaloes having normal physique and reproductive status were subjected to deworming prior to this study with broad spectrum anthelmentic Fenbendazole at dose rate of 1.5 gm once and found negative for parasitic infestation were taken for the experiment.

The selected buffaloes were treated with LutalyseTM (PGF2α analogue) at the dose rate of 25 mg, intramuscular per animal. The day of administration of the drug was taken as day 0. The animals showing signs of estrus after the treatment were discarded from present investigation and allowed for insemination. Out of 38 animals 2 buffaloes that had shown signs of estrus have been dropped and they were discarded from further investigation. Thus out of 36 animals, only 24 animals were selected randomly and divided into four groups having six animals in each group. The animals of control group were treated with 2 ml normal saline (N.S.) on day 39, 43, 47 and 51 while the animals of Group T1, T2 and T3 were treated with 1 ml progesterone (DuraprogenTM 250 mg) intramuscularly on day 39. The animals of Group T1 were further treated with 1 ml progesterone (DuraprogenTM 250 mg) on day 41, 43, 45, 47 and 49 (i.e. on alternate day). While those under Group T2 were further treated with 1 ml progesterone (DuraprogenTM 250 mg) on day 43, 47 and 51 (i.e. three day interval) and those under Group T3 were further treated on day 46 (i.e. six days interval).


The effect of progesterone (Duraprogen

TM250 mg, 1 ml) on cyclicity was summarized in Table 2 and 3. In control, no animal showed sign of estrus. In group T1, five out of 6 animals showed signs of estrus. Two animals showed signs of estrus


100

Table 1. Experimental protocol.

GroupNo. of animal

Treatment

Control 6 Injection of normal saline 2 ml I/M on every 4th day (i. e. 39 th, 43 th, 47 th and 51 th)

T1 6 Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on alternate day (i.e. day 39th, 41st, 43rd, 45th, 47th and 49 th)

T2 6 Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on every 4th day (i.e. day 39th,43rd, 47th and 51st)

T3 6 Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on every 7th day after administration of 1st injection (i.e. day 39th and 46th)

*DuraprogenTM having 17-α-Hydroxyprogesterone Caproate.

on day 44 while other three showed the estrus sign on day 45, 46 and 49. The intensity of estrus was moderate in 2 animals and strong in 3 animals. In group T2, three out of 6 animals showed signs of estrus. Two animals showed sign of estrus on day 46 and one on day 50. The intensity of estrus was moderate in 2 animals and strong in 1 animal. In group T3, one out of 6 animals showed sign of estrus on day 43 and the intensity was moderate.

Detection of estrus in 5 (83.33%) out of 6 buffaloes of Group T1 receiving 250 mg of progesterone on alternate day, suggests sensitization of hypothalamo-hypophyseal-gonadal axis to release its respective hormones ultimately to trigger the mechanism of folliculogenesis and subsequent fertile estrus. The positive response of consistent administration of even lower dose of progesterone through parentral route release of progesterone through intra-vaginal implant and ear implant, might expert depressing effect on hypothalamo-hypophyseal-gonadal axis; and their withdrawal released the very axis from the negative effect and thereby set to function for release the tropic hormones indirectly or directly responsible for

folliculogenesis expression of estrus symptom and ovulation (Hafez and Hafez, 2000). Animals under Group T2 though received four injections each of 250 mg progesterone at 3rd day interval only three animals were detected in estrus. This might be due to continuous administration of higher doses of progesterone. Detection of estrus in 1 out of 6 buffaloes in Group T3 indicates that the injection of 250 mg progesterone might not have been sufficient to modulate the hypothalamo hypophyseal-gonadal axis. Thakur et al. (1989) and Kumar et al. (2000) reported successful induction of estrus in anestrous buffaloes with administration of 500 mg of progestrone and estradiol combination, while Singh et al. (1983) and Singh (2003) induced estrus in anestrous buffaloes with only progestrone; supports our observation of induction of estrus in 1 out of 6 buffaloes under group T3 treated with high dose of Progestrone. However, attempt to induce estrus in buffalo could not achieve height because the experiment was conducted in the months of April to June, the period which is well known to keep buffalo away from breeding.

The buffaloes of Group T1, Group T2 and


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Table 2. Response of anestrus buffaloes to progesterone administration on resumption of estrus cyclicity.

TreatmentNo. of

AnimalNo. of Animal

showing sign of estrusIntensity of estrus

Insemination Remark

Group Control- Injection of Normal saline I/M

6 0 -

Group T1 -Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on alternate day (i.e. day 39th, 41st, 43rd, 45th, 47th and 49th )

6 5Moderate to strong Natural Pregnant(3)

Group T2 -Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on every 4th day (i.e. day 39th,43rd, 47th and 51st)

6 3Moderate to strong Natural Pregnant(3)

Group T3 - Injection of progesterone (Duraprogen TM250 mg,1ml) I/M on every 7th day after administration of 1st injection (i.e. day 39th and 46th)

6 1 Moderate Natural Pregnant(1)

Table 3. Effect of progesterone on anestrus buffaloes.

Group No. of animal

No. of animal in

estrusNo. of animal

in anestrusDay on which sign of estrus

observed

Control 6 0 6 -T1 6 5 1 Day 44, 44, 45, 46 and 49T2 6 3 3 Day 46, 46 and 50T3 6 1 5 Day 43


102

Group T3 that were having higher progesterone concentration on day ‘40’ were detected in estrus between 4 and 9 days in group T1 and between 6 and 8 days in Group T2 of last progestrone injection. The buffaloes of group T1 receiving 1 ml (250 mg) progesterone on alternate days although were having similar higher serum total cholesterol concentration exhibited estrus between day 4 of 2nd progesterone injection and similarly the buffaloes of group T2 having higher serum cholesterol on day progesterone injection also exhibited the estrus between 6 and 8 days after the two doses of progesterone treatment. The reproductive behaviour of these two groups revealed clearly that these buffaloes were although having developing follicle on the ovary might be secreting elevated level of estradiol but could not exhibit the overt sign of estrus and it took 6 to 8 days after first progesterone injection for exhibition of estrus. It might be due to suppression of the mechanism responsible for the final development of graffian follicles, secretion of higher concentration of estradiol by the developed follicle and changes in female genitalia and behaviour of the buffaloes due to the decrease in estradiol-progesterone ratio in the circulation and imbalance in the co-ordination in the functioning of hypothalamo-hypophyseal-gonadal system required for bringing the animal in estrus (Hafez, 1982). The observation suggest that in both the groups the mechanism responsible for steroid metabolism and its disintegration might have taken 5 to 9 days to reduce the concentration of progesterone in circulation increasing the estradiol-progesterone ratio to bring the animal to estrus.

The dose related response of progesterone administration has also been observed during present experimentation in terms of synchronization of estrus by progesterone treatment in anestrus

buffaloes. It has been observed through present experimentation, that the administration of 250 mg progesterone to buffaloes as a single injection did not have any influence on estrus cyclicity. It may be presumed that administration of single injection of 250 mg progesterone might not be sufficient to sensitize the hypothalamo-hypophyseal-gonadal system to establish co-ordination in the organs to integrate the functional activity therein. As minimum threshold level of any hormone is required to activate its target organ (Hafez, 1982 and Mc. Donald, 1980). The injection of 500 mg progesterone twice have influenced the system similar to the single dose responsible to bring the animals to estrus cyclicity even then the excess higher dose of progesterone does not have positive response to bring the animal to estrus. The observation of present experimentation suggest that either 250 mg progesterone followed with 500 mg of progesterone or 500 mg progesterone either as single or double injection in anestrus buffaloes are sufficient to set the possible mechanism to bring the animal to estrus. However, before drawing any conclusion on the efficacy of the synchronization of estrus in buffaloes by using prostaglandin and progesterone combination various trial of progesterone at different doses and frequency on large number of animals are required.

On critical analysis it may be concluded that repeated dosages of progesterone (Duraprogen 250 mg, 1 ml) on alternate day had better effect than administered at three day interval or at six day interval. It needs further investigation to know the effect of progesterone at different dose and at different frequency on resuming the estrus cyclicity on post-partum anestrus buffaloes.


103

REFERENCES

Hafez, E.S.E. 1982. Reproduction in Farm Animals, 4th ed. Lea and Febiger Philedelphia, USA.

Hafez, E.S.E. and B. Hafez. 2000. Reproduction in Farm Animals, 7th ed. Lea and Febiger, Philadelphia, USA.

India, 2004. Publication Division. Ministry of Information and Broadcasting, Government of India. 81p.

Kumar, N., S. Mahmood, L.P. Singh and L.N. Purbey. 2000. Induction of estrus and ovulation in postpartum anestrus crossbred cows with short term steroid treatment. Indian J. Anim. Reprod., 21: 53-54.

Kumar, S., M.C. Sharma and S.K. Dwivedi. 1986. Calcium, phosphorous and serum electrolyte changes in anestrus and repeat breeding cows and heifers. Cheiron, 15: 133-136.

Kurien, M.O. and E. Madhavan. 1985. Clinical evaluation of clomiphens citrate and or combination of MGA and ethenyl estradiol for treatment of anestrus cattle. Indian J. Anim. Reprod., 6: 14-18.

McDonald, L.W. 1980. Veterinary Endocrinology and Reproduction, 3rd ed. Lea and Febiger, Philadelphia, London.

Roberts, S.J. 1971. Veterinary Obstetrics and Genital Diseases (Theriogenology), 2nd ed. Publication, Ithaca, New York.

Singh, C. 2003. Response of anestrus rural buffaloes (Bubalus bubalis) to intravaginal progesterone implant and PGF2α injection in summer. J. Vet. Sci., 4: 137-141.

Singh, G., G.B. Singh, R.D. Sharma and A.S. Nanda. 1983. Experimental treatment of summer anestrus in buffaloes with norgestomet and PRID. Theriogenology, 19: 323-329.

Sinha, B.P., S.N. Sinha and B. Singh. 1987. Incidence of anestrus in crossbred cattle in field and farm condition. Lives. Adv., 12: 43-48.

Thakur, M.S. 1989. Synchronization of estrus in postpartum anestrus buffaloes (Bubalus bubalis) with short term steroid treatment. Indian J. Anim. Reprod., 10: 19-21.


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ABSTRACT

The present study was conducted to find out testicular biometry and its correlation with body weight, scrotal circumference semen production. Semen sample were collected from six sexually mature Murrah buffalo bulls, aged 4 to 12 years, maintained in bull station of College of Veterinary Sciences and Animal Husbandry, Kumarganj, Faizabad, Uttar Pradesh, India. Biometrical evaluation was performed by caliper in all experimental bulls. The biometric testicular parameters (scrotal circumference, testicular length, testicular width and testicular volume) were analyzed. Present findings indicate strong correlations between scrotal circumference, testicular volume and body weight.

The body weight was significantly (P<0.01) and positively correlated with scrotal circumference (r=0.98), testicular volume (r=0.97), sperm concentration (r=0.94), concentration/ejaculate (r=0.64), initial motility (r=0.90), live count (r=0.89), whereas it was significantly (P<0.01) negatively correlated with total sperm abnormality (r=-0.79). The overall mean (±SE) scrotal circumference of Murrah bulls were ranges between 31.02±0.39 to 46.33±0.43 cm. A significant (P<0.01) variation in S.C. was reported among the bulls. The scrotal circumference was significantly (P<0.01) and positively correlated

with body weight (r=0.98), testicular volume (r=0.96), testicular parenchyma (r=0.98), sperm concentration (r=0.91), concentration/ejaculate (r=0.63).

Keywords: Murrah bulls, biometry, body weight, testes, sperm output

INTRODUCTION

Buffalo, through their potential for producing milk, meat and draft power, contribute significantly to the agricultural economy of many developing countries including India. One of the major constraints in maximizing the production of buffalo is their inherent low reproductive efficiency. Proper bull selection is the most rapid way to make genetic improvements to the herd. Performance testing provides valuable information that can be used in selection of superior breeding animals.

Testicular biometry is an important component of monitoring the testis for normality and gauging potential sperm production (Paula et al., 2001). Biometric parameters, such as scrotal circumference (S.C.), testicular weight (T.W.) and testicular length (T.L.), and testicular volume (T.V.) and testicular parenchyma (T.P.) are essential measurements in the andrological evaluation of a breeding animal. Among these parameters, S.C. is

TESTICULAR BIOMETRY AND ITS CORRELATION WITH BODY WEIGHT AND SEMEN OUTPUT IN MURRAH BULL

Sanjeet Kumar and Sushant Srivastava*

Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Kumarganj, Faizabad, Uttar Pradesh, India, * E-mail: [email protected]

Original Article


106

used most often because it is easy to measure and displays a high correlation with body weight and reproductive capacity (libido), particularly sperm production (Brito et al., 2004), while the biometric data related to S.C. helps to define the reproductive parameters for a species. S.C. alone should not be used for the selection of breeders, rather, a complete andrological evaluation (a breeding soundness examination), including an evaluation of semen quality, should be performed to certify the reproductive capacity of a male (Ohashi et al., 2007). The biometric parameters of buffalo testicles can be established using comparative studies with other species. Investigations into the mammalian body and testicular biometrics are important for various aspects of reproduction; these studies help to characterize puberty and sexual maturity and enable inferences about spermatogenesis (Assis Neto et al., 2003).

According to the Society for Theriogenology (SFT), bull breeding soundness examination comprises general and reproductive physical examination, scrotal circumference indexed for age, semen motility and sperm morphology examinations (Alexander, 2008). The information on body and testicular development has been well studied in dairy bulls (Coulter and Foote, 1977), beef bulls (Sosa et al., 2002), and buffalo bulls (Ahmad et al., 2010).

The aim of this study was to identify the reproductive characteristics of Murrah buffalo bulls using testicular biometric parameters and their correlations with body weight and semen output.


AnimalsThe study was carried out during October

2014 to April 2015. A total of six adult sexually mature male Murrah buffalo bulls, aged 4 to 12 years were used. They were maintained in bull station of College of Veterinary Sciences and A.H., Kumarganj, Faizabad, U.P., India. All these buffalo bulls were in good health. They were maintained in nearly identical nutritional and managerial condition throughout the period of study. Biometrical evaluation was performed using caliper in all 6 Murrah bulls. Semen was also collected using artificial vagina from these 6 Murrah bulls. All the experimental animals were examined for general health status and the appearance of genitalia. The scrotal skin was observed for any kind of lesions. The testes were palpated and observed for their size, shape, free movement and position in the scrotum.

Body weight and scrotal circumference measurements:

The body weight of each bull was recorded in Kilogram (kg) in the morning before meal. The weights were taken with a top loading balance.

Scrotal circumference was measured as per method recommended by the Society of Theriogenology (Ball et al., 1983). The testes were first retracted into the lower part of the scrotum for measurement of scrotal circumference. To prevent separation of the two testes, the thumb and the fingers were placed on the sides rather than on the front or back of the scrotum. Then a measuring tape (scrotal tape) was looped and placed around the greatest diameter of the scrotum and pulled snugly so that the tape was firmly in contact with the entire circumference. Repeated measurements were done


107

and the mean of the measures was recorded to ensure the accuracy.

Testicular size measurements (In vivo)Testicular measurements were made while

bulls were restrained in standing position. The testes were brought into the distal part of scrotum and the greatest testis length and width were measured with the help of a flexible measuring tape. Testicular volume was determined using the formula of Fields et al. (1979): VT = 2[(r2) × π × L], where r2 = testis width (radius), π = correction factor (3.14) and L = testis length.

Semen collection and evaluationSemen samples were collected from six

buffalo bulls by using artificial vagina maintained at temperature between 38oC and 41oC. The semen was usually collected early in the morning, before feeding. Semen sample were collected twice a day and twice a week intervals from 6 buffalo-bulls. A total of two ejaculates were taken with a minimum interval of 30 minutes. Each semen sample was examined for routine semen parameters (volume, total sperm concentration, pH, mass motility, initial progressive motility, per cent live count, abnormal sperm as per standard methods described earlier (Salisbary et al., 1985).

Sperm concentration per ml and initial progressive motility (percent)

Concentration of spermatozoa (million/ml) in the neat semen was determined by the haemocytometer method adopting RBCs counting procedure (Salisbury et al., 1985). A drop of semen was placed on a pre-warmed glass slide and covered with a cover slip. The percentage of motile spermatozoa was assessed subjectively at 37oC, using a heated stage, by viewing 5 to 6 fields

per slide with the aid of a closed-circuit television attached to a phase contrast microscope (40X). A spermatozoon that moved due to swimming, regardless of its speed, was considered as to be motile.

Percentage of live spermatozoa and abnormal spermatozoa

The percentage of live and dead spermatozoa in fresh ejaculates as well as in pre freeze and cryopreserved semen was estimated by differential staining technique using Eosin-Nigrosin stain (Campbell et al., 1953). The smears were prepared in duplicate after mixing a small drop of neat semen with four drops of stain on a clean grease free microscopic slide at 37oC. Hundred spermatozoa were counted under the oil immersion, objective (100X) of a phase contrast microscope for estimating the percentage of live (unstained) spermatozoa. The pinkish (eosinophilic) and partially stained spermatozoa were classified as dead.

The same slide made for live and dead count was also used for the morphological study of sperm to find out sperm abnormalities.

Sperm membrane integrity (percent)Hypo-osmotic swelling test (HOST)

was performed after slight modification of the experiment carried out in human being (Jayendran et al., 1984) to assess the functional integrity of the sperm tail membrane which gives idea of the spermatozoal fertilizing capacity in vitro. Sodium citrate (0.73 g; Merck) and fructose (1.351 g; Merck) were dissolved in 100 ml distilled H2O to prepare HOS solution (osmotic pressure ~150 mOsmol/kg) and maintained at 37oC for 5 minutes before use 0.1 ml of each semen sample was mixed with 0.9 of HOS solution and incubated at 37oC for


108

60 minutes. After incubation, place a small droplet on glass slide and put cover slip and Observation for swollen tail under high power magnification of phase contrast microscope. One hundred sperm were assessed for their swelling ability in HOS. The swollen sperm characterized by coiling of the tail were considered having an intact plasma membrane.

Statistical analysisData were presented as mean and standard

error of the mean (SEM). Analysis of variance (ANOVA) was used to assess differences among the bulls. When the F ratio was significant (P<0.05). Descriptive analyses of the mean and standard deviation for each testicular biometric parameter were also performed with the Graph Pad Prism 5 software.


Body weight and scrotal circumferenceThe overall mean (±SE) body weight (Kg)

of Murrah bulls was observed in the range between 454.5±2.19 to 611.3±3.04 Kg. Average body weight of each bull is presented in Table 1. It differed significantly (P<0.01) among the bulls. The body weight was significantly (P<0.01) and positively correlated with scrotal circumference (r=0.98), testicular volume (r=0.97), sperm concentration (r=0.94), concentration/ejaculate (r=0.64), initial motility (r=0.90), live count (r=0.89) and HOS (r=0.80) whereas it was significantly (P<0.01) negatively correlated with total sperm abnormality (r=-0.79).

Relationship of age to body weight and scrotal circumference in Murrah bulls is presented in Table 1. In general, both mean SC and body

weight increased (P<0.05) in a curvilinear manner. The mean scrotal circumference was increase with body weight in ND5, ND9, ND7, ND2, ND1 and ND4 respectively.

For Nd5 the maximum SC was 46.33 cm and minimum was 31.02 cm for ND4 bull and other ND9, ND7, ND2 and ND1bulls 40.07cm, 37.97 cm 33.38 cm, and 32.6 cm, respectively. The correlation between body weight and scrotal circumference were 0.94, 0.94, 0.60, 0.94, 0.81 and 0.94 for ND1, ND2, ND4, ND5, ND7 and ND9 respectively. Mean SC followed the same pattern as that of body weight.

Similar findings were also observed by Pant et al. (2003) and lower than the observation reported by Asghar et al. (1985); Heuer et al. (1987) and Koonjaenak et al. (2007) but higher than those recorded by the Luz et al. (2012).

Body weight was highly (P<0.01) positive correlated with testicular volume, scrotal circumference which is comparable to the findings of Silva et al. (1999) (Pig) and Caurot et al. (1970) (Ram and Cattle) but lower correlation with these parameters was observed by Luz et al. (2012). The strength of correlation obtained suggested that scrotal circumference and testicular volume are useful parameter for the selection of breeding bulls.

Testicular volumeThe testicular volume mean (±SE)

of experimental bulls was recorded between 700.03±4.70 to 1796.0±9.23 cm3. The average testicular volume of each bull is given in Table 1. Lower T.V. was observed Pant et al. (2003) and Sequeira et al. (2007). The testicular volume differed significantly (P<0.01) among experimental bulls.

Testicular volume of bulls was significantly (P<0.01) and positively correlated with body weight


109

Tabl

e 1.

Tes

ticul

ar b

iom

etry

(Cal

liper

and

Ultr

asou

nd) a

nd se

min

al a

ttrib

utes

in fr

esh

Mur

rah

bulls

.

Bul

l No

ND

-1N

D-2

ND

-4N

D-5

ND

-7N

D-9

Pool

B.W

. (K

g)47

9.5e ±

2.51

481.

8ed±3

.05

454.

5f ±2.

1961

1.3a ±

3.04

511.

7c ±2.

9458

3.7b ±

1.94

520.

4±9.

78S.

C. (

Cm

)32

.60e ±

0.75

33.3

8ed±0

.53

31.0

2f ±0.

3946

.33a ±

0.43

37.9

7c ±0.

3740

.07b ±

0.53

36.8

9±0.

91T.

V. (C

m3 )

932.

4e ±16

.36

990.

7d ±14

.68

700.

3f ±4.

7017

96.0

a ±9.

2313

79bc

±7.7

113

79b ±

7.71

1196

±61.

22C

onc.

(M/m

l)14

28cd

±38.

0214

06de

±22.

5112

33f ±

11.3

017

48a ±

20.6

814

76c ±

25.3

616

19b ±

30.6

114

85±2

5.87

Vol.

(ml)

5.49

±0.2

94.

80±0

.34

5.05

±0.3

25.

19±0

.32

5.11

±0.2

95.

35±0

.26

5.17

±0.1

2C

/E. (

M/m

l)78

43ab

c ±49

7.7

6970

ce±5

85.8

6239

def ±

427.

291

01a ±

640.

375

52bc

d ±57

0.4

8685

ab±5

04.3

7731

±252

.5IM

(%)

55.6

3e ±0.

4258

.25d ±

0.49

53.1

3f ±0.

6466

.88a ±

0.61

60.0

0c ±0.

6563

.25b ±

0.53

59.5

2±0.

70LC

(%)

79.0

0e ±0.

7183

.00d ±

0.85

73.2

5f ±0.

8889

.63a ±

0.42

84.6

3c ±0.

5787

.25b ±

0.53

82.7

9±0.

83A

b. (%

)14

.75b ±

0.31

14.0

0bc±0

.46

16.8

8a ±0.

5211

.63f ±

0.26

13.3

8cd±0

.18

12.8

8de±0

.35

13.9

2±0.

28H

OS

(%)

34.0

0e ±0.

6039

.00cd

±0.4

631

.75ef

±0.7

343

.25a ±

0.59

40.8

8abc ±

0.72

42.0

0ab±0

.57

38.4

8±0.

66

Mea

n be

arin

g di

ffere

nt su

pers

crip

t (a,

b, c

, d, e

, f) i

n a c

olum

n di

ffere

d si

gnifi

cant

ly (P

<0.0

5), s

epar

atel

y fo

r eac

h at

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110

(r=0.97), concentration (r=0.90), concentration/ejaculate (r=0.55), initial motility (r=0.89), live count (r=0.88) and HOS reactive spermatozoa (r=0.81) whereas, significantly (P<0.01) negatively correlation with total sperm abnormality (r=-0.81).The findings of present study on correlation between various testicular indices were in agreement with the earlier study carried out on Murrah bulls. (Pant et al., 2003).

Semen qualityRelationship of age to semen quality

(ejaculate volume, percent motility, sperm concentration, plasma membrane integrity, live sperm, and sperm abnormalities) is presented in Table 1. Average ejaculated volume (mean±SE) of semen was observed in the range between 4.80±0.34 to 5.49±0.29 ml. Volume of semen was differed significantly (P<0.01) between the ejaculate of same bull as well as among all experimental Murrah bulls. The mean value of ejaculate volume was comparable to those reported by Pillai, (1965); Dhami, (1992); Srivastava, (2011) and Maurya et al. (2013).

The average sperm concentration of (million/ml) of Murrah buffalo bulls were recorded between 1233±11.30 to 1748±20.68. The present finding regarding sperm concentration (106/ml) was similar to the observation of Patil, (1981) and higher than those reported by Pillai, (1965); Kumar et al. (1993); Gokhale and Bhatt et al. (1996); Bhakat et al. (2011) and Srivastava, (2011) but lower than those reported by Bhakat et al. (2015). The wide variation in the sperm concentration has been attributed to factors like season, individuality, age of bull, sexual excitement frequency of collection etc. (Tomar, 1986).

The average per cent initial progressive motility, live spermatozoon, sperm abnormality

and HOS reactive spermatozoa in semen of Murrah buffalo bull under experimental condition was ranges between 53.13±0.64 to 66.88±0.61 %, 73.25±0.88 and 89.63±0.42, 11.63±0.26 to 16.88±0.52 % and 31.75±0.73 to 43.25±0.59, respectively.

The correlation of 0.96 between scrotal circumference and testicular volume is similar to the values (0.92 to 0.97) reported in several studies (Hahn et al., 1969; Van Demark, 1986; Pant et al., 2003; Gober et al., 1998), whereas, positively lower correlation reported by Luz et al. (2012). The correlation between scrotal circumference and sperm output was recorded higher in all the bulls.

Others have reported similar trends in dairy bulls (Willet and Ohms, 1957; Hahn et al., 1969 and Dhage et al., 2010).These results emphasis the importance of scrotal circumference in selecting breeding bull for future semen production.

In conclusion, Higher correlation of body weight with testicular biometry and seminal attributes clearly indicate that higher body condition score may directly affect the performance of breeding bulls.

REFERENCES

Ahmad, N., S. Umair, M. Shahab and M. Arslan. 2010. Testicular development and establishment of spermatogenesis in Nili-Ravi. Theriogenology, 73(1): 20-25.

Alexander, J.H. 2008. Bull breeding soundness evaluation: A practitioner’s perspective. Theriogenology, 70: 469-472.

Asghar, A.A., M.A. Chaudhry and J. Iqbal. 1985. Productive and reproductive performance of Nilli-Ravi buffaloes under optimal feeding and management conditions. Sixth Annual


111

Report (1984-85). LPRI Bahadurnagar, Okara, Pakistan.

Assis Neto, A.C., M.A.M. Carvalho, M.I.V. Melo, M.A. Miglino, M.F. Oliveira, M.M. Almeida, P.C. Papa, Jr.J.R. Kfoury. 2003. Biometric aspects of the testicular and corporal development of Agoutis (Dasyprocta aguti) rised in captivity. Braz. J. Vet. Res. Anim. Sci., 40: 154-160.

Ball, A.B. 2008. Diagnostic methods for evaluation of stallion subfertility: a review. J. Equine Vet. Sci., 28: 650-664.

Bhakat, M., T.K. Mohanty, V.S. Raina, A.K. Gupta and H.M. Khan. 2011. Frozen semen production performance of Murrah buffalo bulls. Buffalo Bull., 30(2): 157-162.

Bhakat, M., T.K. Mohanty, S. Singh, A.K. Gupta, A.K. Chakarvarty and P. Singh. 2015. Influence of semen collector on semen characteristics of Murrah buffalo and Crossbred bulls. Advances in Animal and Veterinary Sciences, 3(4): 253-258.

Brito, L.F., A.E. Silva, M.M. Unanian, M.A. Dode, R.T. Barbosa, J.P. Kastelic. 2004. Sexual development in early and late maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil. Theriogenology, 62: 1198-1217.

Campbell, R.G., J.L. Hancock and L. Rothscild. 1953. Counting live and dead spermatozoa. J. Expt. Biol., 30: 44.

Coulter, G.H. and R.H. Foote. 1977. Relationship of body weight to testicular size and consistency in growing Holstein bulls. J. Anim. Sci., 44: 1076-1079.

Courot, M., M.T. Hochereau-De Revivers and R. Ortavant. 1970. Spermatogenesis. p. 339-432. In Johnson A.D., W.R. Gomes and N.L. Vandemark. (eds.). The Testis. New

York, Academic Press.Dhami, A.J. 1992. Comparative evaluation of

certain procedures in deep freezing of cattle and buffalo semen under trophical climate. Ph.D. Thesis. Indian Veterinary Research Institute, Izatnagar.

Fields, M.J., W.E. Burns and A.C. Warnick. 1979. Age, season and breed effects on testicular volume and semen traits in young beef bulls. J. Anim. Sci., 48: 1299-1304.

Gabor, G., R.G. Sasser, J.P. Kastelic, M. Mezes, G. Falkay, S. Bozo, J.C. Vrlgyi, I. Bárány, A. Hides, J.F. Szfisz and G. Borest. 1998. Computer analysis of video and ultrasonographic images for evaluation of bull testes. Theriogenology, 50: 223-228.

Gokhale, S.B., M. Mushtaque, N.L. Phadke and G.S. Ambhore. 2003. Studies on the effect of hydrogen ion concentration of extender on semen characters of Murrah buffalo bulls. Indian J. Anim. Reprod., 24(2): 158-160.

Hahn, J., R.H. Foote and Jr.G.F. Seidel. 1969. Testicular growth and related sperm output in dairy bulls. J. Dairy Sci., 29: 41-47.

Heuer, C., M.N. Tahir and H. Amjad. 1987. Effect of season on fertility of frozen buffalo semen. Anim. Reprod. Sci., 13: 15-21.

Jeyendran, R.S., H.H. Vander-Ven, M. Perez-Pelaez, B.G. Crabo and L.J.D. Zanevld. 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characters. J. Repord. Fertil., 70: 219-228.

Jeyendran, R.S., H.H. Vander-Ven, M. Perez-Pelaez, B.G. Crabo and L.J.D. Zanevld. 1984. Development of an assay to assess the functional integrity of the human sperm


112

membrane and its relationship to other semen characters. J. Repord. Fertil., 70: 219-228.

Koonjaenak, S., V. Chanatinart, S. Aiumlamai, T. Pinyopumontr and H. Rodriguez-Martinez. 2007. Seasonal variation in semen quality of swamp buffalo bulls (Bubalus bubalis) in Thailand. Asian J. Androl., 9: 92-101.

Kumar, S., K.L. Sahni and G.S. Bistha. 1993. Cytomorphological characteristics of motile and static semen of buffalo bulls. Buffalo Journal, 9(2): 117-127.

Luz, P.A.C., P.R.S. Santos, S. Andrighetto, A.M. Jorge and A.C. Assis-Neto. 2012. The Correlation between Age, Body Weight and Testicular Parameters in Murrah Buffalo Bulls Raised in Brazil. J. Reprod. Develop., 59(1): 14-17.

Maurya, K., O.P. Singh and S. Srivastava. 2013. Seminal plasma ascorbic acid level and its relationship to sperm characteristics in Murrah buffalo bulls. Indian J. Anim. Sci., 83(5): 498-501.

Ohashi, O.M., M.S. Miranda, M.S. Cordeiro, S.S. Socorro and D. Santos. 2007. Reproductive development of male buffalo: testicular biometry, espermatic activity and endocrinology. Rev. Bras. Repr. Anim., 31: 299-306.

Pant, H.C., R.K. Sharma, S.H. Patel, H.R. Shukla, A.K. Mittal and J.R. Kasira. 2003. Testicular development and its relationship to semen production in Murrah buffalo bulls. Theriogenology, 60: 27-34.

Patil, D.S. 1981. Deep freezing of buffalo bull semen in various diluters. M.V.Sc. Thesis. Kokan Krishi Vidyapeeth, Dapoli, India.

Paula, T.A.R and R.D. Navarro. 2001. Testicular componentes of peccaries (Tayassu pecari)

and collared peccary (Tayassu tajacu). Rev. Bras. Reprod. Anim., 25: 206-207.

Pilai, K.V. 1965. Observation on certain semen characteristics of Murrha buffalo bulls. M.V.Sc. Thesis, Bombay University, Bombay.

Salisbury, G.W., N.K. VanDemark and J.R. Lodge. 1985. Semen Evaluation, Physiology of Reproduction and Artificial Insemination of Cattle, 2nd ed. CBS Publishers and Distributors, 485, Shahdara, Delhi, India.

Silva, S.M., M.I.V. Melo, I.R. Scheid, E.F. Nascimento and G.D. Cassali. 1999. Establishment of spermatogenesis in pigs of Large White and Landrace breeds with different growth with emphasis on puberty. p. 301-302. In Congresso Brasileiro de Veterinários Especialistas em Suínos, Belo Horizonte, Brazil.

Siqueira1, J.B., E. Oba1, R.O. Pinho, E.F. Castilho, R.F. Bittencourt, S.S. Assaf1 and J.D. Guimarães. 2007. Gonadic Sperm Reserve in Buffaloes Created under Tropical Conditions. Ital. J. Anim. Sci., 6(2): 615-618.

Sosa, J.M., P.L. Senger and J.J. Reeves. 2002. Evaluation of American Wagyu sires for scrotal circumference by age and body weight. J. Anim. Sci., 80: 19-22.

Srivastava, S. 2011. Effect of Amino acids incorporation of post thaw seminal attributes and in vitro fertility assay of Murrah bull spermatozoa. Ph.D. Thesis, Dr. Bhim Rao Ambedkar University, Agra, Uttra Pradesh, India.

The Society for Theriogenology. 1983. Manual for Breeding Soundness Examination of Bulls. 12p.

Tomar, N., 1986. Artificial Insemination and


113

Reproduction of Cattle and Buffaloes, 3rd ed. Saroj Prakashan Allahabad, India.

Van Der Ven, H.H., R.S Jeyendran, S. Al-Hasani, M. Perez-Pelaez, K. Diedrich and L.J.D. Zanaveld. 1986. Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization. J. Androl., 7: 190-196.

Willet, E.L. and J.I. Ohm. 1957. Measurement of testicular size and its relation to the production of spermatozoa by buck. J. Dairy Sci. 40: 1559-1569.


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ABSTRACT

A study was conducted on Murrah buffalo male calves born during 1997 to 2012, reserved for breeding at Indian Council of Agricultural Research, National Dairy Research Institute, Karnal, Haryana, India to assess the influence of season and period of birth on semen production characteristics of buffalo bulls. Chi-square analysis revealed that neither season nor period had significant effect on these parameters. The result depicted that 36.47% males donated semen and 28.68% males produced freezable quality semen out of reserved males. Overall least squares means of age at first successful semen collection (AFSC), age at first successful freezing (AFSF), age at last successful freezing (ALSF), age at last successful semen collection (ALSC), semen production period (SPP), frozen semen production period (FSPP) and age at disposal (AD) were 1073.5±28.41, 1196.05±28.18, 1600.26±143.18, 1570.33±101.55, 644.28±112.05, 712.33±159.08 and 1668.25±128.53 days, respectively. Effect of season and period of birth was not found significant for all the traits except for ALSC which showed declining trend over the periods. Significant effect

(P<0.05) of age group showed that the bulls donating semen for the first time at younger age also produced semen of freezable quality at a younger age and remained in the herd producing semen for a longer duration. The age at first semen donation in Murrah males can be reduced by introducing the young male calves to training at an early age, which could increase the doses of semen obtained from each male.

Keywords: semen, AFSC, AFSF, ALSF, ALSC, Murrah

INTRODUCTION

Murrah buffalo is the most efficient producer of milk and known for its better adaptability throughout India. The breeding tract of Murrah breed lies in Rohtak, Hisar and Jind district of Haryana and Nabha and Patiala districts of Punjab in India. The germplasm and bulls of this breed are used extensively for upgrading native buffalo stock of many countries including Thailand, Malaysia, Indonesia, Philippines, Madagaskar and Brazil. It has been anticipated

EFFECT OF NONGENETIC FACTORS ON SEMEN PRODUCTION CHARECTERISTICS OF MURRAH BUFFALO BULLS AT ORGANIZED SEMEN STATION

Pushp Raj Shivahre1,*, A.K. Gupta1, A. Panmei1, A.K. Chakravarty1, M. Bhakat1, S.K. Dash2, S.K. Sahoo1, V. Kumar3 and M. Singh1

1Dairy Cattle Breeding Division, Indian Council of Agricultural Research, National Dairy Research Institute, Karnal, India, *E-mail: [email protected] of Animal Genetics and Breeding, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India3Department of Animal Genetics and Breeding, Guru Angad Dev Veterinary and Animal Sciences University, Mathura, Uttar Pradesh, India

Original Article


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that buffalo is the future hope to meet the milk and meat demond in the densely populated countries like India and China (Ranjhan and Qureshi, 2006). The buffaloes are kept under peri-urban and rural farming systems with the primary aim to produce milk for urban and rural populations. Since artificial insemination (AI) was introduced to the dairy industry in the 1950s, it has become the preferred method of breeding in most developing countries of the world. The optimum serving capacity and seminal profile are the crucial parameters to assess the breeding soundness of bovine bulls. Age of the bull affects semen volume and sperm concentration (Amann and Almquist, 1976; Rustenev, 1989 and Siratskii, 1990). Through seasonal variation in semen production has been reported (Graffer et al., 1988), but specific causes are not understood. A better knowledge of the influence of age of the bull at collection, season of collection, and frequency of collection on semen production will help the AI industry to adapt management of bulls to improve semen output. Differences in age at puberty may be attributed to breed composition, management, and environment in which bulls were being reared (Brito et al., 2004). To gain a better understanding of semen production of Murrah bulls, 16 years of data from Artificial Breeding Research Centre, National Dairy Research Institute, Karnal were analyzed.


Data on Murrah buffalo males born during the period from 1997 to 2012 were collected from Artificial Breeding Research Center, Indian Council of Agricultural Research-National Dairy Research Institute, Karnal to study the effect of season of birth, period of birth and age group on

semen production traits of Murrah males. The following traits were generated i.e. Age at disposal (AD), Age at first successful semen collection (AFSC), Age at first successful freezing (AFSF), Age at last successful freezing (ALSF), Age at last successful semen collection (ALSC), Frozen semen production period (FSPP) and Semen production period (SPP). Data were classified into four periods viz., P-1 (1997 to 2000), P-2 (2001 to 2004), P-3 (2005 to 2008), P-4 (2009-2012); four seasons viz., winter, summer, rainy and autumn i.e. S-1 (December to March), S-2 (April to June), S-3 (July to September), S-4 (October to November), respectively; and age groups D1 (<2.5 years), D2 (2.5 to 3 years), D3 (3 to 3.5 years) and D4 (>3.5 years); to study semen production characteristics of Murrah males calves reserved for breeding. The semen production traits of breeding bulls in different seasons and periods of birth were calculated by proportion using descriptive statistics. To study the differences in number of males reaching semen production stage, Chi-square value was calculated (Snedecor and Cochran, 1968).

The statistical model used for least square analysis to study effect of season of birth and period of birth on age at first semen collection (AFSC) was as under:

Yijk = µ + SBi + Pj+ eijk

Where Yijk = kth observation of a bull born in ith

season and jth periodµ = overall meanSBi = Effect of ith season of birth (i=1to 4)

Pj = Effect of jth period of birth (j=1to 4)Eijk = Random error NID (0, σe

2 )

The model used to study the effect of age,


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season and period on AFSF, ALSF, ALSC and AD was:

Yijkl = µ + SBi + Pj +Dk + eijkl

Where Yijkl = lth observation in in ith season and jth period and belonging to kth age group µ = overall mean SBi = Effect of Ith season of birth (i=1to 4) Pj = Effect of jth period of birth (j=1to 4) Dk = Effect of kth age group of the bull donating semen for the first time (k=1 to 4) eijkl = Random error NID (0, σe

2 )


During study period 1029 male animals were born out of which 244 (23.71%) males were reserved for breeding purpose on the basis of dam’s best 305 day lactation yield. Average dam’s best 305 days lactation yield was 2879.93 kg for selected vs 2311.45 kg for all the males born. Out of total reserved males 28.68% bulls produced semen and out of which 78.57% bulls were able to produce freezable quality semen. Period-4 was excluded from the data set as a number of bulls from this period could not reach the semen donation stage. Analysis of production of breeding bulls from males reserved for breeding purpose revealed that percent bulls reached upto semen donating stage and successful freezing out of total males reserved for breeding was 31.55 and 25.40% respectively whereas only 80.51% bulls gives freezable quality semen out of total bulls reaching the semen donation stage. Chi-square analysis revealed that season and period of birth had no significant effect on AFSC and AFSF. In Period-2, 46.55% males reached the semen donation followed by Period-1

(42.11%) and Period-3 (31.70%), this may be due to managemental difference in the farm during the periods. Whereas male born in rainy season, 40.20% reached into AFSC i.e. maximum compared to other seasons. If we compare the males reaching AFSF stage out of males reaching the semen donation stage, it was seen that maximum 85.71% males reached to AFSF, born in winter season and minimum (62.50%) in summer season. The results showed that the males born during summer months, very few of them reached successful freezing stage prior to being considered for inclusion in progeny testing programme. It may be due to winter born males got quality feed and fodder during their growth period than summer born males.

Overall least squares means for age at first semen collection (AFSC), age at first successful freezing (AFSF), age at last successful freezing (ALSF), age at last semen collection (ALSC), semen production period (SPP), freezable semen production period (FSPP) and age at disposal (AD), were 1073.5±28.41, 1196.05±28.18, 1600.26±143.18, 1570.33±101.55, 644.28±112.05, 712.33±159.08 and 1668.25±128.53 days respectively (Table 3). Higher values of AFSC were reported by Suryaprakasam and Rao (1993) (1326±1.2) and Khate (2005) (1179.02±27.25) in Murrah. Kodagali et al. (1980), while studying the culling percentage of Surti buffalo bulls, reported the mean age at disposal (AD) and breeding tenure as 2,330 and 480 days, respectively. Mukhopadhyay et al. (2010) had reported higher values of AFSC, AFSF, SPP and AD but lower value of FSPP. Similarly Khatun et al. (2013) had reported higher values of all these traits. This may be due to variation in managemental conditions or geographical locations of farm differences. The results indicated that average age at first semen collection was on higher side, therefore management of males from


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Table 1. Number of Murrah buffalo males reserved, donated semen and produced freezable quality semen.

Breed No. born Reserved Produced semenProduced freezable

quality semenMurrah 1029 244 (23.71) 70 (28.68) 55 (78.57)

Figure in parentheses indicate percentage.

Table 2. Effect of season and period of birth on AFSC (Bulls donating semen) and AFSF (Bulls producing freezable quality semen) of Murrah buffalo bulls.

Period No. born No. reserved % AFSC

out of reserved

% AFSF out of AFSC

Overall % AFSF out of

reserved P-1 (1997-2000) 275 57 42.10 (24) 95.83 (23) 40.35P-2 (2001-2004) 256 58 46.55 (27) 70.37 (19) 32.75P-3 (2005-2008) 252 82 31.70 (26) 76.92 (20) 24.39P-4 (2009-2012) 246 47 - -Overall 1029 244 31.55 (77) 80.51 (62) 25.40Chi-square value 1.50 0.60 2.10SeasonS-1 (Dec-Mar) 311 59 35.59 (21) 85.71 (18) 30.50S-2 (Apr-Jun) 135 33 24.24 (8) 62.50 (5) 15.15S-3 (Jul-Sep) 389 97 40.20 (39) 84.61 (33) 34.02S-4 (Oct-Nov) 194 55 38.18 (21) 71.42 (15) 27.27Overall 1029 244 36.47 (89) 78.65 (70) 28.68Chi-square value 1.4 0.40 2.60

Figure in parentheses indicate number of bulls.


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Table 3. Effect of season, period and age group on Least squares means of semen production characteristics of Murrah buffalo bulls.

Effects AFSC (days) AFSF (days) ALSF (days) ALSC (days) SPP (days) FSPP (days) AD (days)

No. of bulls 89 71 53 69 54 38 72

Overall 1073.5± 28.41 1196.05± 28.18 1600.26± 143.18 1570.33± 101.55 644.28± 112.05 712.33± 159.08 1668.25± 128.53

Period of birth

P-1 (1997-00) 1090.88± 48.77 1203.98± 41.38 1806.35± 152.53 1871.18± 134.21a 726.7±146.88 732.56± 198.92 1951.97± 138.69

P-2 (2001-04) 1016.16± 44.59 1186.87± 45.69 1742.08±166.56 1648.19± 124.10ab 651.06± 148.48 788.53± 210.44 1794.07± 127.77

P-3 (2005-08) 1171.81± 44.12 1278.80± 36.16 1648.58± 153.26 1629.79± 123.01ab 555.09± 156.26 615.9± 184.88 1773.01± 133.36

P-4 (2009-12) 1015.17± 65.05 1114.55± 57.91 1204.02± 379.98 1132.17± 269.68b - - 1153.93± 399.67

Season of birth

Winter season 1096.18± 49.11 1204± 46.37 1629.41± 189.06 1666.41± 155.97 680.03± 151.30 695.02± 174.75 1670.97± 172.77

Summer season 1066.55± 78.77 1226.27± 76.12 1420.28± 373.82 1399.69± 236.09 502.18± 290.41 568.71± 512.94 1695.81± 265.34

Rainy season 1101.90± 36.12 1196.38± 29.18 1735.80± 130.45 1685.08± 106.30 859.26± 110.68 801.42± 117.50 1673.35± 131.56

Autumn season 1029.40± 48.06 1157.25± 42.26 1615.53± 194.76 1530.14± 153.25 535.67± 188.69 784.18± 228.56 1632.84± 179.87

Age Group

D1 (<2.5 yrs) - 1088.52± 47.33ac 1749.36±209.65 1711.97± 180.94 1211.7± 175.11a 1014.05± 186.21 1793.38± 205.13

D2 (2.5-3 yrs) - 1093.71± 35.28a 1737.46± 159.75 1610.41± 118.23 808.8± 120.62a 891.42± 166.61 1832.26± 141.42

D3 (3-3.5 yrs) - 1200.07± 37.28c 1482.18± 159.90 1488.71± 133.22 475.81± 143.40b 674.35± 209.69 1642.53± 166.14

D4 (>3.5 yrs) - 1401.91± 68.59b 1432.02± 281.80 1470.23± 213.54 80.81± 305.56ab 269.51± 368.72 1404.80± 229.27

Mean with similar superscript do not differ significantly (*P<0.05). AFSC = Age at first semen collection; AFSF = Age at first semen freezing; ALSF = Age at last semen freezing; ALSC = Age at last semen collection; SPP = Semen production period; FSPP = Frozen semen production period; AD = Age at disposal


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young age in scientific line as well as introducing them at appropriate age for semen donation. Lower age at disposal and frozen semen production period could be due to early fulfillment of desired number of frozen semen doses from the breeding bulls.

The results showed that AFSC, AFSF, ALSF and ALSC were the lowest in bulls born in Period-4 (1015.17±65.05, 1114.55±57.91, 1204.02±379.98 and 1132.17±269.68 days) whereas, AFSC and AFSF was highest in Period-3 (1171.81±44.12 and 1278.80±36.16 days) while ALSF, ALSC, SPP and AD was highest in Period-1 (1806.35±152.53, 1871.18±134.21, 726.7±146.88 and 1951.97±138.69). FSPP was found to be the highest in Period-2 i.e. 788.53±210.44 days, reflecting the changes in management from time to time at Artificial Breeding Research Centre. The effect of period of birth on ALSC was found significant (P<0.05). Chauhan et al. (2010) conducted a study on Karan Fries and found effect of period of birth to be significant for AFSC, AFSF, ALSC, and AD. Similarly Mukhopadhyay et al. (2010) found effect of period of birth to be significant for AFSC, AFSF, SPP, FSPP and AD in Karan Fries, Sahiwal cattle and Murrah bulls.

Effect of season of birth on AFSC and AFSF showed that in autumn it was the lowest i.e. 1029.40±48.06 and 1157.25±42.26 days it may be due to feed and fodder availability resulted in better nutrient availability to male born in autumn season. Whereas male born in summer showed lowest ALSF, ALSC, SPP and FSPP values i.e. 1420.28±373.82, 1399.69±236.09, 502.18±290.41 and 568.71±512.94, respectively. The bulls born in autumn season reached AFSC and AFSF earlier than those born in other seasons. The bulls born during rainy season were the last to donate their first semen whereas males born in summer season stopped donating semen earlier than the bulls born

in other seasons. Males born in autumn season performed better in term of semen donation as well as semen freezing while males born on other seasons are not performing upto mark. Therefore management decision can take care of this to manipulate the breeding condition of the female herd to optimize maximum number of birth of males offspring in favorable season. The season of birth had no significant effect on AFSC, AFSF, ALSF, ALSC SPP, FSPP and AD. Season of birth has its effect up to a first few months of age of the animal and gradually diminishes. In similar line Chauhan et al. (2010) and Mukhopadhyay et al. (2010) also found no significant effect of season on any of these traits. The effect of age at sexual maturity was significant (P<0.05) on AFSF and SPP. The males donating semen at an early age reached in freezable quality semen production stage i.e. 1088.52±47.33 and it increased in case of males donating semen at later ages. ALSF, ALSC, SPP and FSPP was found maximum in males donating semen at earlier age i.e. 1749.36±209.65, 1711.97±180.94, 1211.7±175.11 and 1014.05±186.21 days, respectively and the estimates revealed decreasing trend with the bulls donating semen at higher age. Age of disposal (AD) was found maximum in D-2 (2.5 to 3 years) age group and minimum in D-4 (>3.5 years) age group which may be due to poor semen quality donation. The effect of age of the bull at first collection was important for young bulls, likely because of physiological changes that occur during their growth to the stage of sexual maturity (Mathevon et al., 1998). Chauhan et al. (2010) reported that age group had significant effect on AFSF.


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CONCLUSION

Semen donation parameters, like age at first semen collection, age at first semen freezing, semen production period and freezen semen production period were not significantly affected by period and season of birth in Murrah buffalo bulls. The age at first semen donation in Murrah males can be reduced by introducing the young male calves to training at an early age, which could increase the doses of semen obtained from each male. Better managemental practices and regular monitoring of breeding soundness may improve the reproductive performance of Murrah buffalo breeding bulls.

ACKNOWLEDGEMENTS

The authors are thankful to the Head, DCB Division and Incharge, ABRC of NDRI, India for providing necessary information. The authors are grateful to the Director, ICAR-NDRI, Karnal, India for providing the financial assistance during the research work. It is also certified that there is no conflict of interest among the authors.

REFERENCES

Amann, R.P. and J.O. Almquist. 1976. Bull management to maximize sperm output. p. 1-15. In Proceedings of 6th Tech. Conf. Artificial Insemination Reprod. Natl. Assoc. Anim. Breed., Columbia.

Brito, L.F.C., A.E.D.F. Silva, M.M. Unanian, M.A.N. Dode, R.T. Barbos and J.P. Kastelic. 2004. Sexual development in earlyand late-maturing Bos indicus and Bos indicus

× Bos taurus crossbred bulls in Brazil. Theriogenology, 62: 1198-1217.

Chauhan, I.S., A.K. Gupta, K. Khate, A. Chauhan, T.K.S. Rao, S. Pathak, R. Hazra and M. Singh. 2010. Genetic and non-genetic factors affecting semen production traits in Karan Fries crossbred bulls. Trop. Anim. Health Prod., 42: 1809-1815.

Graffer, T., H. Solbu and O. Filseth. 1988. Semen production in artificial insemination bulls in Norway. Theriogenology, 30: 1011-1021.

Khate, K. 2005. Studies on multistage selection of dairy bulls, M.V.Sc. Thesis, National Dairy Research Institute, Karnal, Haryana, India.

Khatun, M., S.K. Kanchan and C.S. Mukhopadhyay. 2013. Subfertility problems leading to disposal of breeding bulls. Asian Austral. J. Anim., 26(3): 303-308.

Kodagali, S.B., B.K. Bhavsar and F.S. Kavani. 1980. Age at and reasons for disposal of AI buffalo bulls. Indian J. Anim. Health, 19: 31-34.

Mathevon, M., M.M. Buhr and J.C.M. Dekkers. 1998. Environmental, management and genetic factors affecting semen production in Holstein bulls. J. Dairy Sci., 81: 3321-3330.

Mukhopadhyay, C.S., A.K. Gupta, B.R. Yadav, K. Khate, V.S. Raina, T.K. Mohanty and P.P. Dubey. 2010. Subfertility in males: an important cause of bull disposal in bovines. Asian Austral. J. Anim., 23(4): 450-455.

Ranjhan, S.K. and S. Qureshi. 2006. Buffalo development in Asia. Asian Buffalo Magazine, 3(2): 4-11.

Rustenev, A.P. 1989. Reproductive ability and quality of progeny of improver bulls. Byull. Vses. Nauch. Issled. Inst. Razveden Genet Sel’ Skokhozyaistvennykh Zhivotnykh, 109:


122

30-32.Siratskii, Z.I. 1990. Inheritance of reproductive

ability of bulls. Tsitol. Genetics, 24: 28-34.Snedecor, G.W. and W.G. Cochran. 1968. Statistical

Methods. Oxford and IBD Publishing Co., Calcutta.

Suryaprakasam, T.B. and A.V.N. Rao. 1993. Studies on breeding life and disposal pattern of AI sires in Andhra Pradesh. Indian Vet. J., 70: 1022-1024.


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ABSTRACT

To evaluate immunologic activities of neutrophils, blood samples were collected from 6 high yielding (HY) and 6 low yielding (LY) Murrah (MU) buffaloes on -15, -7, -5, -3, -2, -1 days prepartum, at calving and on 1, 2, 3, 5, 7 and 15 days postpartum. Blood Total leucocyte counts (TLC) and neutrophil percent increased at calving in both the groups, but the levels were found to be significantly (P<0.05) high in HY MU buffaloes. The number of bend neutrophils were also significantly (P<0.05) higher in HY buffaloes. Significant (P<0.05) immunosuppression in relation to PA was found for HY MU buffaloes throughout the peripartum period with lowest immunosuppression at calving in both the groups. Cortisol levels were significantly (P<0.01) higher during calving and negatively correlated with neutrophilic functions. The difference between two groups also remained significant (P<0.05) as higher level of cortisol found in HY MU buffaloes. Elastase, collagenase and cathepsin were significantly (P<0.05) decreased during parturition. Elastase of HY buffalo neutrophil was reduced 2 times than 1.5 times for LY buffalo at calving. Collagenase and cathepsin levels were significantly (P<0.05) higher in LY buffaloes. At 7 day pre

calving and 7 and 15 days post calving, expression of TLR-2 gene were significantly (P<0.05) lower in HY buffaloes. Expression of TLR-4 and IL-8 genes were significantly (P<0.05) lower on days 15 pre and post caving in HY buffaloes. Decreased blood neutrophilic functions in buffaloes having high production potential provides lower disease resistance and make them more susceptible to infection around peripartum.

Keywords: buffaloes, cortisol, gene expression, neutrophil activity, peripartum

INTRODUCTION

Buffaloes are found mostly in the Indian subcontinent and some part of South America, Southern Europe, Middle East and Northern America. Buffalo is the major source of milk production contributing 12.1% in world, 38% in Asia and 55% in India’s total milk production (FAO STAT, 2007). Buffaloes are highly resistant to various infections as compared to cows (El-Wishy, 2007). Milk production potential also affects the immunity of animals. Buffaloes with higher production potential are more prone to infection as compared to low producing buffaloes.

COMPARATIVE EVALUATION OF FUNCTIONAL ACTIVITY OF NEUTROPHIL IN HIGH AND LOW YIELDING MURRAH BUFFALOES DURING PERIPARTUM PERIOD

M.M. Pathan1,*, M. Kaur2, A.K. Mohanty3 and A.K. Dang2

Original Article

1Department of Veterinary Physiology and Biochemistry, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, India, *E-mail: [email protected] Cattle Physiology Division, National Dairy Research Institute, Karnal, Haryana, India3Animal Biotechnology Division, National Dairy Research Institute, Karnal, Haryana, India


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The immunological basis behind this has not been completely elucidated in buffaloes in specific during peripartum period (Nanda et al., 2003). Keeping this objective in mind present study was formulated to explore the immune physiological basis of resistance of buffaloes and also to understand the immune related factors which affect the health status of high producing buffalo and make them more prone to infection. For that, blood neutrophil is taken as the cell of interest and one of the major components of immune axis as they are considered as first line of cellular defence which form an integral part of innate immune system. Neutroophils mediate killing of bacterial pathogens by phagocytosis through a cascade of proteases, antimicrobial peptides, and free redicals (Segal, 2005). Eventually, neutrophils undergo apoptosis in the tissues or at the sites of inflammation. Primary role of neutrophils is the participation in inflammatory response by producing cytokines, eccosanoids and cell signalling molecules. The complex interplay between all these leads to neutrophilic activity causing host cell protection (Serhan et al., 2005).

The critical period of calving was selected as neutrophils play a vital role in the onset of disease around parturition (Kehrli et al., 1989). Phagocytic and respiratory burst activity of neutrophil reduces around parturition (Hoeben et al., 2000), which may lead to mastits, metritis and retained placenta like postpartum disease (Kehrli and Harp, 2001). According to Burton et al. (2005), blood neutrophils exhibits expression of glucocorticoids receptors and these receptors respond to high plasma cortisol concentrations in bringing out altered neutrophil signalling and functioning around parturition.

Literature lacks information in high and low yielding buffaloes regarding the activity of neutrophils in terms of phagocytosis (Dang et

al., 2009, 2012). Also, there are no reports on the enzymatic activity and differential expression of neutrophilic genes during calving period and specific to production potential. With this overview, the present study was undertaken to elucidate and compare the neutrophilic activities in both high and low producing Murrah buffaloes around parturition.


Selection of animalsTwelve Murrah buffaloes in their advance

stage of gestation i.e. at 15 days before the expected date of calving were selected from the National Dairy Research Institute experimental herd. They were further divided into two subgroups, high yielding (HY) and low yielding (LY) based on their production potential of previous lactation. HY MU buffaloes (n=6) were producing above 2157±102.45 liter per lactation whereas LY MU buffaloes (n=6) were producing below 1632.53±77.53 liter per lactation. All the buffaloes were offered adlib green fodder and calculated amount of concentrate mixture. Fresh tap water was also made available adlib at all times of the day. All the experimental buffaloes were healthy and free from any anatomical, physiological and infectious disorders.

Collection of samples and analysisBlood samples were collected from all

the buffaloes during -15, -7, -5, -3, -2 and -1 days prepartum, on the day of calving and 1, 2, 3, 5, 7 and 15 days postpartum. Calving in all the animals occurred within ±5 days of the expected date of calving.

Blood total leukocyte counts (TLC) and differential neutrophil counts were estimated microscopically from all the group of animals. In


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vitro phagocytic activity of blood neutrophils by nitro blue tetrazolium (NBT) assay (Dang et al., 2012) and plasma cortisol levels were also estimated by ELISA (Endocrine Technologies, USA) during both the pre and postpartum days as indicated above. The minimum detectible concentration of cortisol by this assay was estimated to be 0.1 ng/ml. Coefficient of Variation (CV) were calculated from the calculated concentrations. Inter-assay % CV was found to be 2.59 and intra-assay % CV was found to be 0.05.

Activities of enzymes Elastase 2, Collagenase and Cathepsin G were measured by ELISA kits (WEKA MED and Wuhan Eiaab Science Co., Ltd., China) from blood samples collected during -7, -3 days prepartum, on the day of calving and 3 and 7 days postpartum. For preparing lysate of neutrophils, the isolated neutrophils were dissolved in 1 ml PBS. Glass beads were added to neutrophil suspension and shock was given for 25 seconds by Bead beater (Unigenetics Instrument Pvt. Ltd., India). Put it in ice for 1 minute, then again shock was given for 25 seconds. It was centrifuged at 1000 x g for 10 minutes. Supernatant was taken in 2 ml eppendorf tubes and were stored at -20oC till further estimation. Percent CV was calculated from the calculated concentrations. Inter-assay %CV was found to be 6.12, 5.33, 6.21 and intra-assay %CV was found to be 3.52, 2.11 and 1.88 for Elastase, Collagenase and Cathepsin G respectively.

Relative expression of neutrophilic genesAll solutions were prepared using DEPC

treated RNase free plastic wares and water. Total RNA from the blood neutrophils was extracted using Trizol method as per Chonczynski and Sacchi, (1987). The RNA pellet was air dried for 15 to 30 minutes and dissolved in 25 μl of RNA

storage solution and stored at -80oC till further use. Quality of RNA was checked by Agarose gel electrophoresis using 0.8% gel (in 1X TAE buffer, pH 8.0) of high quality molecular biology grade agarose (Sigma, USA). Ethidium bromide was used as fluorescence dye at the rate of 0.5 µg/ml of gel, whereas, bromophenol blue was used as tracking dye at the rate of 3 µl mixed with RNA during time of loading of sample in to well of the gel. Electrophoresis was carried out at 8 V/cm for half an hour. After completion of electrophoresis, the gel was examined under UV transilluminator. DNase treatment was done by using DNA free Kit (Ambion, UK) according to manufacturers’ instructions. Total RNA was quantified, and OD260nm/OD280nm was determined with ND-3300 flurospectrophotomer (NanoDrop Technologies, UT) and purity of RNA was judged on the basis of optical density ratio at 260:280 nm. Reverse transcription was performed from 1 µg of RNA using Novagen first strand cDNA synthesis kit (La Jolla, CA).

Real Time PCR for TLR-2, TLR-4 and IL-8 and two housekeeping genes (Glyceraldehydes 3-phosphate dehydrogenase - GAPDH and β- actin) was carried out using Roche Light Cycler-480, Germany. The above housekeeping genes were selected as they had been shown to be the most stably expressed in the neutrophils (Robinson et al., 2007). The sequence information of gene was retrieved from NCBI database and suitable primers were designed using primer-3 web interfaces. Details of primer specification are given in the Table 1. Broadly for each real-time quantitative PCR (qPCR), 1 µg cDNA was added to a 20 µl mix containing primers, IQ SYBER-green supermix (Bio-Rad) and nuclease free water. PCR conditions were 300s at 95oC, 45 cycles of 20s at 95oC, 20s at appropriate annealing temperature (Table 1),


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20s at 72oC. A melting curve for each qPCR with a single peak at the correct melting temperature was indicative of reliable and desired PCR product. mRNA abundance on 0 day (a day of parturition) was taken as calibrator to whom relative expression were seen. Calculation was done using the 2-ΔΔCt

method (Livak and Schmittgen, 2001).

Statistical analysisStatistical analysis was performed using

least square model through SYSTAT software (sigma plot 11.0, Chicago, IL, USA). The model used for analysis was Yij = µ + Gi + Dj + Ti (Dj) + Eij, where Yij was an observation of dependent variable; µ was the population mean for the variable; Gi was the effect of the group; Dj was the effect days; Gi (Dj) was the interaction between the group and days and Eij was the random error associated with observation. The means were separated and compared using Tukey test as post hoc test, because this test is able to control the errors of multiple comparisions simultaneously. Further, the effect of different treatments on 15 days of prepartum was not used as covariate for subsequent analysis as our main interest was to differentiate the effect of two different treatments.

RESULTS

Total leukocyte counts was measured from blood of HY and LY MU buffaloes during pre and postpartum period and shown in Table 2. Highest (P<0.001) level of TLC was found on the day of calving in both the groups but the levels were found to be significantly (P<0.05) high in HY MU buffaloes as compared to LY MU buffaloes. After calving TLC was reduced significantly (P<0.01) on 3, 5 and 7 day after calving as compared to on the

day of calving in both HY and LY MU buffaloes. Difference in TLC between the day of calving and 15 days after calving was highly significant (P<0.001). Between HY and LY MU buffaloes, significant changes were found in all postpartum days except day 7 and 15 of postpartum. Overall mean of TLC remained significantly (P<0.05) higher for HY than LY MU buffaloes.

High yielding MU buffaloes found significantly (P<0.05) higher neutrophil count than LY MU buffaloes. However, blood neutrophils was found highest (P<0.001) on the day of calving in both the groups. After parturition, significant (P<0.01) reduction in neutrophil counts were observed just one day after calving in both the groups (Table 3). We also observed an increase in percentage of band neutrophils and decrease in segmented neutrophil percentage on the day of caving in both the groups of buffaloes. But the percentage of immature or band neutrophils were significantly (P<0.05) higher in HY buffaloes then LY buffaloes.

Neutrophil PA was estimated in both the groups of buffaloes during peripartum period (-15 to +15 days). The PA was represented in terms of optical density due to formation of formazan crystals (Table 4). Lowest neutrophilic PA was observed on the day of calving in both HY and LY MU buffaloes but in between two groups, significant (P<0.05) immunosuppression was found for HY MU buffaloes as compared to LY MU buffaloes throughout the peripartum period. After calving, sluggish increase in PA was observed upto 3 day postpartum in HY MU buffaloes and upto 5 days postpartum in LY MU buffaloes which became significant (P<0.001) on 15 days postpartum as compared to that observed on the day of calving in both HY and LY MU buffaloes.

Plasma cortisol level were measured by


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ELISA during peripartum period (-15 to +15 days) and presented in Table 5. Levels of plasma cortisol were always found to be significantly (P<0.001) higher in HY MU buffaloes as compared to LY MU buffaloes during peripartum period. A steady rise in plasma cortisol was observed in both the groups of animals with peak observed on the day of calving. Level of cortisol observed on the day of calving was approximately four and 2.5 times higher for HY and LY MU buffaloes respectively as compared to level of cortisol observed on day 15 preparrtum. After parturition, steady significant (P<0.001) decline in cortisol levels was observed on 1 day after calving in both groups of buffaloes. Thereafter, cortisol levels increased or decreased but the differences were non significant upto 15 day postpartum as compared to 1 day after calving in both HY and LY MU buffaloes.

Neutrophilic enzymes that are important in combating infection like Elastase, cathepsin G and Collagenase were estimated byELISA in both groups of MU buffaloes and depicted in Table 6. Two and 1.5 time reduction in level of elastase 2 was observed on the day of calving in HY and LY MU buffaloes respectively as compared to prepartum level. After calving, rapid increase in elastase 2 was observed on 3 day postpartum in both the groups of buffaloes. The levels of elastase 2 observed during whole peripartum period were remained significantly (P<0.05) higher for HY MU buffaloes as compared to LY MU buffaloes. But on the day of caving the significantly (P<0.05) lower level was observed in HY MU buffaloes than LY MU buffaloes. Levels of collegenase were also reduced up to 2 fold on the day of calving in both groups of buffaloes (Table 6). However, significant (P<0.05) reduction was seen in HY than LY MU buffaloes. Lowest level of cathepsin G was also observed on the day of calving in both HY and

LY MU buffaloes during whole peripartum period (Table 6). Between HY and LY MU buffaloes, significant (P<0.05) difference were observed only on the day of calving with significantly lower level in High yielder as compared to low yielder Murrah buffaloes. The result for the relative expression of the important neutrophilic genes TLR-2, TLR-4 and IL-8 have been presented in Table 7. Significantly (P<0.05) lowest expression of TLR-2, TLR-4 and IL--8 genes were observed on the day of calving as compared to all peripartum days in both HY and LY MU buffaloes. Expression of these genes remained lower in HY MU buffaloes as compared to LY MU buffaloes. Significant (P<0.05) change in expression of TLR-2 gene between HY and LY MU buffaloes found throughout peripartum period except 15 days before calving, whereas, TLR-4 gene expression differed only at 15 days before parturition in between two groups. Expression of IL-8 gene was differed significantly between HY and LY MU buffaloes on 15 days before and after calving.

DISCUSSION

White blood cells are involved in defence against pathogens. The blood TLC and neutrophil counts reflect the immune status of animal in HY and LY MU buffaloes. Values of blood TLC and neutrophil counts observed around the peripartum period in both groups of buffaloes were within the normal range as reported by Abd Ellaha et al. (2013) in midterm pregnant buffaloes. An increase in TLC around calving is coupled with rise in cortisol level in both the groups. It is believed that TLC increases around calving as a result of antipartum rise in cortisol level. However, TLC decreases during


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postpartum period and it is coupled with migration and recruitment of blood neutrophils towards uterine lumen and mammary tissues (Preisler et al., 2000). The study also signifies that higher cortisol stimulates larger amount of neutrophil release around calving. Neutrophils are known as first line defence, they migrate first from blood into an inflamed area for phagocytosis and intracellular killing by engulfing bacteria with two distinct mechanisms, the respiratory burst and by digestion through lysosomal enzymes (Jain, 1986). Mature segmented neutrophils only have the complete machinery to phagocytose bacteria and so they must be high in circulation (Paape et al., 2003) but, we observed significantly higher numbers of band (immature) neutrophils at calving in all buffaloes. Larger number of bend neutrophils in high producing buffaloes as compared to low producing buffaloes may be due to stress on the mammary tissues to produce more milk. The rapid increase of circulating neutrophils was attributed mainly to an influx of neutrophils from the hematopoietic system and not from a marginal pool of mature leukocytes. A higher level of cortisol around calving is the reason behind an increase in immature neutrophils. Mature neutrophils are more sensitive to cortisol as compared to immature neutrophils and have more number of glucocorticoid receptors as compared to immature neutrophils (Burton et al., 2005). Migration of mature neutrophils from bone marrow reduces due to reduction in number of adhesion receptors (L-selectin and CD18). Immature neutrophils are less affected due to lower effect of cortisol in response to lower number of glucocorticoid receptors. So, they marginate more from hematopoietic reserve as compared to mature neutrophils (Paape et al., 2003; Burton et al., 2005)

In vitro analysis of neutrophil function

provides a very effective tool for the study of natural disease resistance. We observed decreased PA of blood neutrophils around calving. Diminished neutrophil functions and compromised host resistance mechanisms during peripartum period in dairy animals have also been observed by Meglia et al. (2001) and Dang et al. (2012), Poor activity of neutrophils may be due to more numbers of immature neutrophils which are coming in circulation which have no proper machinery to fight or phagocytose against infection as also observed by us from 3 day prepartum to 5 day post partum, whereas, reduction in phagocytic activity was highly significant on the day of calving as compared to 15 day before calving. This suppression in the PA might be due to a sharp increase in the cortisol levels. We also found a significant negative correlation between PA of blood neutrophils and plasma cortisol levels. There were increased neutrophil numbers during parturition yet phagocytic activity remained lower. Parturition reflex causes higher plasma cortisol level that causes hyper stimulation of red bone marrow for the faster release of neutrophils. As a result of this, there is release of more number of immature band neutrophils and a less number of matured segmented neutrophils. That is why the phagocytic activity of neutrophils decreases as evident in our study (Paape et al., 2003). During prepartum period (15 day before calving) animals are in dry stage so, there is no stress of milk production but at parturition, animals have to face stress of calving, synthesize colostrum (up to 3 days) and milk. Further, milk production potential is higher in high producing buffaloes than low producing; therefore, they exhibited more stress and low PA as compared to low yielding buffaloes.

Glucocorticoids are a class of steroid hormones that bind to the glucocorticoid receptor, and are part of the feedback mechanism in the


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immune system that down regulate the immune activity. Cortisol is released in response to stress and a low level of blood glucose. Glucocorticoid suppresses the immune system, increase blood sugar through gluconeogenesis, and aid in metabolism of protein, fat and carbohydrate. We observed a significantly higher level of cortisol at calving as compared to 15 days before and 15 days after calving in both the groups. A similar observation was reported in cattle (Prakash and Madan, 1985; Goff and Horst, 1997) and cross bred goats (Khan and Ludri, 2002). However, the cortisol values reported at calving were more than those reported by above authors. We also found a significantly higher level of cortisol in HY than LY KF cows. The cows during peripartum period are under various types of stressful conditions like stress of providing nutrition to its growing calf, stress of labor, to synthesize colostrum and then milk. A social stress of being isolated is also there. Overall effects of stress increased cortisol level, produced neutrophilia with decreased functional capacity of neutrophils, immunosupression and ultimately the high producing cows become more prone to mastitis and other infections (Kehrli et al., 1991). In agreement with our finding high levels of cortisol at calving have also been reported to act as powerful immunosuppressive agent (Goff and Horst, 1997).

Neutrophils mediate phagocytosis through a complex cascade of enzymes and their interrelated pathways. The release of enzymes is specifically regulated by cytokine network and their signaling to neutrrophils via cytokine receptors. Elastase 2, collagenase and cathepsin G are granular enzymes that are stored in neutrophil cytoplasm. The timely and net release of these enzymes determines the ultimate fate of neutrophil activities in terms of phagocytosis and resolution of inflammatory

cascades. Granules are stored in neutrophils and are store house of variety of enzymes that are released to extra cellular space during inflammation and midiate pathogen inactivation and killing. Soluble agent like fMLP (N-formyl-methionine-leucine-phenylalanine), chemotaxins and C5a are the regulators of granule release from neutrophils. Neutrophils release elastase and cathepsin, which are serine proteases during inflammation to bring out destruction of pathogens (Belaaouaj et al., 2000).

Decreased neutrophil enzyme levels observed in this study may be because at calving more immature neutrophils are released that are poor in synthesis of granular enzyme as well as due to high cortisol are not able to release granular enzyme. During any disease condition the levels of these enzymes increased, which provide immunity to animals (Haddadi et al., 2006). During calving when animals go down in concentration of these enzymes due to higher level of cortisol make animal more susceptible to postpartum diseases (mastitis, matritis etc.). Higher levels of these enzymes in LY MU buffaloes then HY MU buffaloes indicate that the LY buffaloes are more resistant as compared to HY buffaloes to postpartum infections. The animals which give more milk found to have fewer amounts of neutrophilic enzymes which make them more prone to diseases.

Detection of pathogens in neutrophils is mediated by a variety of pattern-recognition systems, foremost amongst which are the Toll-like receptors (TLRs), and thus these receptors are likely to have important roles in the regulation of neutrophil function (Parker et al., 2005). Our study is the first to indicate a down regulation of blood PMN expression of immune genes due to increased endogenous blood plasma cortisol during peripartum period in buffaloes. We observed a significantly

http://en.wikipedia.org/wiki/Cortisol

http://en.wikipedia.org/wiki/Stress_(medicine)

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higher expression of TLR-2, TLR-4 and IL-8 on 15 day before and after calving as compared to the day of calving. During the periparturient period animal experience negative energy balance (NEB) from 3 day before to 3 days after calving. (Ingvartsen and Andersen, 2000). The higher level of cortisol helps to provide energy demand by increasing lypolysis and gluconeogenesis, which results in an increase in the ratio of unsaturated fatty acid to saturated fatty acid. Saturated fatty acid induces the activation of TLR-2 and 4, whereas, unsaturated fatty acids inhibits (Lee and Hwang, 2006). Decreased expression of these genes during calving might be due to increased levels of unsaturated fatty acids.

Similar role is played by IL-8, which is considered as the central regulator of neutrophil signalling (Burton et al., 2005). During the study, we estimated the expression of (IL-8). It is a potential chemoattractant factor for neutrophil which mediate transendothelial migration of neutrophils to tissue spaces to destroy bacterial pathogens (Kehrli and Harp, 2001). IL-8 regulates the recruitment of neutrophils as well as T-lymphocytes to the site of infection (Wang et al., 2007). Activation of neutrophils during inflammation is a key event which is mediated by IL-8 (Galligan and Coomber, 2000). In our study, there was a significantly (P<0.05) higher expression of IL-8 on 15 day before and 15 day after parturition as compared to the day of calving. This indicates that the neutrophils of high producing buffaloes having lower ability to migrate to the site of infection then low producing buffaloes. With this finding the statement comes out that, neutrophils of buffaloes having higher production potential are less immunocompetent then buffaloes with low production potential.

CONCLUSIONS

Present study was planned to evaluate the relative competency of blood neutrophils in high and low producing Murrah buffaloes during peripartum period. The range experiment conducted during the period were mainly confined to understand some of the basic features of blood neutrrophils in terms of their activities and gene expression that are associated with the regulation of immune physiological responses. We observed a decrease in the PA of HY buffalo blood neutrophils as compared to LY buffaloes. The relative higher circulating concentration of cortisol is another determining factor that keeping up lower PA of HY buffalo neutrophils. Higher content of neutrophilic enzymes in the LY buffalo neutrophils strongly supported that immunocompetency of LY buffalo neutrophils is more than that of HY buffaloes. Eventually, we also reported a decrease in the mRNA expression of TLR-2, TLR-4 and IL-8 genes in HY MU buffaloes. Altogether, these findings make us to frame a conclusion that the LY buffalo neutrophils are more potent as compared to the HY buffalo neutrophils. It can be a probable explanation behind the fact that HY buffaloes are less resistant to infections during transition period as compared to the LY buffaloes. This study although carried out on some of the neutrophilic functions, clearly indicated the degree of immune suppression occurring in two different yielders of same species around peripartum. These results will help in understanding the physiology of neutrophils at calving and help to develop strategies to improve the immune functions around this period. Also, further studies are required to employ genetic and proteomic tools to find out the exact mechanism of neutrophil action in buffaloes.


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ACKNOWLEDGMENTS

The authors are thankful to the Department of Biotechnology, Ministry of Science and Technology, Government of India for providing financial assistance for carrying out this research.

REFERENCES

Abd Ellaha, M.R., M.I. Hamedb and R.I. Derarc. 2013. Serum biochemical and haematological reference values for midterm pregnant buffaloes. J. Appl. Anim. Res., 41(3): 309-317.

Belaaouaj, A., K.S. Kim and S.D. Shapiro. 2000. Degradation of outer membrane protein in Escherichia coli killing by neutrophil elastase. Science, 289(5482): 1185-1188.

Burton, J.L., S.A. Madsen, L.C. Chang, P.S.D. Weber, K.R. Buckham, R.V. Dorp, M.C. Hickey and E. Bernadette. 2005. Gene expression signatures in neutrophils exposed to glucocorticoids: A new paradigm to help explain “neutrophil dysfunction” in parturient dairy cows. Vet. Immunol. Immunop., 105: 197-219.

Chonczynski, P. and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156-159.

Dang, A.K., S. Kapila, M. Purohit and C. Singh. 2009. Changes in colostrums of Murrah buffaloes after milking. Trop. Anim. Heath Pro., 41: 1213-1217.

Dang, A.K., S. Prasad, K. De, S. Pal, J. Mukherjee, I.V.R. Sandeep, G. Mutoni, M.M. Pathan, M. Jamwal, S. Kapila, R. Kapila, H. Kaur,

S. Dixit, A.K. Mohanty and B.S. Prakash. 2012. Effect of supplementation of vitamin E, copper and zinc on the in vitro phagocytic activity and lymphocyte proliferation index of peripartum SW (Bos Indicus) cows. J. Anim. Physiol. An. N., 97(2): 315-321.

El-Wishy, A.B. 2007. The postpartum buffalo: I. Endocrinological changes and uterine involution. Anim. Reprod. Sci., 97(3-4): 201-215.

FAOSTAT, 2007. Statistics Division, Food and Agriculture Organization, UNO, RRome, Italy.

Galligan, C.L. and B.L. Coomber. 2000. Effects of human IL-8 isoforms on bovine neutrophil function in vitro. Vet. Immunal. Immunop., 74: 71-85.

Goff, J.P. and R.L. Horst. 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci., 80: 1260-1263.

Haddadi, K., C. Prin-Mathieu, F. Moussaoui, G.C. Faure, F. Vangroenweghe, C. Burvenich and Y. Le Roux. 2006. Polymorphonuclear neutrophils and Escherichia coli proteases involved in proteolysis of casein during experimental E. coli mastitis. Int. Dairy J., 16: 639-647.

Hoeben, D.E., G. Monfardini, C. Opsomer, H. Burvenich, A. Dosogne, De Kruif and J.F. Beckers. 2000. Chemiluminescence of bovine polymorphonuclear leucocytes during the periparturient period and relation with metabolic markers and bovine pregnancy-associated glycoprotein. J. Dairy Res., 67: 249-259.

Ingvartsen, K.L. and J.B. Andersen. 2000. Integration of metabolism and intake regulation: a review focusing on


132

periparturient animals. J. Dairy Sci., 83: 1573-1597.

Jain, N.C. 1986. In: Schalm’s Veterinary Hematology, Lea and Febiger, Philadelphia. 821-837.

Kehrli, M.E. and J.A. Harp. 2001. Immunity in the mammary gland. Vet. Clin. North Am Food Anim. Pract., 17(3): 495-516.

Kehrli, M.E., B.J. Nonnecke and J.A. Roth. 1989. Alterations in bovine lymphocyte function during the periparturient period. Am. J. Vet. Res., 50: 215-220.

Kehrli, M.E., K.A. Weigel, A.E. Freeman, J.R. Thurston and D.H. Kelley. 1991. Bovine sire effects on daughter’s in vitro blood neutrophil functions, lymphocyte blastogenesis, serum complement and conglutinin levels. Vet. Immunol. Immunop., 27: 303-319.

Khan, J.R. and R.S. Ludri. 2002. Hormone profile of crossbred goats during the periparturient period. Trop. Anim. Health Prod., 34: 151-162.

Lee, J.Y. and D.H. Hwang. 2006. The modulation of inflammatory gene expression by lipids: Mediation through Toll- like Receptors. Mol. Cells., 21: 174-185.

Livak, K.J. and T.D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT Method. Methods., 25(4): 402-408.

Meglia, G.E., A. Johannisson, L. Petersson and K.P. Waller. 2001. Changes in some blood micronutrients, leukocytes and neutrophil expression of adhesion molecules in periparturient dairy cows. Acta Veterinaria Scandinavia., 42: 139-150.

Nanda, A.S., P.S. Brar and S. Prabhakar. 2003. Enhancing reproductive performance

in dairy buffalo: major constraints and achievements. Reprod. Suppl., 61: 27-36.

Paape, M.J., D. Douglas, X.Z. Bannerman and J.W. Lee. 2003. The bovine neutrophils: Structure and function in blood and milk. Vet. Res., 34: 597-627.

Parker, L.C., M.K. Whyte, S.K. Dower and I. Sabroe. 2005. The expression and roles of Toll-like receptors in the biology of the human neutrophil. J. Leukocyte Biol., 77: 886-892.

Prakash, B.S. and M.L. Madan. 1984. Radioimmunoassay of cortisol in peripheral blood plasma of buffaloes puerperium, Theriogenology, 22(3): 241-246.

Preisler, M.T., P.S. Weber, R.J. Tempelman, R.J. Erskine, H. Hunt and J.L. Burton. 2000. Glucocorticoid receptor down-regulation in neutrophils of periparturient cows. Am. J. Vet. Res., 61: 14-19.

Robinson, T.L., I.A. Sutherland and J. Sutherland, 2007. Validation of candidate bovine reference genes for use with real-time PCR. Vet. Immunol. Immunop., 115: 160-165.

Segal, A.W. 2005. How neutrophils kill microbes. Annu. Rev. Immunol., 23: 197-223.

Serhan, C.N. and J. Savill. 2005. Resolution of inflammation: the beginning programs the end. Immunology, 6: 1191-1197.

Wang, X., S. Xui., X. Gao., H. Ren and J. Chen. 2007. Genetic polymorphism of TLR4 gene and correlation with mastitis in cattle. J. Genet. Genomics., 34(5): 406-412.

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term= Meglia%2BG%5bauth%5d

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term= Johannisson%2BA%5bauth%5d

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term= Petersson%2BL%5bauth%5d

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term= Waller%2BKP%5bauth%5d

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=PubMed&term= Waller%2BKP%5bauth%5d


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ABSTRACT

Genetic disorders constitute a menacing threat whose consequences often become evident only after several generations of breeding, when short-term, low cost solutions are no longer possible. The present study involved screening of 135 buffaloes viz. Murrah buffalo (n=106) and Surti buffalo (n=29) bulls for autosomal recessive genetic disorders such as Bovine Leukocyte Adhesion Deficiency (BLAD), Deficiency of Uridine Monophosphate Synthase (DUMPS), Bovine Citrullinemia (BC) and Factor XI Deficiency (FXID) using PCR based techniques. Genomic DNA was extracted from blood by High Salt Method and the fragments of genes of interest were amplified by PCR technique. The amplified PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. Bulls were screened for FXID based on PCR conformation. The screening of Murrah and Surti bulls revealed that none of the buffaloes screened were carrier for BLAD, DUMPS, BC and FXID.

Keywords: genetic disorders, BLAD, DUMPS, bovine Citrullinemia, FXID, Buffaloes, PCR-RFLP

INTRODUCTION

Buffalo, a triple purpose animal, provides milk, meat and mechanical power to mankind. Buffalo was originated from Asian wild buffalo which has been domesticated since pre-historic times in Asia particularly in Indo-Pak subcontinent. Riverine buffalo, Bubalus bubalis, was domesticated nearly 5000 years ago in Iran, Iraq and Indo-Pak subcontinent, whereas domestication of swamp buffalo, Bubalus carabensis, took place in China and other part of Southeast Asia after 1000 years (Bruford et al., 2003). India has 57% of the world’s buffalo population. Riverine buffaloes contribute enormously to the rural economy and have adapted to the existing ecosystem over the years and have gained eminence as an important dairy animal in Indian subcontinent. The importance of this species to the Indian dairy industry is immense; buffalo (Bubalus bubalis) constitute 35% of the bovine population in India but they contribute more than 52.6% to the total milk production (BAHS, 2010).

Recessive autosomal genetic disorders are found at very low frequencies in livestock, but they have a disproportionate economic impact on livestock agriculture. Most of the genetic diseases in domesticated species are inherited as autosomal recessive traits, and carriers generally give no outward indications, the undesirable trait can be spread widely and covertly. Artificial insemination

SCREENING FOR GENETIC DISORDERS IN INDIAN MURRAH AND SURTI BUFFALO (BUBALUS BUBALIS) BULLS

K.P. Ramesha*, Akhila Rao, Rani Alex, G.R. Geetha, M. Basavaraju, M.A. Kataktalware,D.N. Das and S. Jeyakumar

Dairy Production Section, Southern Regional Station, National Dairy Research Institute, Adugodi, India, *E-mail: [email protected]

Original Article


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has, since its inception, revolutionised the buffalo breeding programme there is, however, an ever-present danger in its widespread application; undiagnosed genetic defects might spread rapidly through the use of a carrier bull’s semen.It has become necessary to screen all animals to minimize the risk of spreading these diseases to next generation. Understanding the molecular basis of a genetic defect makes it possible to detect carriers directly at the DNA level and more important, early in the animal’s life. The diagnosis method based on PCR and PCR-RFLP based analysis is more reliable and useful method for extensive screening for genetic disorder.

Some of the known genetic disorders in bovines are Bovine Leukocyte Adhesion Deficiency (BLAD), Bovine Citrullinemia (BC), Deficiency of Uridine Monophosphate Synthase (DUMPS), Complex Vertebral Malformation (CVM) and Factor XI deficiency (FXID). BLAD is an autosomal recessive genetic disorder which results in death of homozygous animals. It is characterized by reduced level of expression of the adhesion molecules on neutrophils called as β–integrins, a complex of CD 11/CD 18 family of proteins that helps the neutrophils to migrate to the site of inflammation (Nagahata et al., 1987). BLAD is caused mainly due to point mutation (A→G) at the position 383 of CD 18 gene located on the first chromosome of bovine, which causes an aspartic acid to glycine substitution at amino acid 128 (D128G) in glycoprotein.

There is also existence of silent point mutation (C→T) at position 775 in the CD 18 gene (Shuster et al., 1992). Afflicted animals show series of severe symptoms, including impaired wound healing and stunted growth, persistent marked neutrophilia, chronic diarrhea, ulcers on oral mucous membranes, chronic pneumonia, gingivitis,

loss of teeth, high fever and other infections (Kherli et al., 1990; Shuster et al., 1992). The enzymatic deficiency for Uridine-5-monophosphate synthase (DUMPS) is recessive genetic disorder that interferes with the biosynthesis of pyrimidines. UMPS is mapped in bovine chromosome 1 which catalyzes the conversion of orotic acid into Uridine monophoshate, precursor for all other pyrimidines and normal constituent of the milk in cow and other ruminants.(Robinson et al., 1993). DUMPS is caused by point mutation of C→G in codon 405 of exon5 (Harlizius et al., 1996). Animals homozygous for DUMPS do not survive to birth and usually die early in gestation. The embryos abort approximately 40 days after conception, leading to repeated breeding problems (Lee et al., 2002). Citrullinemia is a rare metabolic disorder characterized by serious neurologic symptoms in newborn calves (Harper et al., 1986). It is caused by C86G transition within exon 5 in the gene coding for arginino succinate synthetase (ASS) enzyme which leads to error of urea metabolism. This conversion results in a truncated peptide product (85 amino acids long rather than the normal 412 amino acids) that lacks enzyme activity. This mutation also eliminates a restriction site for the enzyme AvaII which will cut the normal gene but not cleave the mutant gene (Dennis et al., 1989). It is characterized by high levels of citrulline, and more seriously, of ammonia in plasma.

After birth, these calves, display neurological problems that become progressively worse. Depression is observable within a day; followed by unsteady gait, head pressing, aimless wandering, apparent blindness, collapse, convulsions, and death within one week (Healy et al., 1990; Harper et al., 1986). FXI Deficiency is an autosomal recessive bleeding disorder which causes increased susceptibility to infectious


135

diseases, mastitis, metritis and pneumonia, low calving and survival rates (Liptrap et al., 1995). The mutation consists of insertion of poly adenine tract (76bp AT(A)28TAAAG(A)26GGAAATAATAATTCA ) into exon 12 of FXI on chromosome 27 which introduces a premature stop codon leading to synthesis of non functional protein (Marron et al., 2004). A very low incidence of BLAD were observed in earlier studies carried out in Holstein population in Brazil, Iran, Turkey and India (Nagahata et al., 1997; Ribeiro et al., 2000; Rahimi et al., 2006; Meydan et al., 2007; Arpita et al., 2012). Earlier studies showed that all the buffaloes screened were normal for BLAD (Muraleedharan et al., 1999; Rajesh et al., 2007). DUMPS carrier cases have been reported in different countries in America and Europe in cattle (Citek and Blahova, 2004). Very low incidence of genetic disorders for DUMPS were observed among cattle population in Poland (Kaminski et al., 2005), India (Rajesh et al., 2006), Turkey (Akyuz et al., 2009; Meydan et al., 2010) and Iran (Rahimi et al., 2006). None of the males were either carrier or affected for Bovine Citrullinemia in cattle population (Meydan et al., 2010; Rajesh et al., 2006; Citek et al., 2006) and for FXID (Cyrus et al., 2011; Saeed et al., 2012). Earlier workers reported that all the buffaloes screened for Bovine Citrullinemia (Muraleedharan et al., 1999) and FXID (Saeed et al., 2012; Rajesh et al., 2007) were normal and none of them were carriers.


Animals and extraction of DNA India possesses the best River milk breeds

in Asia which include Murrah, Nili-Ravi, Surti and Jaffarabadi, which originated from the north-

western states of India and have a high potential for milk and fat production apart from their use as a work animal and as a supplementary stock for use as meat production (Sethi, 2003). A total of 135 buffalo bulls belonging to Murrah (n=106) and Surti (n=29) breeds maintained at different State frozen semen stations in Karnataka, India were utilized for the study. Blood sample (10 ml) was collected aseptically from each bull by jugular veinipuncture into vacutainer tubes containing EDTA and was stored at 4oC till further use. Within 24 h after collection of blood, genomic DNA was extracted by High Salt Method as described by Miller et al. (1988) with minor modifications. Agarose gel electrophoresis and spectrophotometric methods were used to determine quality, quantity and purity of DNA. The samples showing an optical density (OD) ratio (260 nm/280 nm) of between 1.8 to 2.0 were stored at -20oC and used for further analysis and diluted to 100 ng-µl for PCR analysis work.

Screening of genetic disorders based on PCR–RFLP technique

The fragments of genes of interest were amplified by PCR technique. Primers, PCR product size, annealing temperature and restriction enzymes (RE) used in the PCR for identification of Bovine Leukocyte Adhesion Deficiency (BLAD), Deficiency of Uridine Monophosphate Synthase (DUMPS), Bovine Citrullinemia (BC) and Factor XI Deficiency (FXID) are presented in Table 1. DNA was amplified with initial denaturation at 94oC for 5 minutes, followed by 35 cycles consisting of denaturation at 94oC for 1 minute, specific annealing temperature for 1 minute, extension at 72oC for 1 minute, with final extension 72oC for 5 minutes. Genotypes were determined using agarose gel electrophoresis (1.5%) stained with ethidium bromide. The genotypes for BLAD,


136

Tabl

e 1.

Prim

ers,

PCR

pro

duct

siz

es, A

nnea

ling

tem

pera

ture

and

rest

rictio

n en

zym

es (R

E) u

sed

for i

dent

ifica

tion

of B

ovin

e Le

ukoc

yte

Adh

esio

n D

efici

ency

(BLA

D),

Defi

cien

cy o

f Urid

ine

Mon

opho

spha

te S

ynth

ase

(DU

MPS

), B

ovin

e C

itrul

linae

mia

(BC

) and

Fac

tor X

I Defi

cien

cy (F

XID

).

Gen

etic

di

sord

erPr

imer

sequ

ence

(5’→

3’)

PCR

pr

oduc

t si

ze (b

p)

Ann

ealin

g te

mpe

ratu

re

(˚C

)

Res

tric

tion

enzy

me

used

R

efer

ence

BLA

DF:

CC

TTC

CG

GA

GG

GC

CA

AG

GG

CT

R: C

TCG

GTG

ATG

CC

ATTG

AG

GG

C

136

bp57

°CTa

qI a

t 650 C

fo

r 5 h

Cite

k et

al.,

200

6

DU

MPS

F: A

GG

GTC

TTA

GTG

GA

GC

AG

GT

R: G

GC

TTA

CC

TCC

TGC

TTC

TAA

CTG

28

2 bp

54°C

AvaI

at 3

70 C

for o

vern

ight

Des

igne

d ba

sed

on

ENSE

MB

L nu

mbe

r EN

SBTA

G00

0000

1372

7

BC

F:

GG

CC

AG

GG

AC

CG

TGTT

CAT

TGA

GG

AC

ATC

R

:TTC

CTG

GG

AC

CC

CG

TGA

GA

CA

CAT

AC

TTG

198

bp57

°CAv

aII a

t 370 C

fo

r ove

rnig

ht

Gru

pe S

, et a

l., 1

996

FXID

F: C

CC

AC

TGG

CTA

GG

AAT

CG

TT

R: C

AA

GG

CA

ATG

TCAT

ATC

CA

C

320

bp55

°CD

irect

PC

R

assa

yM

arro

n et

al.,

200

4


137

DUMPS and BC were identified by using PCR-RFLP analysis. The PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS and BC, respectively. The FXID genotypes were detected by PCR analysis by running the amplified product on 1.5% agarose gel. 10 μl of PCR products of BLAD, DUMPS and BC were digested with particular enzyme. The digested products were separated on 3% agarose gel and analyzed by visualizing the gels under Gel doc system (Bio-Rad, USA). Representative samples were sequenced and sequences were confirmed by Amnion Bioscience Private Ltd, Bangalore, India.


A total of 135 buffaloes belonging to Murrah (n=106) and Surti (n=29) breed were screened for autosomal recessive genetic disorders viz. BLAD, DUMPS and BC using PCR-RFLP technique and FXID was screened by PCR conformation. The primers used in the study successfully amplified the DNA fragments of 136 bp for BLAD, 282 bp for DUMPS, 198 bp for BC and 320 bp for FXID. In case of BLAD, carrier animal produces three fragments of 136 bp, 108 bp and 28 bp, affected animal shows one band of 136 bp. In the present study the amplified product of CD 18 gene upon digestion by TaqI, yielded two fragments of 108 bp and 28 bp in all buffalo bulls showing normal homozygote animals hence no animal were found to be either affected or carrier for BLAD (Figure 1). The amplified product of uridine monophosphate synthase upon digestion by AvaI, yielded two bands of 213 bp and 69 bp for normal animals. Carrier animals produce three bands of (282 bp, 213 bp and 69 bp) and affected animals produce single band of 282

bp. Among the screened buffalo, all animals were found to be normal in case of DUMPS (Figure 2). To detect the point mutation in gene coding for argininosuccinate synthase (ASS), the amplified product upon digestion by AvaII yielded two bands of 109 and 89 bp for normal animals (Figure 3). Carrier animals produce three bands of 198, 109 and 89 bp and affected produce single band of 198 bp. None of the buffaloes screened were neither affected nor carrier for BC. In FXID, unaffected animals produce a fragment of 244 bp, carrier animals produce two fragments of 320 bp and 244 bp and affected animal produce a fragment of 320 bp. All the screened buffaloes were found to be negative for FXID (Figure 4).

The spread of genetic disorders in cattle and buffalo have been increased in recent years. In the present study all the screened animals were found to be normal and none of them were carrier for BLAD, DUMPS, BC and FXID. Similar results were observed in earlier studies carried out in buffalo for BLAD (Muraleedharan et al., 1999; Rajesh et al., 2007), Bovine Citrullinemia (Muraleedharan et al., 1999) and FXID (Saeed et al., 2012; Rajesh et al., 2007). Similar results of very low or lack of incidence of BLAD were observed in earlier studies carried out for Holstein population (Nagahata et al., 1997; Ribeiro et al., 2000; Rahimi et al., 2006; Meydan et al., 2007; Arpita et al., 2012). DUMPS allele has been reported in different countries in America and Europe (Citek and Blahova, 2004), Poland (Kaminski et al., 2005), India (Rajesh et al., 2006), Turkey (Akyuz et al., 2009; Meydan et al., 2010) and Iran (Rahimi et al., 2006). Previous studies in cattle for Bovine Citrullinemia showed none of the males to be either carrier or affected (Meydan et al., 2010; Rajesh et al., 2006; Citek et al., 2006) and for FXID (Cyrus et al., 2011; Saeed et al., 2012). The present study suggests that the


138

Figure 1. PCR-RFLP result of CD18 gene: Lane 1 to 12 showing normal homozygote two fragments, 108bp and 28bp (not visible in gel); Lane 13 - 100 bp DNA Marker.

Figure 2. PCR-RFLP result of gene coding for uridine monophosphate synthase: Lane 1 to 12 normal homozygote animal showing two fragments of 213bp and 69bp; Lane 13 - 100bp DNA Marker.


139

Figure 3. PCR-RFLP result of Arginosuccinate synthase gene: Lane 1 to 12 normal homozygote showing two fragments, 109bp and 89bp; Lane 13 - 100bp DNA Marker.

Figure 4. Polymerase Chain Reaction (PCR) genotyping of FXID: Lane 1 to 10 showing product size 244bp, normal homozygote genotype; Lane 11 - 100bp DNA Marker.


140

PCR and PCR-RFLP based technique could be employed as an efficient technique for screening of various genetic disorders for identification of carriers or affected animals before using them in breeding programmes to minimize the spread of defective allele. Molecular markers are identified as powerful tool which helps in diagnosis of genetic disorders at a very early stage. Hence it is required to establish the screening methods allowing breeders to test their animals to minimize the spread of disease in a population.

REFERENCES

Akyuz, B. and B.C. Kul. 2009. Detection of deficiency of uridine monophosphate synthase (DUMPS) in female Holstein cattle in Turkey. Vet. J. Ank. Uni., 56: 231-232.

Arpita, R., K.P. Rajesh, K. Rosaiah, A. Radhika and K. Sanghamitra. 2012. New cases of Bovine Leukocyte adhesion deficiency (BLAD) carriers in Indian Holstein Cattle. Int. J. Vet. Sci., 1: 80-82.

Bruford, M.W., D.G. Bradley and G. Luikart. 2003. DNA markers reveal the complexity of livestock domestication. Nat. Rev. Gen., 4: 900-909.

Citek, J. and B. Blahova. 2004. Recessive disorders-a serious health hazard. J. App. Biomed., 2: 187-194.

Citek, J.V., J. Rehout, J. Hajkova and J. Pavkova. 2006. Monitoring of the genetic health of cattle in the Czech Republic. Vet. Med., 51: 333-339.

Cyrus, E., A. Cyrus, E.K. Naser, C. Mohammad and F. Jamal. 2011. Study of factor XI deficiency in Khuzestan cattle population of

Iran. Afr. J. Biotechnol., 10: 718-721.Dennis, J.A., P.J. Healy, A.L. Beadudet and

W.E. O’Brien. 1989. Molecular definition of Bovine Argininosuccinate Synthase Deficiency. Proceedings of the National Academy of Sciences, USA, 86: 7947-7951.

BAHS. 2010. Basic Animal Husbandry Statistics (BAHS). Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Government of India. India.

Grupe, S., G. Diet and M. Schwerin. 1996. Population survey of citrullinemia on German Holsteins. Livest. Prod. Sci., 45(1): 35-38.

Harlizius, B., S. Schrober, I. Tammen and T. Simon. 1996. Isolation of bovine uridine monophospate synthase gene to identify the molecular basis of DUMPS in cattle. J. Anim. Breed. Genet., 113(1-6): 303-309.

Harper, P.A.W., P.J. Healy, J.A. Dennis, J.J. O’Brien and H.D. Rayward. 1986. Citrullinaemia as a cause of severe neurological disease in neonatal Friesian calves. Aust. Vet. J., 63: 378-379.

Healy, P., P.A.W. Harper and A. Dennis. 1990. Bovine citrullinemia: a clinical, pathological, biochemical and genetic study. Aust. Vet. J., 67(7): 255-228.

Kaminski, S., G. Grybowski and B. Prusak. 2005. No incidence of DUMPS carriers in Polish dairy cattle. J. Appl. Genet., 46: 395-397.

Kherli, M.E., F.C. Schmalstieg, D.C. Anderson, M.J. Van Der Maaten, B.J. Hughes, M.R. Ackerman, C.L. Wilhelmsen, G.B. Brown, M.G. Stevens and C.A. Whestone. 1990. Molecular definition of the Bovine Granulocytopathy Syndrome-Identification of deficiency of the Mac-1 (CD 1 lb / CD 18) glycoprotein. Am. J. Vet. Res., 51: 1826-


141

1836.Lee, Y.K., K.W. Chang, I.S. Nam, W.K. Chang, T.Y.

Tak, K.N. Kim and K.J. Lee. 2002. Studies on the detection of congenital genetic disorder in Holstein proven and candidate bulls. J. Anim. Sci. Tech., 44(3): 279-288.

Liptrap, R.M., P.A. Gentry, M.L. Ross and E. Cummings. 1995. Preliminary findings of altered follicular activity in Holstein cows with coagulation factor XI deficiency. Vet. Res. Commun., 19(6): 463-471.

Marron, B.M., J.L. Robinson and P.A. Gentry. 2004. Identification of a mutation associated with factor XI deficiency in Holstein cattle. Anim. Genet., 35: 454-456.

Meydan, H., F. Ozdil, Y. Gedik and M.A. Yildiz. 2007. Detection of BLAD, DUMPS and factor XI by PCR-RFLP. In Proceedings of the 5th Animal Science Congress, Yuzuncu Yil University, Van, Turkey.

Meydan, H., M.A. Yildiz and J.S. Agerholm. 2010. Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey. Acta Vet. Scand., 52: 1-8.

Miller, S.A., D.D. Dykes and H.F. Polesky. 1988. A simple salting out procedure for extracting DNA from humane nucleated cells. Nucleic Acids Res., 16(3): 1215.

Muraleedharan, P., V. Khoda, G. Sven, P.N. Mukhopadhyaya, S. Manfred and H.K. Mehta. 1999. Incidence of hereditary Citrullinemia and bovine Ieucocyte adhesion deficiency Syndrome in Indian dairy cattle (Bos taurus, Bos indicus) and buffalo (Bubalus bubalis) Population. Arch Tierz., 42(4): 347-352.

Nagahata, H., H. Noda, K. Takahashi, T. Kurosawa and M. Sonoda. 1987. Bovine granulocytopathy syndrome: Neutrophil dysfunction in Holstein Friesian calves. Zentralbl Veterinarmed A., 34(6): 445-451.

Nagahata, H., T. Miura, K. Tagaki, M. Ohtaki, H. Noda, T. Yasuda and K. Nioka. 1997. Prevalence and allele frequency estimation of bovine leukocyte adhesion deficiency (BLAD) in Holstein-Friesian cattle in Japan. J. Vet. Med. Sci., 59(4): 233-238.

Rahimi, G., A. Nejati-Javaremi and K. Olek. 2006. Genotyping BLAD, DUMPS and CSN loci in Holstein young bulls of the National Animal Breeding Center of Iran. Pakistan Journal of Biological Sciences, 9(7): 1389-1392.

Rajesh, K.P., M.S. Krishna, J.S. Kalpesh, B.C. Jenabhai, R.S. Krothapalli and S. Rao. 2006. Lack of carriers of citrullinaemia and DUMPS in Indian Holstein cattle. J. Appl. Genet., 47(3): 239-242.

Rajesh, K.P., M.S. Krishna, J.S. Kalpesh, B.C. Jenabhai, R.S. Krothapalli and S. Rao. 2007. Low incidence of bovine leukocyte adhesion deficiency (BLAD) carriers in Indian cattle and buffalo breeds. J Appl. Gen., 48: 153-155.

Rajesh, K.P., J.S. Kalpesh, B.C. Jenabhai, M.S. Krishna, R.S. Krothapalli and S. Rao. 2007. Factor XI deficiency in Indian Bos taurus, Bos indicus, Bos taurus × Bos indicus crossbreds and Bubalis bubalis. Genet. Mol. Biol., 30(3): 580-583.

Ribeiro, L.A., E.E. Baron, M.L. Martinez and L.L. Coutinho. 2000. PCR screening and allele frequency estimation of bovine leukocyte adhesion deficiency in Holstein and Gir cattle in Brazil. Genet. Mol. Biol., 23: 831-


142

834.Robinson, J.L., R.G. Popp, R.D. Shanks, A.

Oosterhof and J.H. Veerkamp. 1993. Testing for deficiency of uridine monophosphate synthase among Holstein Frisian cattle of North America and Europe. Livest. Prod. Sci., 36: 287-298.

Saeed, B., A. Cyrus, C. Mohammad, A. Mahdi, A.S. Ali and R.S. Hamid. 2012. Identification of factor XI deficiency in Khuzestan buffalo population of Iran. Global Veterinaria, 8(6): 598-600.

Sethi, R.K. 2003. Buffalo breeds of India. In Proceedings of 4th Asian Buffalo Congress, New Delhi, India.

Shuster, D.E., B.T. Bosworth and M.E. Kehrli. 1992. Sequence of the bovine CD-18 encoding cDNA: comparison with the human and murine glycoproteins. Genetics, 114(2): 267-271.


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ABSTRACT

Incidence S. nasale (causative agent of nasal schistosomiasis) in graded murrah buffaloes was studied. 4(8%) among healthy and 46(92%) among suspected buffaloes were diagnosed with nasal schistosomiasis. Area under investigation was highly irrigated giving ample scope for exposure of animals to cercaria of S. nasale. Buffaloes showing signs of reduced water intake, reduced milk yield, normal body temperature and normal feed intake were suspected with S. nasale and the results obtained are in agreement with these observations. It is concluded that buffaloes showing above signs may be suspected with nasal schistosomiasis, especially in highly irrigated areas.

Keywords: S. nasale, graded murrah buffaloes, incidence, signs, irrigation

INTRODUCTION

Nasal schistosomiasis (also called snoring disease) is a disease affecting mainly large ruminants and to a little extent small ruminants and horses (Latchumikanthan et al., 2014). The causative blood fluke for Nasal schistosomiais, Schistosomiais nasale was first identified by Rao in 1933. Nasal schistosomiais is recognized as

the 5th major helminthosis of domestic animals in Indian subcontinent (Sumanth et al., 2004). This trematode infection is majorly transmitted by fresh water snails belonging to Indoplanorbis exustus and Lymnea luteola, carrying Cercariae indicae, larval form of parasite. Shape of the egg is generally referred as Palanquin (Ravindran and Kumar, 2012) or boomerang (Sundar et al., 2004) with presence of terminal spine. Most common signs noticed in cattle are rhinitis, sneezing, dyspnoea, mucopurrulent nasal discharge, leading to snoring. Granulomatous growth of nasal epithelium (Polyps) with small abscess may occur in cattle, whereas only congestion and pin head size eruptions are observed in buffaloes in chronic manifestation of the disease (Latchumikanthan et al., 2014). No major zoonotic problems were reported except for the possibility of cercarial dermatitis as reported by Sundar et al. (2004). Even though there are many epidemiological as well as outbreak reports regarding occurrence of nasal schistosomiasis in cattle, there is dearth of reports in case of buffaloes (Ravindran and Kumar, 2012). In this regard a study was carried out to assess the incidence of S. nasale in buffaloes in Gudivada of Krishna district in Andhra Pradesh, India.

INCIDENCE OF NASAL SCHISTOSOMIASIS IN GRADED MURRAH BUFFALOES

Hareesh Didugu* and Ch. E. Narasimha Reddy

Animal Disease Diagnostic Laboratory, Vijayawada, Andhra Pradesh, India, *Email: [email protected]

Original Article


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100 she-buffaloes of different age groups from Gudivada region of Krishna district, Andhra Pradesh, India were selected for the study. Among 100 animals, 50 apparently healthy animals and 50 suspected animals showing symptoms of sudden drop in milk yield and reduced water intake were selected for diagnosis of S. nasale infection. From all the animals deep nasal scrapings were collected with the help of a scoop, pressed between two slides and examined directly under microscope for presence of palanquin/ boomerang shaped eggs.


Results revealed that 4 (8%) among healthy and 46 (92%) among suspected buffaloes were positive for nasal schistosomiasis. In general nasal schistosomiasis in buffaloes is mostly subclinical and often goes unnoticed (Latchumikanthan et al., 2014). In agreement with the above statement, we observed low levels of incidence (8%) in apparently healthy animals. Similar results of 8.51% in cattle and 6.28% in buffaloes with and overall incidence of 7.56% in Andhra Pradesh (Rao, 2005) was reported, whereas higher incidences of 11.1% and 23.4% in cattle and buffaloes in slaughter house samples, respectively in Wayanad, Kerala (Ravindran and Kumar, 2012), 12.6% in Sri lanka (De Bont et al., 1989), 17.7% in Maharastra (Kolte et al., 2012), 44% in Madhya Pradesh (Banerjee and Agrawal (1992), 60.3% among nondescript bullocks and 68.9% among Hallikar bullocks in Andhra Pradesh (Sreeramulu, 1994), 69.2% in slaughter house samples in Karnataka (Sumanth et al., 2004) were also reported.

It is also observed that suspected buffaloes presented with symptoms of decreased water intake, decreased milk yield, normal feed intake and normal body temperature were found to have high incidence of nasal schistosomiais (92%). Symptoms like reduced water intake observed in this study may be due to irritation and inflammation caused to the nasal mucosa, leading to sudden drop in milk yield. These findings need further detailed investigation. Even though many other diseases will occur with same symptoms, infestation with S. nasale should be kept in mind while diagnosing the cause of disease, especially in highly irrigated areas. Rajamohanan and Peter (1975), after studying both cattle and buffaloes, suggested buffaloes are suitable hosts compared to cattle. Most of the field veterinarians would examine a nasal washing only in case of observing specific sign “snoring” in bovines, which only occur when the disease is severe, developed to polyps and obstructing the air way. Treating at this stage takes a lot of time and economically tolls a heavy loss to owners because of drop on production. In support of our findings, treating animals with these specific signs with lithium antimony given fruitful results. High incidence observed in this study (92% in suspected animals) was in agreement with Sundar et al. (2004), who reported outbreak of nasal schistosomiasis in bovines. High incidence of S. nasale in buffaloes in this study may be attributed to continuous exposure of the animals to cercaria on green grass, which is available throughout the year as area in this study is highly irrigated. In agreement with De Bont et al. (1989) all the suspected animals didn’t show any prominent signs like snoring or any polyp like growths in nasal mucosa. Similar findings were reported by authors like Dutt and Srivatsava (1968), who reported that buffaloes have innate resistance, causing lack of


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profound signs compared to cattle and Ravindran and Kumar (2012), who reported minor infection in all positive cases. The incidence is more in winter and old animals are highly effected in agreement with Kolte et al. (2012); Rao (2005). From this study it is concluded that nasal schistosomiasis can be suspected in buffaloes showing signs of reduced water intake and sudden drop in milk yield, especially in highly irrigated areas.

REFERENCES

Banerjee, P.S. and M.C. Agrawal. 1992. Epizootiological studies in bovines on fluke infections with special reference to schistosomiasis. Indian Vet. J., 69(3): 215-220.

De Bont, J., D. V.an Aken, J. Vercruysse, J. Fransen, V.R. Southgate and D. Rollinson. 1989. The prevalence and pathology of Schistosoma nasale Rao, 1933 in cattle in Sri Lanka. Parasitology., 98(2): 197-202.

Dutt, S.C. and H.D. Srivastava. 1968. Studies on Schistosoma nasale Rao 1933 II Molluscan and mammalian hosts of the blood–fluke. Indian J. Vet. Sci. Anim. Husb., 38: 210-216.

Kolte, S.W., N.V. Kurkure, D.K. Maske and S. Khatoon. 2012. Prevalence of Schistosoma nasale infection in bovines from eastern Vidharbha (Maharashtra) vis-à-vis infection in Indoplanorbis exustus. J. Vet. Parasitol., 26(2): 140-143.

Latchumikanthan, A., P. Pothiappan, D. Ilayabharathi, S.S. Das, D. Kumar and C. Ilangovan. 2014. Occurrence of Schistosoma nasale infection in bullocks of Puducherry. J. Parasit. Dis., 38(2): 238-240.

Rajamohanan, K. and C.T. Peter. 1975. Pathology of nasal schistosomiasis in buffaloes. Kerala Journal of Veterinary Science., 6: 94-100.

Rao, T.B. and M. Hafeez. 2005. Prevalence of nasal schistosomiasis in bovines in East Godavari District of Andhra Pradesh. Intas Polivet., 6(2): 305-307.

Ravindran, R. and A. Kumar. 2012. Nasal schistosomiasis among large ruminants in Wayanad, India. Se. Asian J. Trop. Med., 43(3): 586-588.

Sreeramulu, P. 1994. Epizootiology of Nasal Schistosomiasis in bovines in Andhra Pradesh. Indian Vet. J., 71(10): 1043-1044.

Sumanth, S., P.E. D’Souza and M.S. Jagannath. 2004. A study of nasal and visceral schistosomosis in cattle slaughtered at an abattoir in Bangalore, South India. Rev. Sci. Tech. OIE., 23(3): 937-942.

Sundar, N., D. Kathiresan, S. Sivaseelan, S. Vairamuthu, V. Purushothaman and R. Rajavelu. 2004. An outbreak of nasal schistosomiasis among cattle and buffaloes in Tamil Nadu. Indian J. Anim. Sci., 74(4): 369-370.


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ABSTRACT

The present study was conducted to Assess the efficacy of various diagnostic techniques for that all the suspected cases of buffaloes coming to TVCC, U.P. Pandit Deen Dayal Upadhyaya pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura were examined by parasitological and serological methods. (viz. giemsa stained thin blood smear, buffy coat method and TELAT) for the diagnosis of trypanosomiasis in naturally infected buffaloes. The order of decreasing diagnostic efficacy during present investigation was found as: TELAT (52.02%) > Buffy Coat method (45.62%) > Giemsa stained thin blood smear (38.20%).

Keywords: Trypanosomiasis, TELAT, buffaloes, efficacy

INTRODUCTION

Trypanosomiasis is one of the most important hemoprotozoan diseases of bovines in India. This is caused by unicellular, flagellated protozoa of the genus Trypanosoma. It is an important vector born disease occurring in tropical and subtropical countries including India (Da Silva

et al., 2009). Diagnosis of trypanosomiasis is based on clinical signs and demonstration of the parasites in the blood supplemented by hematological, biochemical and serological test. The clinical manifestations of Surra, although indicative, are not pathognomonic enough to confirm the disease without laboratory diagnosis (Dia et al., 1997). When there is high parasitaemia, the examination of wet blood films, stained blood smears and lymph node materials reveals the trypanosomes but in chronic cases such as the carrier status, examination of thick blood smears as well as methods of parasite concentration are required. The standard diagnostic test for T. evansi infection is the giemsa-stained thin blood-smear, which has a sensitivity of ~105 trypanosomes ml−1 of blood (Paris et al., 1982). The diagnostic capability can be significantly improved by adopting simple, low-cost alternatives, such as HCT (hematocrit centrifugation technique), which has a sensitivity of ~85 Trypanosomes ml−1 blood (Reid et al., 2001). The sensitivity of parasite detection can be enhanced by approximately tenfold when using buffy coat (Reid et al., 2001). In India, a monoclonal antibody based latex agglutination test (MAb-TELAT) with immense field applicability was developed to detect T. evansi antigens (Rayulu et al., 2007).

ASSESSMENT OF DIAGNOSTIC EFFICACY OF VARIOUS METHODS IN DETECTION OF TRYPANOSOMA EVANSI INFECTION IN BUFFALOES

A.P. Singh, A.K. Tripathi, Ajit Singh*, A. Srivastava and Rakesh Singh

Department of Veterinary Clinical Medicine Ethics and Jurisprudence, College of Veterinary Science and Animal Husbandry, Uttar Pradesh, Pandit Deen Dayal Upadhyaya Pashu Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan (DUVASU), Mathura, India,*E-mail: [email protected]

Original Article



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The study was performed at Teaching Veterinary Clinical Complex, Uttar Pradesh Pandit. Deen Dayal Upadhyaya Pashu-Chikitsa Vigyan Vishwavidyalaya Evam Go Anusandhan Sansthan, Mathura (TVCC, DUVASU, Mathura) from June 2013 to May 2014. Animals came to TVCC with history of suffering with fever, anorexia, loss of production and loss of body condition were selected for study. Blood samples were drawn from animal by usual technique of collection from ear tip and Jugular vein aseptically with a sterilized disposable syringe and needle. The sample from jugular vein was collected in two clean, dry blood collection vials containing EDTA as anticoagulant. In present study all the suspected cases of buffaloes were examined by parasitological and serological methods.

Diagnosis on the basis parasitological examinations in the suspected cases was done by thin blood smear examination and buffy coat examination technique. For thin blood smear examinations, a drop of blood was placed 20 mm from one end of a clean microscopic slide and a thin film was drawn. The film was air-dried briefly, fixed in absolute methanol for 2 minutes and allowed to dry. The smears were then stained by giemsa (one drop giemsa +1 ml PBS, pH 7.2) for 25 minutes. This preparation was poured off; the slide was washed in tap water and dried. Slides were visualized under microscope at 100x using immersion oil.

In buffy coat technique, blood was collected into micro-hematocrit centrifuge tubes containing anticoagulant and sealed with clay and centrifuged in microhaematocrite centrifuge at 12,000 rpm for 5 minutes. A smear was prepared by scratching and breaking the capillary tube 1mm

below the surface of the buffy-coat and one drop of the buffy coat was expelled onto microscope slide, smeared and covered with a cover slip and examined under microscope at 40x.

A monoclonal antibody-based latex agglutination test for the diagnosis of Surra in domestic, zoo and wild animals (Invented at LLRUVAS, Hisar). It involves the detection of T. evansi circulating antigens in sera samples from infected animals. Standard protocol was followed as proposed by inventor. Reagent was shaked well before use and aliquot of required volume (20 µl per test sample) was transferred into the empty vial supplied in the kit. The stock reagent was transferred back in refrigerator at 4ºC. The reagent (20 µl) was put on a clean glass cavity slide and mixed with equal amount of serum sample obtained from the suspected animal. Similarly positive and negative serum control were used, ensuring that reagent should not auto agglutinate. Observed for up to five minutes for agglutination to occur in strong positive serum samples. The reagent was mixed intermittently (for 15 seconds preferably 3 to 4 minutes after start of the test) by swirling motion of the slide for agglutination to occur. Agglutination was indicated by appearance of granules or curdle-like aggregates in the solution mixed with the serum sample that contained T. evansi circulating antigen. The blue reagent turned watery with blue granules settling out from the solution. The samples showing agglutination within 5, 10 and 15 minutes were marked as positive. All other samples which did not show the agglutination within 15 minutes were declared as negative.Assessment of diagnostic efficacy

Diagnostic efficacies of giemsa stained thin blood smear; buffy coat method and TE-LAT were evaluated on the basis of % positivity shown by individual diagnostic test.


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In present study giemsa stained thin blood smear examinations in suspected cases of trypanosomiasis in buffaloes revealed the presence of T. evansi with an efficacy of 38.20%. The present findings are more or less similar with the findings of Mandal et al. (1977) who reported 36.9% occurrence of surra in buffaloes by blood smear examination after giemsa staining. Carlos et al. (1990) reported 45.6% efficacy with giemsa stained blood smear which is higher than the present findings. Laha et al. (2009) detected 5.3%, 9.4% and 40.6% infections in cattle, buffalo and horses by examination of giemsa-stained blood smears. The present findings are much higher than the findings by the work done by Das et al. (1998) who reported 2.63%, Dhami et al. (1999) reported 1.34%, Agarwal et al. (2003) reported 7.49% prevalence, Awandkar et al. (2004) reported 1.73%, Muraleedharan et al. (2005) reported 0.04% and by Shahzad et al. (2010) finding was 3.5% by the giemsa stained thin blood smear. It may be due to the reason that most of the cases that came to TVCC, DUVASU, Mathura, were in acute stage which were showing high degree of parasitaemia with prominent clinical signs. It may also be due to the fact that owners usually came to the TVCC only when their animals started showing prominent clinical signs like high fever, respiratory distress with loss of production etc. Mathura and its surrounding areas are more prone for exposure to biting flies (Tabanid flies); because of the fact that in this area there is higher vector density due to its agro climatic condition and Yamuna belt, therefore, the animals might be getting acute infection during grazing time.

Buffy coat examination in suspected cases of trypanosomiasis in buffaloes revealed

the presence of T. evansi with efficacy 45.62%. similar findings were earlier reported by Hollanda (2001) found the case sensitivity of the buffy-coat technique (BCT) to be 38.6%, Carlos et al. (1990) found an efficacy of 63.4%. The buffy coat technique detected more number of cases of T. evansi infection compared with giemsa stained blood smears examination. It could attribute to the reason that in most of the hosts, T. evansi can induce mild clinical or subclinical carrier infections with low parasitaemia and in such conditions concentrations methods like buffy coat technique become necessary. The application of parasite concentration methods like buffy coat techniques is recommended (OIE, 2000) to diagnose the T. evansi infection as an alternative method including the serological techniques. Dwivedi (2004) stressed the importance of buffy coat technique for the identification of subclinical or carrier state of T. evansi infection in bovines.

In present study TE-LAT examination in suspected cases of trypanosomiasis in buffaloes revealed the presence of circulating T. evansi parasite antigen in 463 cases showing an efficacy of 52.02%. All the blood smear and buffy coat positive cases were also found positive with TE-LAT however, healthy control showed negative reaction indicating the higher sensitivity of TE-LAT than other techniques applied. The findings of present investigations are in accordance with the findings earlier reported by Shyma et al. (2012); Rayulu et al. (2007). Reema et al. (2012) inferred that TE-LAT as being simple to perform, rapid, convenient and cost-effective could be quite suitable for diagnosis of trypanosomiasis at field level.

In order of decreasing diagnostic efficacy during present investigation the results obtained were as follows: TE-LAT > Buffy Coat method


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Table. 1. Percent positivity (efficacy) of various diagnostic tests in the diagnosis of trypanosomiasis in buffaloes.

Diagnostic testsBuffaloes (Total- 890)

No. positive % positive BLOOD SMEAR 340 38.20 BUFFY COAT 406 45.62 TELAT 463 52.02

Figure 1. Showing comparative efficacy of three diagnostic tests.


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Figure 2. Blood smear showing T. evansi. Figure 3. Capillary tube showing buffy coat.

Figure 4. TE-LAT (Positive). Figure 5. TE-LAT (Control).


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> Giemsa stained thin blood smear. Similar kind of findings were earlier reported by Shyma et al. (2012) who found that diagnostic sensitivity of TE-LAT was more than WBF and PCR. Carlos et al. (1990) performed a comparative study between different parasitological methods for diagnosis of T. evansi and found that case sensitivity in order of mouse inoculation test > haematocrit centrifuge technique > buffy coat method > wet blood films > giemsa-stained smears. However, Paris et al. (1982) evaluated the order of diagnostic sensitivity as follows: dark ground buffy coat technique > haematocrit centrifuge technique > thick film > thin film > wet film.

In present study the diagnostic efficacy of giemsa stained thick blood smear was found less in comparison with the buffy coat and TE-LAT. It is may be due to the fact that microscopic detection of parasites in the blood are not always effective since trypanosomes are frequently absent from peripheral blood (Kendrick, 1968). In the present study, the buffy coat technique detected more number of cases of T. evansi infection compared with the giemsa stained thin blood smears examination. It may be due to the reason that, T. evansi can induce mild clinical or subclinical carrier infections with low parasitaemia and in this conditions concentrations methods like buffy coat technique become necessary. Therefore, it can be said that serological test like TE-LAT is of immense value in the detection of T. evansi infections than the parasitological examination methods.

ACKNOWLEDGMENT

Authors are highly thankful to Dr Ajit Singh, Professor Immunology, LLRUVAS, Hisar for providing Technical support and TELAT

reagent to carry out this study.

REFERENCES

Agrawal, R., R. Singh, M. Kumar and A.K. Upadhyay. 2003. Epidemiological features of bovine trypanosomosis and babesiosis in Durg district of Chhattisgarh state. Indian Vet. J., 80: 314-317.

Awandkar, S.P., V.H. Shende and D.K. Maske. 2004. Prevalence of haemoprotozoan infection in domestic animals of Nagpur region, p. 25-27. In Proceeding national Symposium. on “Application of molecular biology in parasitic diseases for rural development” and XV national congress of veterinary parasitology” Govind Ballabh Pant University of Agriculture and Technology, Pantnagar.

Carlos, M.M., A.M. Orlando and P.R. Juan. 1990. Comparison between six parasitological methods for diagnosis of T. evansi in the subtropical area of Argentina, Vet. Parasitol., 36(1-2): 141-146.

Da Silva, A.S., R.A. Zanette, P. Wolkmer, M.M. Costa, H.A. Garcia, S.T.A. Lopes, J.M. Santurio, M.M.G. Teixteria and S.G. Monteiro. 2009. Diminazene aceturate in the control of T. evansi infection in cats. Vet. Parasitol., 165: 7-540.

Das, A.K., N.C. Nandi and M.O.R. Kumar. 1998. Prevalence of bovine Surra in Guntur district, Andhra Pradesh. Indian Vet. J., 75: 526-529.

Dhami, D.S., P.D. Juyal and L.D. Singla. 1999. Sero-epidemiology of T. evansi by using CATT. Indian Vet. J., 76: 842-844.

Dia, M.L., M.N. Van, E. Magnus, A.G. Luckins,


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C. Diop, A. Thiam, P. Jacquiet and R. Hamers. 1997. Evaluation de 4 tests de diagnostic: Frottis sanguins, CATT, IFI et ELISA-Ag dans l’étude de l’épidémiologie de latrypanosomose cameline à T. evansi en Mauritanie. Rev. Elev. Med. Vet. Pays Trop., 50(1): 29-36.

Dwivedi, S.K. 2004. Trypanosomiasis - Recent advances in diagnosis and therapy. Indian J. Vet. Pract., 41-47.

Hollanda, W.G., F. Claesc, L.N. My, N.G. Thanhb, P.T. Tamb, D. Verlooc, P. Büscherc, B. Goddeerisd and J. Vercruyssea. 2001. A comparative evaluation of parasitological tests and a PCR for T. evansi diagnosis in experimentally infected water buffaloes, Vet. Parasitol., 97(1, 9): 23-33

Kendrick, K.R. 1968. The diagnosis of Trypanosomosis of livestock: A review of current techniques. Vet. Bull., 38: 191-197.

Laha, R. and N.K. Sasma. 2009. Detection of T. evansi infection in clinically ill cattle, buffaloes and horses using various diagnostic tests. Epidemiol. Infect., 137(11): 1583-1585.

Mandal, A.K., R.A. Madhusudhan and N.C. Nandi. 1977. Outbreak of Surra in bovines from Krishna district, Andhra Pradesh. Indian J. Anim. Health., 16: 100.

Muraleedharan, K., S.K. Ziauddin, M.P. Hussain, B. Puttabyatappa, G.B. Mallikarjun and S.J. Seshadri. 2005. Incidence of Anaplasma sp., Babesia sp. and Trypanosoma sp., in cattle of Karnataka. J. Vet. Parasitol. 19(2): 135 137.

Office International des Epizooties/World Organization for Animal Health, (OIE). 2000. Manual of Standard diagnostic tests and vaccines.

Paris, J., M. Murray and F. McOdimba, 1982. A comparative evaluation of the parasitological techniques currently available for the diagnosis of African trypanosomiasis in cattle. Acta Trop., 39: 307-316.

Rayulu, V.C., A. Singh and S.S. Chaudhri. 2007. Monoclonal antibody based immunoassays for the detection of circulating antigens of T. evansi in buffaloes, Ital. J. Anim. Sci., 6(2): 907-910.

Reema, S.S. Chaudhri and A. Singh. 2012. Examination of cross-reactivity among T. evansi, T. annulata and B. bigemina by Te-Mab LAT and PCR, Indian J. Anim. Sci., 82(8): 812.

Reid, S.A., A. Husein and P.D. Compeman. 2001. Evaluation and improvement of parasitological tests for Trypanosoma evansi infection. Vet. Parasitol., 102(4): 291-297.

Shahzad, W., R. Munir, M.S. Khan, M.D. Ahmad, M. Ijaz, A. Ahmad and M. Iqba. 2010. Prevalence and molecular diagnosis of T. evansi in Nili-Ravi buffalo (B. bubalis) in different districts of Punjab (Pakistan). Trop. Anim. Health Prod., 42: 1597-1599.

Shyma, K.P., S.K. Gupta, A. Singh and S.S. Chaudhri. 2012. Efficiency of monoclonal antibody based latex agglutination test in detecting T. Evansi under field conditions for improving the productivity in buffaloes, Buffalo Bul., 31(3): 162-172.


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Original Article

ABSTRACT

The present study was aimed at elucidating the gross morphology, morphometry and biometry of major salivary glands of buffalo during different prenatal as well as neonatal stages. The study was conducted on major salivary glands of twenty-four buffalo foetuses as well as six neonatal buffalo calves. Three pairs of major salivary glands viz., parotid, mandibular and sublingual were distinguished during prenatal and neonatal life in the buffalo. The mandibular gland was the largest among the three major salivary glands in prenatal buffalo, which may be attributed to its early development among all the salivary glands. The sublingual gland was the smallest among the three major salivary glands in prenatal buffalo and measured about half the size of the mandibular gland. At 5.8 cm CVRL (54th day), the mandibular gland began to form an epithelial outgrowth into the mesenchyme, forming the floor of the mouth in the linguo-gingival groove. At 6 cm CVRL (55th day), the sublingual gland arose as a number of small epithelial thickenings in the linguogingival groove and each thickening formed its own canal. Grossly, the superior (polystomatic) and inferior (monostomatic) parts of the sublingual gland were distinguishable at 16.5 cm CVRL (102nd day). At 6.9 cm CVRL (60th day), the parotid gland was seen in the form of a small rod, extending dorsally

from the lateral part of the oral cavity, just below and infront of the external ear and behind the facial nerve. Significant differences in the biometrical parameters of all major salivary glands between foetuses of Group I, II, III as well as neonatal buffalo were observed at P<0.05 and P<0.01 level.

Keywords: ontogeny, major salivary glands, buffalo, gross anatomy, biometry

INTRODUCTION

The study of prenatal development is prerequisite to understand the normal developmental biology of an organ. The documentation of normal foetal growth can serve as a guide for understanding the consequence of harmful influences at various stages of gestation. The study of the salivary glands forms an important link between the anatomy and surgery, as the salivary glands and associated ducts may be affected by inflammation, calculus formation, rupture or neoplasia. The secretion, saliva, contains water, various enzymes, mucopolysaccharides and lubricating glycoproteins. The gland has an important role to provide lubrication for eating and vocalization, aid digestion and supply saliva for pH buffering (Moghaddam et al., 2009). In general, the major salivary glands of the herbivores are better

PRENATAL DEVELOPMENT OF BUFFALO MAJOR SALIVARY GLANDS: GROSS MORPHOLOGICAL AND BIOMETRICAL STUDIES

A.D. Singh and Opinder Singh*

Department of Veterinary Anatomy, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India,* E-mail: [email protected]



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developed than those of the carnivores. Saliva is secreted into the oral cavity via a series of ducts in the ductal system. Dysfunction of salivary secretion (hyposalivation) causes xerostomia (dry mouth) and sequentially leads to severe dental caries as well as oral mucosal disorders (Featherstone, 2000).

The saliva, which plays important role in digestion, is secreted from major and minor salivary glands. The major salivary glands in domestic animals include the parotid, mandibular and sublingual salivary glands while the minor glands are the buccal, labial, lingual, palatine, molar and zygomatic salivary glands (Dellmann and Eurell, 1998). The minor salivary glands were embedded in the subepithelial tissues of the mouth and oro-pharynx. This wide distribution of the minor salivary glands is advantageous for the protection of the oral cavity against pathogens (Singh et al., 2011).

The prenatal development of major salivary glands had been studied in cat (Knospe and Bohme, 1995), human (Chi, 1996), rat (Wolff et al., 2002) and pig (Pospieszny et al., 2010), however, there is no detailed information about the gross morphology as well as biometry of major salivary glands of buffalo during prenatal life, therefore, the goal of this study was to describe the macroscopic ontogenetic events of major salivary glands of buffalo.


The present study was conducted on major salivary glands of twenty-four buffalo foetuses, during different stages of prenatal development, as well as six neonatal buffalo calves. Immediately after collection, the foetus was measured for its

curved crown rump length (CVRL) in centimetres with a calibrated inelastic thread. The approximate age of foetuses was calculated by using the following formula given by Soliman (1975) in buffalo:

Y = 28.66+4.496 X (CVRL <20 cm) Y = 73.544+2.256 X (CVRL ≥20 cm)

Where Y is age in day(s) and X is curved crown rump length (CVRL) in cm(s). Depending upon CVRL, foetuses were divided into three groups with a minimum of eight samples in each group:

Group I : CVRL between 0 to 20 cmGroup II : CVRL >20 to 40 cmGroup III : CVRL >40 cm

Immediately after measuring CVRL, the foetuses as well as head of neonates were fixed in 10% neutral buffered formalin (NBF). After dissection, the skin over each gland was incised and the fascia was transected. The topography of the major salivary glands in relation to other structures was studied. Each gland was carefully freed from the adhering tissue and the length, breadth and weight were measured for each gland on two sides (left and right) and then compared. The data was subjected to statistical analysis for determining correlation between development of major salivary glands and the foetal age by using SPSS statistics software version 22.


Three pairs of major salivary glands viz., parotid, mandibular and sublingual were


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distinguished during prenatal and neonatal life in the buffalo as reported in human beings (Chi, 1996). The gross morphological and biometrical observations were made on major salivary glands of buffalo during prenatal and neonatal life.

Parotid salivary gland The gland was light yellow in colour and was seen in the form of a small rod, extending dorsally from the lateral part of the oral cavity, just below and infront of the external ear and behind the facial nerve at 6.9 cm CVRL (60th day). At this stage, the gland was devoid of superficial lobulations. At 9.2 cm CVRL (70th day), the gland was pyramidal in shape with loosely arranged lobules and situated along the caudal border of the masseter muscle extending from the region of the external auditory canal to the level above the angle of the mandible (Figure 1). The parotid gland was reported to be in the form of a epithelial rod from 56 to 95 days of prenatal life in human beings (Nayeem et al., 2000). The gland gradually attained triangular shape with a broad base and narrow apex at 11.5 cm CVRL (80th day). The base of the gland was superior with a notch placed around the external auditory canal, while the apex was situated little above the angle of the mandible. Well developed parotid duct (Stenson’s duct) was observed from 12.5 cm CVRL (84th day) onwards, leaving the gland at apex and ascending in the groove along the rostral border of masseter muscle along with the facial vessels and ventral buccal nerve. In human beings, Stenson’s duct was observed from 80th day onwards during the prenatal life (Attie and Sciubba, 1981). The left parotid gland of Group I foetuses on average measured 0.58±0.1 cm in length and 0.43±0.1 cm in breadth whereas right one was 0.60±0.1 cm in length and 0.45±0.1 cm in breadth. The mean weight of left and right

parotid glands was 1.39±0.2 gm and 1.40±0.2 gm, respectively (Table 1). The gland was enclosed within a fibro-adipose capsule and reached the space between the base of the ear and vertical ramus of the mandible at 25.9 cm CVRL (132nd day). The facial nerve was seen very superficially and passed through the parenchyma of the gland. The auriculo-temporal nerve and few branches of facial nerve were seen at this stage of gestation (Figure 3). Similar findings were reported in domestic animals by Getty (1975); Barnwal and Sinha (1982); Nayeem et al. (2000). The prenatal parotid gland attained the shape of narrow triangle in mid age groups (Figure 3a). The lobules of the gland were loosely arranged and separated by interlobular septa at 29.5 cm CVRL (140th day). The parotid duct was opened into the mouth cavity at the level of upper 2nd erupting cheek tooth till 32 cm CVRL (145th day). The same was evident at the level of upper erupting 3rd

cheek tooth from 36.2 cm CVRL (155th day) to 80 cm CVRL (254th day) of foetal life. In Group II, morphometric studies revealed that on average left parotid gland was 1.68±0.4 cm in length, 1.45±0.1 cm in breadth and weighed 3.85±0.5 gm whereas right one was 1.70±0.4 cm in length, 1.50±0.1 cm in breadth and weighed 3.90±0.5 gm (Table 1). The adult characteristic features of the parotid gland were attained from 45.2 cm CVRL (175th day) onwards. At 100 cm CVRL (299th day), the colour of the gland varied from light yellow to light brown. The lobules of the gland were distinctly visible (Figure 4a). The upper anterior part of the gland was loosely attached to the parotid lymph node, while the lower anterior part was in close contact with the masseter muscle (Figure 4). The middle part of the gland was penetrated by the maxillary vein from lateral to medial surface throughout the prenatal life. The lateral surface was


158

covered by parotid fascia, developing parotido-auricularis muscle and facial muscles. The medial surface was uneven and related to great cornu of hyoid bone, digastricus, occipito-hyoideus and sterno-mastoideus muscles, external carotid artery, external jugular vein and its tributaries, facial nerve and its branches. The dorsal border was related to the base of the external ear. The anterior border was in contact with the parotid lymph node above and masseter muscle below. The posterior border was related to the posterior auricular vein. Nayeem et al. (2000) reported that the human parotid gland, in later stages of prenatal development, was closely related to 9th, 10th and 11th cranial nerves. The facial nerve was also very superficial. The left parotid gland of Group III foetuses on average measured 2.95±0.6 cm in length and 2.72±0.2 cm in breadth whereas right one was 3.00±0.6 cm in length and 2.80±0.2 cm in breadth. The mean weight of left and right parotid glands was 8.65±1.1 gm and 9.00±1.1 gm, respectively (Table 1). Gradual increase in length, breadth and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation during the foetal stage. In neonatal buffalo, the parotid gland was long, narrow, pinkish brown in colour and triangular in shape with a wide thick dorsal end that reached the region of the temporo-mandibular joint. However, the gland was reported to be rectangular in sheep and goat (Getty, 1975) and pyramidal or triangular in buffalo calves (Barnwal and Sinha, 1982). The gland was located along the caudal border of the masseter muscle and extended from the zygomatic arch to the ramus of the mandible. The ventral aspect followed the caudal border of the mandible and was deeply related to the mandibular gland. The deep surface was related to the angle of the stylohyoid bone as well as the

occipito-hyoideus and digastric muscles. The parotid duct left the gland ventrally with the facial artery as well as facial vein and ascended on the lateral surface of the masseter muscle to open near the posterior upper molar teeth in the oral cavity. The morphometric studies revealed that on average left parotid gland of neonatal buffalo was 5.50±0.8 cm in length, 3.00±0.2 cm in breadth and weighed 19.7±1.5 gm whereas right one was 5.80±0.8 cm in length, 3.10±0.2 cm in breadth and weighed 20.9±1.5 gm (Table 1). There was significant difference in the biometrical parameters of parotid gland between foetuses of Group I, II, III as well as neonatal buffalo at P<0.05 and P<0.01 level. The biometrical studies showed that there was no significant difference in the left and right parotid salivary gland within same group at P<0.05 and P<0.01 level (Graph 1).

Mandibular salivary gland The mandibular gland was the largest among the three major salivary glands in prenatal buffalo, which may be attributed to its early development among all the salivary glands, while the parotid gland was reported to be the largest major salivary gland in human beings during prenatal life (Attie and Sciubba, 1981). The gland was light yellow in colour, long, narrow and curved in shape. It began to form an epithelial outgrowth into the mesenchyme, forming the floor of the mouth in the linguo-gingival groove at 5.8cm CVRL (54th day). At this stage, the gland was devoid of any superficial lobulations. The mandibular gland showed loosely arranged lobules at 9.2 cm CVRL (70th day) (Figure 1). The mandibular duct was well developed at 12 cm CVRL (82nd day). Budras and Habel (2003) reported similar findings in domestic animals. At 14 cm CVRL (91st day), mandibular gland was long, narrow and curved and enclosed


159

within a fibroadipose capsule. It was situated along the medial side of the angle of the mandible. In Group I, morphometric studies revealed that the mean length and breadth of left mandibular gland was 1.09±0.1 cm and 0.39±0.1 cm, respectively and average weight was 1.53±0.3 gm whereas right one was 1.10±0.1 cm in length, 0.40±0.1 cm in breadth and weighed 1.55±0.3 gm (Table 2).

The gland showed lobulations similar to that of parotid gland at 25.9 cm CVRL (132nd day). It extended from the region of tympanic bulla to the angle of the mandible behind the parotid salivary gland (Figure 3). At this stage, it was rough quadrilateral in shape (Figure 3b). The location was caudomedial to the parotid salivary gland in the mid and late foetal age groups. The left mandibular gland of Group II foetuses on average measured 2.38±0.6 cm in length and 1.19±0.1 cm in breadth whereas right one was 2.40±0.6 cm in length and 1.20±0.1 cm in breadth. The mean weight of left and right mandibular glands was 4.76±1.0 gm and 4.80±1.0 gm respectively (Table 2). Sivakumar et al. (2003) reported that in Group II (18 to 25 weeks), the length and breadth of human foetal submandibular gland increased 1.6 times and weight increased 2.4 times as compared to that of Group I.

At 42.7 cm CVRL (170th day), the mandibular gland attained the characteristics of adult gland. At 100 cm CVRL (299th day), the gland was elongated, curved and longer than the parotid gland and covered by fascia and the confluence of linguo-facial, occipital and maxillary veins with the external jugular vein laterally and developing thymus caudally. Medially, it was related to the larynx, division of the common carotid artery, external carotid artery, 9th, 10th, 11th cranial nerves, stylo-hyoideus muscle and great cornu of hyoid. The dorsal border was related to the duct of this gland

and the ventral border was related to the external maxillary vein (Figure 4). Mandibular lymph node was placed above the anterior extremity of the gland. Dense compact lobulation of the gland was also observed at this stage (Figure 4b). In Group III, morphometric studies revealed that the mean length and breadth of left mandibular gland was 4.95±0.7 cm and 1.98±0.2 cm, respectively and average weight was 11.42±1.5 gm whereas right one was 5.00±0.7 cm in length, 2.00±0.2 cm in breadth and weighed 11.50±1.5 gm (Table 2). Gradual increase in length, breadth and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation during the foetal stage. Sivakumar et al. (2003) reported that in Group III (28 weeks-full term), the length and breadth of human foetal submandibular gland increased two times and weight increased five times than that of Group I.

The mandibular duct was seen leaving the gland at lower third of the inferior border between 12.5 cm CVRL (84th day) to 22 cm CVRL (123rd day) and middle of the concave border between 29.5 cm CVRL (140th day) to 78.7 cm CVRL (250th day). The duct running on the floor of the mouth cavity in close association with the sublingual duct and opened closely along the side of monostomatic sublingual duct at the caruncula sublingualis. Similar findings were described in domestic animals by Budras and Habel (2003); Akers and Denbow (2008). Pospieszny et al. (2010) observed that in pig, the location of the mandibular gland changed in respect to the mandibular angle during the prenatal period. With age its location changed from lateral to more medial, and from caudal to more rostral.

In neonatal buffalo, the mandibular gland was larger than the parotid gland. It was pale yellow to pinkish brown in colour, distinctly


160

lobulated and extended in a curve medial to the angle of the mandible from the atlantal fossa to the basihyoid muscle. Caudally, the gland was partly covered by the parotid gland. The mandibular duct left the gland from the middle of its rostral border and extended laterally along the digastric muscle, then passed forward on the deep surface of the mylohyoid and opened lateral to the sublingual caruncle. These observations were in partial agreement with the findings of Rauf et al. (2004) in one day old kid. The left mandibular gland of neonatal buffalo on average measured 9.8±1.0 cm in length and 2.8±0.2 cm in breadth whereas right one was 10.1±1.0 cm in length and 3.0±0.2 cm in breadth. The mean weight of left and right mandibular glands was 28.8±1.5 gm and 30.1±1.5 gm, respectively (Table 2).

Significant differences in the biometrical parameters of mandibular gland between foetuses of Group I, II, III as well as neonatal buffalo were observed at P<0.05 and P<0.01 level. The biometrical studies showed that there was no significant difference in the left and right mandibular salivary gland within same group at P<0.05 and P<0.01 level (Graph 2).

Sublingual salivary gland The sublingual gland was the smallest among the three major salivary glands in prenatal buffalo and measured about half the size of the mandibular gland. The gland arose as a number of small epithelial thickenings in the linguogingival groove and on the outer side of the groove at 6 cm CVRL (55th day). Each thickening formed its own canal. At 9.2 cm CVRL (70th day), the gland was devoid of any superficial lobulations (Figure 2). The left sublingual gland of Group I foetuses on average measured 1.49±0.3 cm in length and 0.20±0.2 cm in breadth whereas right one was

1.50±0.3 cm in length and 0.20±0.2 cm in breadth. The mean weight of left and right sublingual glands was 1.34±0.1 gm and 1.35±0.1 gm, respectively (Table 3). The sublingual gland was composed of two parts, the superior (polystomatic) and inferior (monostomatic) parts. Grossly, the superior and inferior parts of the gland were distinguishable at 16.5 cm CVRL (102nd day). At 25.9 cm CVRL (132nd day), the colour of the superior part varied from light yellow to light brown (Figure 3c). The superior part of the gland was arranged in a chain of lobules and extended from the palato-glossal arch to the incisive part of the mandible. The colour of the inferior part varied from light yellow to light brown. It was elongated and situated beneath the mucosa of the floor of the mouth, above the mylohyoideus muscle, between the mandible laterally, and the muscles of tongue medially. Pospieszny et al. (2010) reported that in the initial developmental periods of pig, the elongation of sublingual salivary ducts was higher than in mandibular gland. In Group II, morphometric studies revealed that the mean length and breadth of left sublingual gland was 3.78±0.5 cm and 0.50±0.3 cm, respectively with mean weight was 1.93±0.1 gm whereas right one was 3.80±0.5 cm in length, 0.50±0.3 cm in breadth and weighed 1.95±0.1 gm (Table 3). At 45.2 cm CVRL (175th day), the sublingual gland attained the characteristic shape, colour and position similar to adult gland. At 100 cm CVRL (299th day), the gland showed distinct lobulations (Figure 5a). The superior part was light brown in colour and extended from the mandibular symphysis to the palatoglossal arch. The ducts of the superior part were not visible during the prenatal period. The inferior part was also light brown in colour and extended from the mandibular symphysis to the level of the last premolar tooth.


161

It was thicker and shorter than the superior part and was situated below it. The inferior part of the sublingual gland was drained by only one duct, which opened along the side of mandibular duct at caruncula sublingualis (Figure 5). These features were in agreement with the reports of Arey (1965) in human beings and Latshaw (1987) in domestic animals. The location of foetuses in the horns of uterus and their sex did not have any significant impact on the morphology and development of salivary glands (Pospieszny et al., 2010). The left sublingual gland of Group III foetuses on average measured 5.98±0.6 cm in length and 1.19±0.3 cm in breadth whereas right one was 6.00±0.6 cm in length and 1.20±0.3 cm in breadth. The mean weight of left and right sublingual glands was 2.86±0.1 gm and 2.90±0.1 gm, respectively (Table 3). Gradual increase in length, breadth and weight of the gland was due to increased proliferation of ducts, increased lobulation and connective tissue formation during the foetal stage.

In neonatal buffalo, the superior part of sublingual gland was composed of a chain of lobules. It was pale yellow in colour and situated under the floor of the mouth. It extended from the incisive area of the mandible to the palato-glossal arch and was drained by many small ducts which opened along rows of long papillae found in the lateral sublingual recess of the mouth. The inferior part was extended from the incisive area of the mandible to the midline of the superior part of gland. A single excretory duct passed forward medial to the gland and accompanied the mandibular duct to the sublingual caruncle located on the floor of the mouth behind the incisor teeth. Collectively, the sublingual glands were related on their lateral aspects to the mylohyoid muscle and the sublingual nerve, medially to the hyoglossus, styloglossus and genioglossus muscles and ventrally to the

geniohyoid muscle. These observations were in total agreement with the findings of Attie and Sciubba (1981) in human beings and Budras and Habel (2003) in domestic animals. The morphometric studies revealed that the mean length and breadth of left sublingual gland of neonatal buffalo was 10.4±1.0 cm and 1.35±0.5 cm, respectively and average weight was 6.7±1.0 gm whereas right one was 10.6±1.0 cm in length, 1.35±0.5 cm in breadth and weighed 6.9±1.0 gm (Table 3).There was significant difference in the biometrical parameters of sublingual gland between foetuses of Group I, II, III as well as neonatal buffalo at P<0.05 and P<0.01 level. The biometrical studies showed that there was no significant difference in the left and right sublingual salivary gland within same group at P<0.05 and P<0.01 level (Graph 3).

CONCLUSION

Three pairs of major salivary glands viz., parotid, mandibular and sublingual were distinguished during prenatal and neonatal life in the buffalo. The mandibular gland was the largest among the three major salivary glands in prenatal buffalo, which may be attributed to its early development among all the salivary glands. The sublingual gland was the smallest among the three major salivary glands in prenatal buffalo and measured about half the size of the mandibular gland. At 5.8 cm CVRL (54th day), the mandibular gland began to form an epithelial outgrowth into the mesenchyme, forming the floor of the mouth in the linguo-gingival groove. The sublingual gland arose as a number of small epithelial thickenings in the linguogingival groove and each thickening formed its own canal at 6 cm CVRL (55th day). The parotid gland was first seen in the form of a small rod,


162

Tabl

e 1.

Gro

ss b

iom

etric

al o

bser

vatio

ns o

n pa

rotid

saliv

ary

glan

d of

buf

falo

foet

uses

and

neo

nate

s.Parotid Salivary Gland

Pren

atal

age

gro

ups

Neo

nata

l gro

upG

roup

I**

Gro

up II

**G

roup

III*

*G

roup

IV**

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

)W

eigh

t(g

m)

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

Wei

ght

(gm

)

Lef

t*0.

58±0

.10.

43±0

.11.

39±0

.21.

68±0

.41.

45±0

.13.

85±0

.52.

95±0

.62.

72±0

.28.

65±1

.15.

50±0

.83.

00±0

.219

.7±1

.5R

ight

*0.

60±0

.10.

45±0

.11.

40±0

.21.

70±0

.41.

50±0

.13.

90±0

.53.

00±0

.62.

80±0

.29.

00±1

.15.

80±0

.83.

10±0

.220

.9±1

.5

Sign

ifica

nt (P

<0.0

5), H

ighl

y Si

gnifi

cant

(P<0

.01)

.*N

on-s

igni

fican

t diff

eren

ce w

ithin

gro

ups a

t 0.0

5 an

d 0.

01 le

vel.

**Si

gnifi

cant

diff

eren

ce b

etw

een

grou

ps a

t 0.0

5 an

d 0.

01 le

vel.

Tabl

e 2.

Gro

ss b

iom

etric

al o

bser

vatio

ns o

n m

andi

bula

r sal

ivar

y gl

and

of b

uffa

lo fo

etus

es a

nd n

eona

tes.

Mandibular Salivary Gland

Pren

atal

age

gro

ups

Neo

nata

l gro

upG

roup

I**

Gro

up II

**G

roup

III*

*G

roup

IV**

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

)W

eigh

t(g

m)

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

)W

eigh

t(g

m)

Lef

t*1.

09±0

.10.

39±0

.11.

53±0

.32.

38±0

.61.

19±0

.14.

76±1

.04.

95±0

.71.

98±0

.211

.42±

1.5

9.8±

1.0

2.8±

0.2

28.8

±1.5

Rig

ht*

1.10

±0.1

0.40

±0.1

1.55

±0.3

2.40

±0.6

1.20

±0.1

4.80

±1.0

5.00

±0.7

2.00

±0.2

11.5

0±1.

510

.1±1

.03.

0±0.

230

.1±1

.5

Sign

ifica

nt (p

< 0

.05)

, Hig

hly

Sign

ifica

nt (p

< 0

.01)

.*N

on-s

igni

fican

t diff

eren

ce w

ithin

gro

ups a

t 0.0

5 an

d 0.

01 le

vel.

**Si

gnifi

cant

diff

eren

ce b

etw

een

grou

ps a

t 0.0

5 an

d 0.

01 le

vel.


163

Tabl

e 3.

Gro

ss b

iom

etric

al o

bser

vatio

ns o

n su

blin

gual

saliv

ary

glan

d of

buf

falo

foet

uses

and

neo

nate

s.

Sublingual Salivary Gland

Pren

atal

age

gro

ups

Neo

nata

l gro

upG

roup

I**

Gro

up II

**G

roup

III*

*G

roup

IV**

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

)W

eigh

t(g

m)

Len

gth

(cm

)B

read

th(c

m)

Wei

ght

(gm

)L

engt

h(c

m)

Bre

adth

(cm

)W

eigh

t(g

m)

Lef

t*1.

49±0

.30.

20±0

.21.

34±0

.13.

78±0

.50.

50±0

.31.

93±0

.15.

98±0

.61.

19±0

.32.

86±0

.110

.4±1

.01.

35±0

.56.

7±1.

0R

ight

*1.

50±0

.30.

20±0

.21.

35±0

.13.

80±0

.50.

50±0

.31.

95±0

.16.

00±0

.61.

20±0

.32.

90±0

.110

.6±1

.01.

35±0

.56.

9±1.

0

Sign

ifica

nt (P

<0.0

5), H

ighl

y Si

gnifi

cant

(P<0

.01)

.*N

on-s

igni

fican

t diff

eren

ce w

ithin

gro

ups a

t 0.0

5 an

d 0.

01 le

vel.

**Si

gnifi

cant

diff

eren

ce b

etw

een

grou

ps a

t 0.0

5 an

d 0.

01 le

vel.


164

Graph 1. Showing variations in mean values of length, breadth and weight of parotid salivary gland of buffalo fetuses. *Non-significant difference within groups at 0.05 and 0.01 level. **Significant difference between groups at 0.05 and 0.01 level.

Graph 2. Showing variations in mean values of length, breadth and weight of mandibular salivary gland of buffalo fetuses. *Non-significant difference within groups at 0.05 and 0.01 level. **Significant difference between groups at 0.05 and 0.01 level.


165

Graph 3. Showing variations in mean values of length, breadth and weight of sublingual salivary gland of buffalo fetuses. *Non-significant difference within groups at 0.05 and 0.01 level. **Significant difference between groups at 0.05 and 0.01 level.


166

M

M

Fig.1

C-1 C-2 C-3

D-1 E-1

Cs

L

C-3

D-2

Fn PM

E

T

SL

Pln

Mm

Pd

P

M

E

E

P

M

PlnFn

Mm

FvFa Pd

Cp Cp

SL

Fig.2

Fig.3

Fig.4 Fig.5

Figure 1. Photograph of 9.2 cm CVRL (70th day) buffalo foetal head showing loosely arranged lobules of parotid (P) and mandibular (M) salivary glands. (Fn- facial nerve; E- external ear).

Figure 2. Photograph of head of 9.2 cm CVRL (70th day) buffalo foetus showing sublingual glands (SL) were devoid of superficial lobulations. (T-tongue).

Figure 3. Photograph of 25.9 cm CVRL (132nd day) buffalo foetal head showing the location of parotid (P) and mandibular (M) glands and their relations. (Pln- parotid lymph node; Mm-masseter muscle; Pd- parotid duct; E- externar ear).

3(a) Photograph of prenatal parotid gland showing roughly triangular shape at CVRL of 25.9 cm (132nd day).3(b) Photograph of prenatal mandibular gland showing roughly quadrilateral shape at 25.9 cm CVRL (132nd day).3(c) Photograph of prenatal sublingual gland showing elongated chain of lobules at CVRL of 25.9 cm (132nd day).

Figure 4. Photograph of head of 100 cm CVRL (299th day) buffalo foetus showing distinct lobulations of well developed parotid (P) and mandibular (M) salivary glands. (Pln- parotid lymph node; Fn- facial nerve; Mm- masseter muscle; Pd- parotid duct; Fa- facial artery; Fv- facial vein; E- externar ear).

4(a) Photograph of prenatal parotid gland showing distinct lobulations at CVRL of 100 cm (299th day).4(b) Photograph of prenatal mandibular gland showing dense compact lobulations at 100 cm CVRL (299th day).

Figure 5. Photograph of 100 cm CVRL (299th day) buffalo foetal head showing distinct lobulations of sublingual glands (SL) in the form of elongated chain of lobules. (Cp- conical papillae; Cs- caruncula sublingualis; L- lower lip).

5(a) Photograph of prenatal sublingual gland showing distinct lobulations at CVRL of 100 cm (299th day).


167

extending dorsally from the lateral part of the oral cavity, just below and infront of the external ear and behind the facial nerve at 6.9 cm CVRL (60th day). The mandibular duct was well developed at 12 cm CVRL (82nd day), however, parotid duct (Stenson’s duct) was observed from 12.5 cm CVRL (84th day) onwards. Grossly, the superior (polystomatic) and inferior (monostomatic) parts of the sublingual gland were distinguishable at 16.5 cm CVRL (102nd day). At 42.7 cm CVRL (170th day), the mandibular gland attained the characteristics of adult gland, whereas in parotid and sublingual glands, the adult characteristic features were attained from 45.2 cm CVRL (175th day) onwards. Significant differences in the biometrical parameters of all major salivary glands between foetuses of Group I, II, III as well as neonatal buffalo were observed at P<0.05 and P<0.01 level. The biometrical studies showed that there was no significant difference in the left and right sets of all major salivary glands within same group at P<0.05 and P<0.01 level.

REFERENCES

Akers, R.M. and D.M. Denbow. 2008. Anatomy and Physiology of Domestic Animals. Blackwell Publishing, Ames, Iowa.

Arey, L.B. 1965. Developmental Anatomy: A Text Book and Laboratory Manual of Embryology, Philadelphia: W.B. Saunders Co. 221-222.

Attie, J.N. and J.J. Sciubba. 1981. Tumors of major and minor salivary glands. In Current Problems in Surgery, Year Book Medical Publishers Inc, Chicago.

Barnwal, A.K. and R.D. Sinha. 1982. Macromorphology of the parotid salivary gland of buffalo. Indian J. Anim. Sci., 52:

744-747.Budras, K.D. and R.E. Habel. 2003. Bovine

Anatomy, Schlutersche, Hannover. 36-37.Chi, J.G. 1996. Prenatal development of human

major salivary glands: Histological and immunohistochemical characteristics with reference to adult and neoplastic salivary glands. J. Korean Med. Sci., 11: 203-216.

Dellmann, H.D. and J.A. Eurell. 1998. Digestive System. In: Text book of Veterinary Histology, 5th ed., William and Wilkins, London. 174-176.

Featherstone, J.D. 2000. The science and practice of caries prevention. J. Amer. Dent. Assoc., 131: 887-899.

Getty, R. 1975. Bovine digestive system. In: Sisson and Grossman’s anatomy of the domestic animals, 5th (ed.), Vol. 1. W.B. Saunders Co., Philadelphia, London, Toronto.

Knospe, C. and G. Bohme. 1995. Prenatal development of the mandibular gland and parotid gland in cats. Anat. Histol. Embryol., 24: 1-6.

Latshaw, W.K. 1987. Salivary gland. In: Vetereinary Developmental Anatomy. BC, Toronto, Philadelphia. 90-93.

Moghaddam, Y.F., J. Darvish, S.N. Mahdavi, A.S. Abdulamir, M. Mousavi and S.K. Daud. 2009. Comparative histological and histochemical inter-species investigation of mammalian submandibular salivary glands. Res. J. Appl. Sci., 4: 50-56.

Nayeem, F., S. Sagaff, G. Krishna and S. Rao. 2000. The prenatal parotid gland. J. Anat. Soc. India, 49: 155-157.

Pospieszny, N., J. Kuryszko, M. Juszczyk and M. Adamski. 2010. Morphological and histological analysis of the mandibular gland and sublingual glands in prenatal


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period of the pig. Bull. Vet. Inst. Pulawy., 54: 351-355.

Rauf, S.M.A., M.R. Islam and M.K. Anam. 2004. Macroscopic and microscopic study of the mandibu ar salivary gland of Black Bengal goats. Bangla. J. Vet. Med., 2: 137-142.

Singh, A.D., R.K. Jain and P. Kumar. 2011. Topographic anatomy of buccal and labial glands in sheep. Haryana Vet., 50: 30-32.

Sivakumar, M., M. Sud and V. Vathsala. 2003. Histogenesis and morphometric study of human foetal submandibular salivary gland. J. Anat. Soc. India, 52: 3-6.

Soliman, M.K. 1975. Studies on the physiological chemistry of the allantoic and amniotic fluids of buffalo at various periods of pregnancy. Indian Vet. J., 52: 106-111.

Wolff, M.S., L. Mirels, J. Lagner and A.R. Hand. 2002. Development of the rat sublingual gland: A light and electron microscopic immunocytochemical study. Anat. Rec., 266: 30-42.


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ABSTRACT

The effect of transition on body condition score (BCS) and ultrasonographic back fat thickness (USG BFT) was examined in 101 multiparous buffaloes grouped according to transition stage i.e., Far off dry (FOD), Close up dry (CUD) and Fresh (F). With BCS 2 to 2.5, the BFT of F period was significantly lower than FOD period. With BCS 3 to 3.5 and 4 to 4.5, the mean BFT at F period was significantly reduced as compared to FOD and CUD periods. Buffaloes with BCS 5, showed a significant reduction in BFT from FOD to CUD and from CUD to F period. BCS 5 at FOD period was characterized by accumulation of ‘fat Pads’ around the buttock. Majority of buffaloes with BCS of 4 to 5 showed reduction in BCS at F period. The overall correlation coefficient between BCS and USG BFT was 82, 83 and 78% for FOD, CUD and F period respectively. The overall correlation coefficient between visual BCS and BCS assessed from digital images for FOD, CUD and F period was 99, 98 and 95% respectively. It was concluded that digital images can be readily used to estimate BCS in buffaloes and USG BFT gives an accurate measure of fat reserves in buffaloes.

Keywords: body condition score, transition period, ultrasonographic back fat thickness

INTRODUCTION

Body condition scoring (BCS) is a subjective technique for assessing the condition of livestock at regular intervals. It is particularly helpful in assessing the body fat reserves of farm animals by visual and manual inspection of the thickness of fat cover and prominence of the bone at the tail head and loin region. The BCS system being non invasive, quick and inexpensive is universally accepted to estimate the degree of fatness (Bittante et al., 2004; Drame et al., 1999) and is also used to assess the post parturient reproductive health (Kadivar et al., 2014). Body condition scoring is particularly useful as an aid to dry cow and pre calving management with the main objective that the cows calve down uneventfully and enter the lactation stage safely. As the dairy cows use body energy reserves in the early lactation to cope up with negative energy balance (NEB) body condition scoring along with a less common method to assess fat reserves in body tissues i.e., measurement of back fat thickness by using real time ultrasound are more promising approaches to ensure an uneventful transition of dairy cows. Various studies on the precision of BCS system including the ultrasonographic assessment of subcutaneous back fat indicated that BCS values were closely related to the actual measurement of

BODY CONDITION SCORING BY VISUAL AND DIGITAL METHODS AND ITS CORRELATION WITH ULTRASONOGRAPHIC BACK FAT THICKNESS

IN TRANSITION BUFFALOES

Randhir Singh1,*, Sarnarinder Singh Randhawa2 and Charanjit Singh Randhawa1

1,*Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, India, *E-mail: [email protected] Angad Dev Veterinary and Animal Sciences University (GADVASU), Ludhiana, India

Original Article


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subcutaneous fat (Zulu et al., 2001). Few studies are available on buffaloes relating BCS to USG BFT (Anitha et al., 2010), but critical information regarding effect of transition period on BCS and USG BFT is lacking. Therefore, the objective of this study was to examine the relationship between BCS and USG BFT in transition buffaloes.


Animals 101 multiparous buffaloes in advanced pregnancy from organised buffalo farm were used for this study, conducted during August 2013 to July 2014. The buffaloes were grouped according to transition stage i.e. Far off dry (FOD): >10 days following dry off and not <30 days expected to calving. Close up dry (CUD): expected calving within 3 to 21 days. Fresh (F): 3 to 30 days in milk.

Body composition Body condition score (BCS) was estimated by the same individual using the five point visual BCS technique (Table 1) with 0.5 increment (Wildman et al., 1982). Some modifications were included in BCS system for buffaloes having BCS above 4.5, keeping in view the consistent and unusual site for fat deposition (Figure 1) and its mobilisation thereafter for meeting energy demands of early lactation after parturition.

Digital BCS The digital photographic images were taken on the day of assessment of visual BCS. Images were taken from behind the buffalo at 0 to

20 degree angle to the tail head and from the bird’s eye view with the help of Sony cyber shot digital camera having 12.1 Megapixels and 4x optical zoom (Figure 2 to 4). The images were evaluated by the same individual who assessed visual BCS but while assigning BCS to digital photographs the visual BCS was not referred.

Ultrasonographic back fat thickness (USG BFT) Subcutaneous back fat thickness was measured by real time ultrasound using a portable Sonosite instrument at 7.5 MHz frequency. Back fat thickness in the rump or thurl area was measured as the thickness of the layer of sub cutaneous fat between skin and the fascia trunci profunda located above the gluteus medius muscle (Figure 5). The transducer was placed vertically to an imaginary line between the pins (tuber Ischia) and hooks (tuber coaxe) at the sacral examination site ( 9 to 11 cm cranial to the pins) (Nanda and Herdt, 2009) after shaving of site and application of coupling gel.

Image measurement and interpretation Images were measured at depth of 4.7. Buffaloes having BCS above 4 were having fat layer deep enough that their interpretation was better done at depth of 9. Captured back fat images were freezed and measured using inbuilt measurement calliper protocol in the instrument.

Both BCS and USG BFT were estimated on the same day at each stage of transition. Visual BCS and USG BFT were measured at all the three periods i.e., FOD, CUD and ‘F’ period in order to observe and calculate any significant change in BCS and USG BFT. The body condition scoring was done as per the characteristics described in Table 1.


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Feeding and management Animals were fed in head to head housing system in mangers. Feeding involved 50 kg green fodder (Maize, Pearl millet and Sorghum during summer; Egyptian clover and Oats during winter), 6 kg of wheat straw and 2 kg of concentrates per day during last 90 days of gestation. During lactation the feeding involved 55 kg green fodder, 8 kg wheat straw and 3 kg of concentrates per day. The proximate analysis from feed and fodder samples was carried out by the methods of AOAC, 1970 (Table 2).

Statistical analysis The statistical analysis was carried out using SPSS (16.0). ANOVA followed by Duncan’s multiple range test (DMRT) was used to estimate significant difference between BFT at different transition stages at P<0.05. The correlation between BCS and BFT was estimated by Microsoft excel.

RESULTS

The results of proximate feed analysis revealed low levels of crude protein (CP) and ether extract (EE). The mean USG BFT of buffaloes with different body condition scores for different time periods (FOD, CUD, F) is presented in Table 3. In buffaloes with BCS 2 to 2.5, the USG BFT of F period was significantly lower than FOD period. The mean USG BFT at F period was significantly reduced as compared to FOD and CUD period in BCS group 3 to 3.5 and 4 to 4.5. In buffaloes with BCS 5, there was a significant reduction in BFT from FOD to CUD and from CUD to F period.

Out of 11 buffaloes with BCS 2 to 2.5 at FOD period, two buffaloes at CUD and F periods had a BCS of <2. Sixty buffaloes had BCS 3-3.5 at

FOD period (Table 4). Out of these, two and seven buffaloes had BCS <2 and 2 to 2.5, respectively at CUD period. Further at F period, 32 out of 60 buffaloes showed reduction in BCS from 3 to 3.5, to 2 to 2.5 (25/60) or <2 (7/60). The majority of buffaloes with BCS 4 to 4.5 showed a reduction in BCS at F period. Twenty two out of 27 buffaloes lost BCS from 4 to 4.5, to 3 to 3.5 at fresh period. All three animals with BCS 5 were characterized by presence of fat pads (Figure 1, 2) at FOD period and had BCS 4 to 4.5 at F period with disappearance of fat pads. Figure 6 represents the USG BFT for different BCS scores of buffaloes. The overall correlation coefficient between BCS and USG BFT was 82, 83 and 78% for FOD, CUD and F period, respectively. The overall cor-relation between visual and digital BCS for FOD, CUD and F period was 99%, 98% and 95%, re-spectively.

DISCUSSION

To our best knowledge this is the first study to evaluate the effect of transition period on BCS and USG BFT in buffaloes. In the present study the BCS was evaluated at three predefined transition stages in buffaloes on 1 to 5 scale with 0.5 increments, as a single BCS does not give any indication of whether a buffalo is gaining or losing body reserves over a period of time. Furthermore, USG BFT was concurrently used in this study to validate the BCS, as during transition it is difficult to judge accurately the real condition of animal due to weight gain associated with fetal growth. In our study the buffaloes with BCS >3.5 were more severely affected with further change in BCS and USG BFT in subsequent stages. Similar to our findings Bernabucii et al. (2005) reported higher


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Table 1. Body condition score (BCS) along with corresponding illustration of buffalo and principal descriptors involved for visual assessment.

BCS Animal Descriptors

1.0-1.5Emaciated / poor

Tail head: deep cavity with no fatty tissue under skin.Loin: prominent spine with sharp horizontal processes.

2.0-2.5Thin / moderate

Tail head: shallow cavity with prominent pin bones. Traces of fat under skin.Loin: horizontal processes can be identified individually with round ends.

3.0-3.5Average / good

Tail head: fat cover over entire area but pelvis can be felt.Loin: end of horizontal process can only be felt with pressure with slight.

4.0-4.5Fat

Tail head: entirely filled, evidence of folds and patches of fat.Loin: processes not felt, completely rounded.

5.0Obese / grossly fat

Tail head: buried in fatty tissue or “fat pads”.Pelvis not palpable even with firm pressure.


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Table 3. USG BFT at different transition stages in buffaloes for different BCS (Mean ± S.E.).

BCS (n) Ultrasonographic Back fat thickness (USG BFT) (in cm) FOD CUD F

1-1.5 (00) - - - 2-2.5 (11) 1.391±0.10a 1.156±0.089ab 1.075±0.104b

3-3.5 (60) 1.750±0.052a 1.657±0.055a 1.384±0.043b

4-4.5 (27) 2.704±0.10a 2.565±0.094a 2.220±0.10b

5 (03) 3.567±0.073a 3.397±0.014b 3.083±0.018c

*The values with different superscripts in a row differ significantly at P≤0.05.

Table 2. Proximate analysis of feed, fodder and wheat straw (% DM basis).

BIS Feed Fodder Wheat strawDM 89 91 30 93CP 20 16 8.4 2.7EE 4.5 2.7 1.5 2.2Ash - 11.1 11.5 10.6ADF 21 20.7 42 47.2NDF 28 40.5 61.4 70.2OM - 90 89.1 90.2

*As per Bureau of Indian specifications (BIS), 2009 for high yielding cows.

Table 4. Effect of transition period on BCS in buffaloes.

BCS FOD CUD F

< 2 2-2.5 3-3.5 4-4.5 5 < 2 2-2.5 3-3.5 4-4.5 5 < 2 2-2.5 3-3.5 4-4.5 5

Number of buffaloes

- 11 - - - 2 9 - - - 2 9 - - -- - 60 - - 2 7 51 - - 7 25 28 - -- - - 27 - - - 6 21 - - - 22 5 -- - - - 3 - - - 1 2 - - - 3 -

*The bold numerical represent the number of buffaloes with reduced BCS from FOD to CUD and or ‘F’.


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Figure 1. Buffalo with BCS 5 having ‘Fat Pads’.


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BCS: 1 BCS: 1.5 BCS: 2

BCS: 2.5 BCS: 3 BCS: 3.5

BCS: 4 BCS: 4.5 BCS: 5

Figure 2. BCS assessment by digital photographs taken from rear of buffaloes.


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Emaciated Good Obese

Figure 3. BCS assessment by digital photographs taken from bird’s eye view.

Emaciated Good Obese

Figure 4. Contour of buffaloes with different body conditions as seen from bird’s eye view.


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Figure 6. USG Back fat thickness (BFT) of buffaloes with different BCS.

Figure 5. USG dipicting various layers of facia, fat and muscle.


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Figure 6. USG Back fat thickness (BFT) of buffaloes with different BCS. (Cont.)


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reduction in high BCS cows from late pregnancy to first 30 days in milk, than the cows with average and good BCS. This may be attributed to increased resistance of adipose tissue to insulin which predisposes the dairy animal to mobilize non esterified fatty acid (NEFA), thus potentially creating a vicious cycle of NEFA mobilization and dry matter intake (DMI) reduction during late prepartum period. This is why high BCS animal have lower DMI and more rapid decrease in BCS during prepartum period than animal of average or good BCS (Grummer et al., 2004).

Our results show there is strong correlation between BCS and USG BFT for all three transition stages. Similar findings were reported by Anitha et al. (2010) with correlation coefficient of 87% between BCS and USG BFT in Murrah buffaloes. Wittek and Furll (2002) and Lubi and Fletcher (1985), also reported a significant correlation coefficient of 91% and 87%, respectively between BCS and USG BFT.

It was observed that 83.33% (25/30) buffaloes having BCS above 4 reduced significantly to a lower BCS over the passage of time during transition, where as only 53.33% (32/60) and 18.18% (2/11) buffaloes having BCS 3 to 3.5 and 2 to 2.5, respectively reduced to lower BCS at F stage (Table 4) over a period of time. Chebel, 2010 reported that only 24.7% of cows entering the dry period with BCS under 3.5 lost BCS during the dry period, whereas 76.6% of cows over 3.75 lost BCS during the dry period. This difference may be due to the feeding management, environmental and species differences. This further indicated that cows with BCS above 3.75 lost almost 45 pounds of body weight. With mobilization of 50 to 60 kg of fat during transition period with majority being in early lactation, adipose tissue quantitatively represents the most critical energy storage and

seems suitable to assess energy balance of dairy cow because the amount of mobilized body fat approximates the energy demand that is lacking for milk output and maintenance (Waltner et al., 1993). This is further justified by our study in which there was significant loss of USG BFT and BCS between FOD and ‘F’ period in buffaloes with BCS > 3.5 and no buffalo remained in BCS group of 5 as compared to initial three buffaloes during the FOD period.

Three buffaloes which were having BCS 5 at FOD period were characterized by accumulation of ‘fat pads’ around the tail and pins (buttock region). At the first instance it was assumed that it may be some sort of edema but ultrasonographic examination revealed it as a fat layer. This layer of ‘fat pads’ subsequently disappeared in later transition stages due to fat mobilization. The assessment of BCS by digital photographs revealed that photographs taken from rear and bird’s eye view can also be used for estimating accurate BCS in buffaloes. This was the first attempt to estimate BCS by digital means in buffaloes and the results depicted a strong correlation coefficient between visual BCS and digital BCS. Previously, Ferguson et al. (2006) correlated visual and digital BCS in cows using four observers and found a strong correlation of 82 to 90 percent among different observers. In our study the correlation was strong which may be due to the fact that in buffaloes there is very little body hair which exposes the full outline of animal’s body in photographs as compared to cows where body has thick layer of hair which could probably mask the body outlines and angles and deter in exact assessment of BCS. Also, in the present study, the assessment of BCS by both the methods was done by the same observer which may have resulted in high correlation even when the assessment was done independently, without


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referring to the scores assigned by other method (visual BCS) in same animal.

CONCLUSION

Ultrasonographic BFT should be concurrently used as an aid to BCS for assessment of body fat reserves in transition buffaloes. BCS assessment by digital means in transition buffaloes can be used with reliable results and buffaloes having BCS 5 tend to accumulate fat around tail and pins which is prominent major descriptor for characterization of buffaloes with BCS 5. Further, disappearance of ‘fat pads’ was the most prominent descriptor when a buffalo with BCS 5 reduced its body condition.

REFERENCES

Anitha, A., R.K. Sarjan, J. Suresh, M.P.R. Srinivasa and R.Y. Kotilinga. 2010. Development of the body condition score system in Murrah buffaloes: validation through ultrasonic assessment of body fat reserves. J. Vet. Sci., 11(1): 1-8.

Association of Official and Agricultural Chemists, AOAC. 1970. Official Methods of Analysis, 11th ed. Association of Official and Agricultural Chemists, Washington, D.C.

Bernabucci, U., B. Ronchi, N. Lacetera and A. Nardone. 2005. Influence of body condition score on relationships between metabolic status and oxidative stress in periparturient dairy cows. J. Dairy Sci., 88: 2017-2026.

Bittante, G., L. Gallo, P. Carnier, A. Comin and M. Cassandro. 2004. Management and breeding of cows using body condition

score. Informatore Agrario, 60: 55-58.Chebel, C.R. 2010. The long lasting impact of

reproductive performance on health and production. Western Dairy News, 10(10): 1-2.

Drame, E.D., C.H. Hanzen, J.Y. Houtain, Y. Laurent. and A. Fall. 1999. Evolution of body condition score after calving in dairy cows. Ann. Med. Vet., 143(4): 265-270.

Furguson, J.D., G. Azzaro and G. Licitra. 2006. Body condition assessment using digital images. J. Dairy Sci., 89: 3833-3841.

Grummer, R.R., D.G. Mashek and A. Hayirli. 2004. Dry matter intake and energy balance in the transition period. Vet. Clin. N. Am-Food A., 20: 447-470.

Kadivar, A., M.R. Ahmadi and M. Vatankhah. 2014. Associations of prepartum body condition score with occurrence of clinical endometritis and resumption of postpartum ovarian activity in dairy cattle. Trop. Anim. Health Pro., 46: 121-126.

Lubis, A. and I.C. Fletcher. 1985. Body Conditionscore in Swamp Buffalo Cows; Research Report. Research Institute for Animal Production, Indonesia, 31.

Nanda, P.J. and T.H. Herdt. 2009. Clinical use of ultrasound for subcutaneous fat thickness measurements in dairy cattle. Current Veterinary Therapy: Food Animal Practice, Saunders, 5: 150-153.

Waltner, S.S., J.P. McNamara and J.K. Hillers. 1993. Relationships of body condition score to milk production variables in high producing Holstein dairy cattle. J. Dairy Sci., 76: 3410-3419.

Wildman, E.E., G.M. Jones, P.E. Wagner, R.L. Boman, J.R. Troutt and T.N. Lesch. 1982. A dairy cow body condition scoring system


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and its relationship to selected production characteristics. J. Dairy Sci., 65: 495-501.

Wittek, T. and M. Fu¨ rll. 2002. Untersuchungen zu Ko¨rperkondition und abdominalen Fettdepots in Beziehung zur Fettmobilisation bei an Labmagenverlagerung erkrankten Ku¨ hen. Tierarztl. Umschau, 57: 302-309.

Zulu, V.C., T. Nakao, M. Moriyoshi, K. Nakada, Y. Sawamukai, Y. Tanaka and W. Zhang. 2001. Relationship between body condition score and ultrasonographic measurement of subcutaneous fat in dairy cows. Asian Austral. J. Anim., 14: 816-820.


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ABSTRACT

Zoologists have introduced buffalo as “future livestock” and claim that their potentials and benefits will be more than any other livestock. River buffaloes play an impressive and crucial role in the economy of rural families. The ability of these animals to produce milk, meat and also their draft power has caused to be kept in rural areas. Mazani buffalo is classified as a river buffalo and no further karyotyping specification was made up to now. Blood samples were taken from ten (5 males and 5 females) Mazani buffaloes from Mazendaran province located in north of Iran. Blood lymphocytes cultured at 37oC for 72 h in presence of colsemid and the metaphase spreads were performed on microscopic slide. Giemsa was applied to stain chromosomes. The current study shows that 2n=50 and fundamental numbers (NF) is 60 in male and female. The types of autosome were 10 submetacentric/metacentric and 38 telocentric. The X chromosome is the largest telocentric and the Y chromosome is one of the smallest telocentric chromosomes. Also, the relative length of chromosomes ranged between 7.2 and 2.17 in Mazani buffalo. All chromosomes were found normal. The karyotype formula of Mazani buffalo is as follow: 2n (50) = 4M+6SM + 38 T+Sex chromosome.

Keywords: karyotype, chromosome, water buffalo, idiogram

INTRODUCTION

Buffalo (family Bovidae and tribe Bovini) can be divided into two main groups: Bubalia and Syncerina. Bubalia is also classified into Arni buffalo, Tamarao buffalo and Anona buffalo. Moreover, Syncerina consist of two subgroups called red buffalo and black buffalo. The arni buffalo is classified further two groups, the river buffalo and the swamp buffalo according to its habitat (Miyake et al., 1980). The diploid number of the swamp buffalo is 48 (Harisah et al., 1989), and the diploid number of the river buffalo is 50 (Murali et al., 2009; Ali et al., 2012).

According to climate conditions, Iranian buffaloes consist of three main categories: 1) Azeri Buffalo (Ardabil, Western and Eastern Azerbaijan provinces); 2) Mazani Buffalo (Gilan and Mazendaran provinces); 3) Khoozestani Buffalo (Khoozestan province) (Naserian and saremi, 2007). Most of these animals are kept in the states of western and eastern Azerbaijan located in northwest and state of Khoozestan located in south (Hasanzadeh and Monazzah, 2011). All of the Iranian buffaloes are riverine (Naserian and

KARYOTYPE OF MAZANI WATER BUFFALO FROM IRAN

M. Pournourali1,*, A. Tarang2 and F. Mashayekhi1

1Department of biology, University of Guilan, Rasht, Iran, *E-mail: [email protected] of Genomics and Animal, Agricultural Biotechnology Research Institute (ABRII), Branch of North Region of Iran, Rasht, Iran

Original Article


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saremi, 2007). River buffaloes have an impressive and crucial role in the economy of rural families (Hasanzadeh and Orojee, 2003). The ability of these animals to produce milk and meat and also their draft power has caused to be kept in rural areas (Mirhoseini et al., 2005). The dollar value of river buffaloes in Iran is nearly equal with a pure Holstein dairy cow. Iranian water buffaloes have close appearance to Iraqi buffaloes. Hence both of them may have been originated from the same ancestor. In addition Iranian river buffaloes in northwest of the country (West Azerbaijan), have same similarity to Mediterranean river buffaloes. So, it’s considered that they have descended from the same ancestor (Naserian and saremi, 2007).

Cytogenetic study is a powerful instrument to determine the normal karyotype of farm animal and to discover more about fundamental basis for abnormalities. Also, the chromosomal analysis is useful in the selection of high productive animals in farm (Ahmad et al., 2004). The chromosomal abnormalities in animals can be recognized and

culled from breeding stock (Ahmad et al., 2004). The present study was undertaken to determine the karyotype of Mazani water buffalo and compare with other river buffaloes in other countries.


Blood samples of buffaloTen Mazani buffaloes (5 males and 5

females) and were used for this chromosomal analysis. The Mazani buffaloes samples were collected from Mazendaran province located in north of Iran. Figure 1, shows the Mazani water buffalo. Peripheral blood samples were aseptically taken from the jugular vein and transferred venojects containing sodium heparin.

Lymphocyte culture4.5 ml of RMPI 1640 medium was prepared

with 2% phytohemagglutinin (PHA) as a mitogen

Figure 1. Mazani water buffalo.


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and transferred in flask. Beside 0.5 ml of blood sample were dropped into a flask, incubated at 37ºC under 5% of CO2 environment and regularly shaken in the day and night. At the 72nd h of incubation, Closemid was added as a mitotic inhibitor and well mixed followed by further incubation for 20 minutes.

Cell harvest and bandingThe mixture of blood samples was

centrifuged and supernatants were discarded. Potassium chloride (hypotonic solution) was applied to the pellet for 35 minutes. KCl was discarded, cells were fixed by cool fixative (3 methanol: 1 glacial acetic acid). Fixative was discarded too, and mixtures were dropped on a clean and foggy slides by micropipette and well dried. Then, the slides stained with 20% Giemsa’s solution for 25 minutes.

Chromosomal counting, karyotyping and idiograming

Chromosome counting was performed on metaphase cells under the light microscope. Fifteen clearly observable spread of each sample selected and then photographed (×1000). The length of the short arm (Ls), length of the long arm (Ll), Length of each chromosome (LT) and centromeric index (CI) were measured by Micro Measure 3.3. Other parameters like relative length (RL) were calculated by Microsoft Excel 2010. The centromeric index and arm ratio were computed to classify the types of chromosomes according to Guerra (1986). The Karyotypes were drawn by Adobe Photoshop CS6 and the idiograms were drawn by Microsoft excel 2010.

RESULTS AND DISSCUSSION

After lymphocyte culturing, cell harvesting, staining, Chromosomal counting, karyotyping and idiograming, the result show that diploid chromosomes (2n) of Mazani buffalo are 2n=50. So they are riverine (Bubalus bubalis bubalis). (Figure 2, and 3).

The present study showed results similar to those of previous reports on river buffalo in Iran (Khavary, 1978) and also on other river buffalo in Brazil (Pires et al., 1997; Rommelt, 1976), India (Murali et al., 2009; Bidhar et al., 1986; Balakrishnan and Yadav, 1984; Ramesha and Hedge 1992; Yadav et al., 1984; Kumar and Yadav, 1991; Gupta and chaudhri, 1978; Joshi and Govindaiah, 1999), Italy (Salerno et al., 1980), Pakistan (Ali et al., 2012), Thailand (Kenthao et al., 2012), Turkey (Ulbrich and Fisher, 1967), Sri lanka (Scheurmann et al., 1974) and Egypt (Ahmed et al., 2004; Cribiu and Obeidah, 1978; Hondt and Ghanam, 1971). Also it showed a similar result to the reported by Halnan (1976) that reported the diploid number is 50. It is not similar to previous reports that reported the diploid number is 2n = 48 in swamp buffalo in Japan (Miyake et al., 1980; Harisah et al., 1989; Dutt and Bhattacharya, 1952), Australia (Toll and Halnan, 1976a), Malaysia (Bongso and Jainudeen, 1979), China (Huang et al., 1987), Vietnam (Balakrishnan et al., 1988) and Thailand (Rommelt, 1977). Fischer and Ulbrich (1978) found that the diploid number of African buffalo is 52.

The fundamental number (NF) of Mazani buffalo is 60 in male and female and it is the same NF for De Hondt and Ghanam (1971); Rommelt (1976); Bongoso et al. (1977); Iannuzzi (1994); Kenthao et al. (2012).

The autosomes consist of 10 submetacentric/


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Figure 2. Chromosome spread (left) and karyotype (right) of male Mazani buffalo (Bubalus bubalis) 2n (diploid) = 50.

Figure 3. Chromosome spread (left) and karyotype (right) of female Mazani buffalo (Bubalus bubalis) 2n (diploid) = 50.


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metacentric (pair Nos. 1 to 5) and 38 telocentric chromosomes (pair Nos. 6 to 24). This is similar to Kenthao et al. (2012) that reported there are 10 submetacentric and 38 telocentric in Mehsani buffaloes. Also, this is not in agreement with Ali et al. (2012) that reported the autosomes of Pakistani river buffalo (Bubalus bubalis bubalis) contains 10 metacentric/submetacentric chromosomes whereas the rest of the autosomes were classified as acrocentric ones. Also this is not in agreement with Cribiu (1987) that reported the autosomes of Egyptian river buffalo (Bubalus bubalis bubalis) contains 5 pairs metacentric/submetacentric chromosomes and 19 pairs acrocentric, like Pakistani river buffalo. Maybe it is because they did not use of MicroMeasure and only predict from slides.

The pair of sex chromosomes was XX in the female and XY in the male. From karyotypes it appeared that the X was the largest telocentric while the Y was one of the smallest telocentric. It is in agreement with Kenthao et al. (2012) that

reported the X is largest telocentric and Y is small telocentric in Mehsani buffaloes from Thailand; However, the X chromosome is the largest acrocentric and Y chromosome is the acrocentric in Pakistani river buffalo (Ali et al., 2012), Indian river buffalo (Murali et al., 2009; Nair et al., 1986; Iannuzzi, 1994), Brazilian river buffalo (Pires et al., 1997) and Egyptian river buffalo (Cribiu, 1978). Also it is not in agreement with Meo et al. (2005) that reported the Y chromosome is acrocentric in river buffalo.

The mean of the short arm (Ls), long arm (Ll), chromosome length (LT), relative length (RL), arm ratio (Ll/Ls) and centromeric index (CI) of Mazani buffalo are shown in Table 1.

The relative length of chromosomes ranged between 7.20 and 2.17 in Mazani buffalo (Table 1), it means difference of range relative length (DRL) is 5.03. It is very different from DRL of Toda buffalo that is 3.95 (Murrali et al., 2009). All chromosomes from this population were found normal. The Mazani buffalo karyotype

Figure 4. Idiogram of Mazani buffalo using the relative length.


188

Table 1. Mean of the short arm (Ls), long arm (Ll), chromosome length (LT), relative length (RL), arm ratio (Ll/Ls) and centromeric index (CI) from metaphase chromosomes of Mazani male and female buffalo.

Chromosome pairs

Ls (µm)

Ll (µm)

LT (µm) CI RL

Arm ratio

(Ll/Ls)

Type of Chromosome

1 5.46 11.53 16.99 0.32 7.20 2.11 Submetacentric2 4.34 11.36 15.70 0.28 6.66 2.62 Submetacentric3 5.17 9.16 14.33 0.36 6.08 1.77 Submetacentric4 6.02 8.07 14.09 0.43 5.97 1.34 Metacentric5 5.16 7.49 12.65 0.41 5.36 1.45 Metacentric6 0.00 9.80 9.80 0.00 4.16 ∞ Telocentric7 0.00 9.56 9.56 0.00 4.05 ∞ Telocentric8 0.00 9.26 9.26 0.00 3.93 ∞ Telocentric9 0.00 8.93 8.93 0.00 3.79 ∞ Telocentric10 0.00 8.74 8.74 0.00 3.71 ∞ Telocentric11 0.00 8.46 8.46 0.00 3.59 ∞ Telocentric12 0.00 8.38 8.38 0.00 3.55 ∞ Telocentric13 0.00 7.88 7.88 0.00 3.34 ∞ Telocentric14 0.00 7.34 7.34 0.00 3.11 ∞ Telocentric15 0.00 7.32 7.32 0.00 3.10 ∞ Telocentric16 0.00 7.26 7.26 0.00 3.08 ∞ Telocentric17 0.00 7.03 7.03 0.00 2.98 ∞ Telocentric18 0.00 6.90 6.90 0.00 2.93 ∞ Telocentric19 0.00 6.40 6.40 0.00 2.71 ∞ Telocentric20 0.00 6.18 6.18 0.00 2.62 ∞ Telocentric21 0.00 5.75 5.75 0.00 2.44 ∞ Telocentric22 0.00 5.54 5.54 0.00 2.35 ∞ Telocentric23 0.00 5.31 5.31 0.00 2.25 ∞ Telocentric24 0.00 5.11 5.11 0.00 2.17 ∞ TelocentricX 0.00 14.52 14.52 0.00 6.16 ∞ TelocentricY 0.00 6.40 6.40 0.00 2.71 ∞ Telocentric


189

can be formula as follow:2n (50) = 4M + 6SM + 38 T + Sex chromosome

The idiogram of Mazani buffalo are presented in Figure 4 and it is similar to the idiogram of Toda buffalo from Indian (Murrali et al., 2009).

Many breeds of the buffalo live in South East of Asia, Australia, North Africa, South America, the Middle East and the Mediterranean coasts. Due to inadequacy of the cytogenetic study and chromosomal analysis of domestic buffaloes, it is not possible to determine the origin of buffaloes in various countries. According to available data, we know that the diploid number is 50 and NF or number of arms is 60 in the river buffalo.

In the future we expect an increase in the number of cytogenetic studies of many kind of buffaloes from many countries, and using the various chromosomal band techniques.

ACKNOWLEDGEMENTS

We are especially grateful to the experts who were integral partner in the preparation of facilities and also we must thank the Deputy and Laboratory staff at agricultural biotechnology research institute of Iran (ABRII).

CONFLICT OF INTEREST

The authors have declared that there is no conflict of interest.

REFERENCES

Ahmad, I., K. Javed and A. Sattar. 2004. Screening

of breeding bulls of different breeds through karyotyping. Pak. Vet. J., 24: 190-192.

Ali, A., M. Abdullah, K. Javed, M.E. Babar, H. Mustafa, N. Ahmad and M. Akhtar. 2012. Cytogenetic and genome studies in Pakistani buffalo (Bubalus bubalis) - A Review. J. Anim. Plant Sci., 22: 225-227.

Balakrishnan, C.R. and B.R. Yadav. 1984. Normal and abnormal chromosomes in the Indian river buffaloes. Buffalo Bull., 3: 13-17.

Balakrishnan, C.R., S. Kumar and B.R. Yadav. 1988. Cytogenetic, biochemical polymorphism and blood groups of the buffalo, p. 16-30. In Buffalo Production and Health: a Compendium of Research Work Done in Indian, Indian Council of Agricultural Research, New Delhi, India.

Bidhar, G.C., G.R. Pattnaik, P.K. Rao and B.N. Patro. 1986. Chromosome number and morphology of paralakhemundi buffaloes in Orissa. Buffalo Bull., 5(3): 54-56.

Cribiu, E.P., A. Obeidah and J. Boscher. 1978. The C-banding pattern of Egyptian water buffalo (Bubalus bubalis). Ann. Genet. Sel. Anim., 10(2): 271-274.

Dutt, M.K. and P. Bhattacharya. 1952. Chromosomes of the Indian water buffalo. Nature, 170(4339): 1129.

Fischer, H. and F. Ulbrich. 1978. Chromosomes of the Murrah buffalo and its crossbreds with the Asiatic swamp buffalo (Bubalus bubalis). J. Anim. Breed. Genet., 84(1-4): 110-114.

Guerra, M.D.S. 1986. Short communication reviewing the chromosome nomenclature of Leavan. Braz. J. genet., 4: 741-743.

Gupta, P. and S.P.R. Chaudhri. 1978. Robertsonian changes in the chromosomes of Indian Murrah buffalo, Bubalus bubalis. The


190

Nucleus, 21: 90-97.Halnan, C.R.E. 1976. A cytogenetic survey of 1101

Australian cattle of 25 different breeds. Ann. Genet. Sel. Anim., 8(2): 131-139.

Harisah, M., T.I. Azmi, M. Hilmi, M.K. Vidyadaran, T.A. Bongso, Z.M. Nav, V. Momongan and P.K. Baasrur. 1989. Identification of crossbred buffalo genotypes and their chromosome segregation patterns. Genome, 32(6): 999-1002.

Hasanzadeh, S. and S. Orojee. 2003. Gross, morphology, histology and histomorphometry of the ileum in river buffalo. Buffalo Journal, 19: 273-282.

Hasanzadeh, S. and S. Monazzah. 2011. Gross morphology, histomorphology and histomorphometry of the jejunum in the adult river buffalo. Iran. J. Vet. Res., 12: 99-106.

De Hondt, H.A. and S.A. Ghanam. 1971. Cytogenetic studies of the Egyptian water buffalo (Bubalus bubalis). Buffalo Journal, 1: 33-40.

Di Meo, G.P., A. Perucatti, S. Floriot, D. Incarnato, R. Rullo, A.C. Jambrenghi, L. Ferretti, G. Vonghia, E. Cribiu, A. Eggen and L. Iannuzzi. 2005. Chromosome evolution and improved cytogenetic maps of the Y chromosome in cattle, zebu, river buffalo, sheep and goat. Chromosome Res., 13(4): 349-355.

Huang, Y.J., M.O. Xie, Y.J. Liu and Z.S. Tan. 1987. Observation of G and C banding karyotypes in water buffaloes. Heriditas, 9: 13-16.

Iannuzzi, L. 1994. Standard karyotype of the river buffalo (Bubalus bubalis L., 2n=50) report of the committee for the standardization of banded karyotypes of the river buffalo. Cytogenet. Cell Genet., 67: 102-113.

Joshi, S.K. and M.G. Govindaiah. 1997. Karyological studies in South Kanara buffaloes of Karnataka. Indian Vet. J., 74: 1037-1039.

Kenthao, A., A. Tanomtong, P. Supanuam, C. Pinyoteppratan, P. Muangprom, P. Buranarom and L. Sanoamuang, 2012. Standardize karyotype and idiogram of Mehsani Buffaloes, Bubalus bubalis by conventional staining, GTG-Banding, CBG-Banding and AG-NOR Banding techniques. Buffalo Bull., 31: 24-39.

Khavary, H. 1978. Le caryotype normal du buffle d’Iran (Bubalus bubalis). Bull. Soc. Sci. Vet. Et. Med. Comparee., 80: 203-205.

Kumar, P. and B.R. Yadav. 1991. Comparative cytogenetical studies in Mehsana, Murrah and Surti buffaloes. Indian J. Dairy Sci., 46: 157-161.

Mirhoseinie, S.Z., S.M.F. Vahidie and B. Gharehyazie. 2005. Survey of efficiency of six microsatellite loci in Iranian indigenous cattle and buffalo populations. Iran. J. Biotechnol., 3: 41-47.

Miyake, Y., H. Kanagawa and T. Ishikawa. 1980. A chromosomal analysis on the G and C band staining techniques of the buffalo (Bubalus bubalis). Jpn. J. Vet. Res., 28: 122-128.

Murali, N., P. Devendran and S. Panneerselvan. 2009. Cytogenetic studies on the chromosomes of Toda buffaloes. Buffalo Bull., 28(2): 95-100.

Nair, P.G., M. Balakrishnan and B.R. Yadav. 1986. The Toda buffaloes of Nilgiris. Buffalo Juornal, 2: 167-178.

Naserian, A.A. and B. Saremi. 2007. Water buffalo industry in Iran. Ital. J. Anim. Sci., 6: 1404-1405.

Pires, R.M.L., R.H. Reichert and S. Kasahara.


191

1998. Cytogenetics of three breeds of river buffalo (Bubalus bubalis L.), with evidence of a fragile site on the X chromosome. Theriogenology, 49: 529-538.

Ramesha, K.P. and B.P. Hegde. 1992. Chromosomes of Surti and nondescript buffaloes of Kamataka. Indian Vet. J., 69: 34-37.

Romelt-Vasters, C., E. Sheurmann and M.R. Jainudeen. 1978. Chromosome of the Asian water buffalo (Bubalus bubalis). Jurnal Kajian Veteriner, 10: 8-14.

Rommelt, C. 1977. Karyotype identification by means of G and C banding techniques in swamp and murrah buffaloes. Thesis, Giessen-Jastus-Liebig University, Giessen.

Salerno, A., D. Valerio, A. Cargiulo and F. Scognamiglio. 1980. C- and G-banding patterns of the Italian buffalo chromosomes. Eur. Colloq. Cytogenet. Dom. Anim., 4: 378-385.

Scheurmann, E., H. Weisner, H. Fisher and M. Jainuddeen. 1974. The karyotype, C-bands and identification of the sex chromosomes in the Ceylon water buffalo. Giessneer Beitr. Erpath and Zuchthyg, 6: 1-7.

Toll, G.L. and C.R.E. Halnan. 1976a. The karyotype of the Australian swamp buffalo (Bubalus bubalis). Can. J. Genet. Cytol., 18: 101-104.

Ulbrich, F. and H. Fisher. 1967. The chromosome of the Asiatic buffalo (Bubalus bubalis) and African buffalo (Syncerus caffer). Z. Tierzuch. Zucht Biol., 83: 219-222.

Vijh, R.K., M.S. Tanita and R. Sahai. 1994. Translocation in Murrah buffalo. Indian J. Anim. Sci., 64: 534-538.

Yadav, B.R., C.R. Balakrishnan and O.S. Tomer. 1984. Chromosomal screening of male cattle and buffaloes. Indian J. Anim. Sci., 54: 519-523.


193

Original Article

ABSTRACT

Twenty four lactating Jaffrabadi buffaloes were randomized and blocked into four groups to receive commercial rumen bypass fat at 0, 10, 20 and 30 g/kg milk production besides meeting their nutrient requirements at ICAR 1985 feeding standards from calving to 180 day. All the buffaloes irrespective of group received bypass fat prior to calving 150 g day. Cost of feeding, cost of milk production, realizable receipts at farm and market prices were calculated and analyzed. Daily feed costs (Rs/head) in T1, T2, T3 and T4 were 96.56±3.80, 111.09±4.23, 106.61±4.58 and 117.47±4.76 respectively and it was statistically lower (P<0.05) in control than those in T2, T3 and T4 which were at par. Daily realizable receipts from sale of milk (Rs/head/day) on prevailing from price and on local market price were 128.58±8.86, 159.21±13.39, 122.94±11.27 and 140.97±7.29 and 205.73±14.17, 254.73±21.58, 196.71±18.03 and 225.54±11.67, respectively. Treatment groups did not differ significantly. Daily return over feed cost (Rs./Head) were 32.02±7.46, 48.12±9.90,16.33±7.51 and 23.49±3.35 on farm

prevailing price and 109.17±12.48, 143.65±17.91, 90.09±14.13 and 108.07±7.45 on local market price for T1, T2, T3 and T4 group respectively. Costs of milk production (Rs./lit.) during the entire experiment period were 15.25±0.85, 14.30±0.84, 17.95±1.43 and 16.73±0.32 in T1, T2, T3, T4 group of buffaloes differences being non significant. This finding indicated that 10 g supplementation of commercial bypass fat per kg milk production has significant realizable benefit as compared to other levels of supplementation of bypass fat.

Keywords: bypass fat, economics of feeding, Jaffrabadi buffalo, cost of milk production

INTRODUCTION

Ruminant production in country is based on crop residues due to shortage of green fodder and concentrates, hence, they fail to derive sufficient energy from these rations for productive purposes, resulting in lower production. This is true in early lactation in high producing buffaloes since the consumption is limited resulting in negative energy

ECONOMICS OF RUMEN BYPASS FAT FEEDING ON COST OF MILK PRODUCTION, FEEDING AND REALIZABLE RECEIPTS IN LACTATING JAFFRABADI BUFFALOES

H.H. Savsani1,*, K.S. Murthy2, P.U. Gajbhiye2, P.H. Vataliya5, K.S. Dutta1, M.R. Gadariya3 and A.R. Bhadaniya4

1Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh, Gujarat, India, *E-mail: [email protected] Breeding Farm, Junagadh Agricultural University, Junagadh, Gujarat, India3Department of Livestock Production Management, 4Department of Veterinary Pathology, 5College of Veterinary Science and Animal Husbandry, Junagadh Agricultural University, Junagadh, Gujarat, India


194

balance. High energy density rations containing rumen protected fats is a solution, though this advantageous feeding practice is not adopted widely under Indian conditions.

Rumen protected fats such as oil seeds, casein-formaldehyde protected fat, crystalline fat, fatty acyl amide’s hydrogenated tallow or triglycerides and calcium salts of long chain fatty acids (LCFA-Ca) are commonly used. LCFA-Ca are relatively less degradable in rumen (Elmeddah et al., 1991) and have the higher intestinal digestibility, besides serving as an additional source of calcium (Anonymous, 2002). Therefore, an experiment was conducted to find out the effect of bypass fat feeding on milk production economics in lactating Jaffrabadi buffaloes.


Twenty four lactating Jaffrabadi buffaloes (1-4 lactation, average body weight of 650 kg and 6 to 8 liter average milk production of previous lactation) from the buffalo herd of Cattle Breeding Farm of Junagadh Agricultural University, Junagadh were randomized and blocked into four groups of six each. All the experimental buffaloes were individually offered a basal diet of 10 Kg. seasonal green and mature pasture grass hay ad lib. Concentrate mixture and cottonseed meal (50:50 w/w) were offered to meet protein requirements (ICAR, 1998). Commercial bypass fat (MAGNAPAC) was provided to buffaloes 0,10 g, 20 g and 30 g/kg milk yield in T1, T2, T3 and T4, respectively. Bypass fat was offered 150 g to all the buffaloes ,irrespective of group prior to 15 days of calving. In all the buffaloes experimental differentiation of supplemental fat was initiated in the second week of lactation and continued up to

180 days of lactation. Since all the experimental animals were not available at a time, they were introduced in the experiment phase wise after calving. Cost of feeding and economics of milk production was worked out at farm prices as well as prevailing market prices and analyzed (Snedecor and Cochran, 1994). FCM was calculated according to Rice et al. (1970).


Mean total milk and FCM production (kg/day) during the experimental period were 6.43±0.44 and 6.63±0.49 in T1, 7.96±0.67 and 8.5±0.72 in T2, 6.15±0.56 and 6.86±0.64 in T3 and 7.05±0.36 and 7.99±0.48 in T4, respectively. Treatment differences were non significant (P>0.05) with regard to milk and FCM production, indicating higher level of bypass supplementation had no additional advantage. Average daily cost of feeding (Rs/day) and cost of milk production per liter of lactating Jaffrabadi buffaloes are given in Table 1. Overall feed cost per day (Rs) per buffalo was 96.56±3.80, 111.09±4.23, 106.61±4.58, 117.47±4.76 in T1, T2, T3 and T4 group respectively. Control feeding was significantly (P<0.05) lower compared to T2, T3 and T4 which were at par. Realizable receipts from sale of buffalo milk was calculated based on prevailing farm prices which was Rs 20/liter and also based on the prevailing market price during time of the experiment which was Rs 32/liter. During the entire experiment period realizable receipts per day per buffalo based on the milk price of Rs 20 per liter is given in Table 1. Mean realizable receipts (Rs/day) for the entire period were 128.58±8.86, 159.21±13.39, 122.94±11.27 and 140.97±7.29


195

respectively, for T1, T2, T3 and T4 with non significant differences. Mean return over feed cost (Rs/buffalo) was 32.02±7.46, 48.12±9.90, 16.33±7.51 and 23.49±3.35, respectively ,for T1, T2, T3 and T4, the means being significant (P<0.05). T1, T3 and T4 were at par while T1 and T2 were at par. Considering the market price of milk, mean realizable receipt for per day per animal was Rs 205.73±14.17, 254.73±21.58, 196.71±18.03 and 225.54±11.67 in T1, T2, T3 and T4, respectively. Mean daily return over feed cost at market price were 109.17±12.48, 143.65±17.91, 90.09±14.13 and 108.07±7.45 in T1, T2, T3 and T4, respectively, and the differences were non significant Considering the cost of milk production during entire experimental period overall means were 15.25±0.85, 14.30±0.84, 17.95±1.43 and 16.73±0.32 for T1, T2, T3 and T4 groups, respectively. All the treatments were at par (Table 1). Cost of feeding was 15.04%, 10.40% and 21.66% higher in T2, T3 and T4 groups of animals, respectively compared to control. At farm price, realizable receipts were higher by 23.82% for T2 group of animals and 9.64% for T4 group of animals, However T3 group of animals realized lesser than 4.34% realizable receipt as compared to control. Return over feed cost was 50.28% higher in T2 group of buffaloes at farm prices. However it was lower by 49% and 26.64% in T3 and T4 compared to control. Considering market price similar trend was observed in terms of realizable receipt as well as return over feed cost. Cost of milk production was 6.22% lower in T2 group of buffaloes compared to T1 group of buffaloes. When bypass fat was offered at higher level, cost of milk production increased by 17.70% and 9.70% in T3 and T4 groups of animals,

respectively. Net returns during the entire experimental period under four treatment groups is given in Table 2. In the present experiment it is evident that T2 group of buffaloes yielded higher returns over feed cost and also higher realizable receipts both at farm and market price. The net return per buffalo under T2 group was Rs. 4409.41 and Rs. 7752.75 at farm and market price determination. Supplementing higher levels of bypass fat at 20 g, 30 g per liter of milk production did not have any positive economic benefits. Costs of feeding cited by different research worker pertain to those respective periods during which experiment was conducted. Any comparison in terms of economics of feeding may not complement present findings.

Garg et al. (2002) reported increase income of Rs 10.18 on feeding 1 kg protected fat and protein supplement per cow per day. Garg et al. (2007) observed an increase in net daily income by 4.92% and 11.20% by feeding 0.5 kg and 1 kg bypass fat and protein supplementation to lactating cows compared to control. Cost of feeding per kg milk and kg FCM production were Rs. 7.65 and 7.41 and 7.33 and 6.90, respectively in cross bred cows that were offered control and by pass diets (Shankpal et al., 2009). Vidhate et al. (2006) observed that by feeding bypass fat 150 g per day to cross bred cows, milk production though decreased, proportionate increase in fat content exhibited additional benefits of Rs. 18.21/animal/day.

Bhorania (2009) reported that daily feed cost (Rs. Per head) in T1 and T2 was 87.63±3.17 and 95.34±1.87, respectively and was statistically higher (P<0.05) in T2 than T1 group, when commercial bypass fat was offered as a supplement compared to unfed group. The data on daily realizable receipt from sale of milk (Rs. Per head)


196

Tabl

e 1.

Eco

nom

ics o

f fee

ding

of e

xper

imen

tal a

nim

als.

Trea

tmen

tD

aily

feed

cost

(Rs/

head

)

Rea

lizab

le r

ecei

pts a

tfa

rm p

reva

iling

pri

ce(R

s 20

/ lite

r)

RO

FC a

t far

mPr

evai

ling

pric

e(2

0 R

s / li

ter)

Rea

lizab

le r

ecei

pts a

tlo

cal m

arke

t pri

ce(R

s 32

/ lite

r)

RO

FC a

t loc

al

mar

ket p

rice

(32

Rs /

lite

r)

Cos

t of m

ilk

prod

uctio

n (K

g/da

y)T

196

.56c ±

3.80

128.

58±8

.86

32.0

2ab±7

.46

205.

73±1

4.17

109.

17±1

2.48

15.2

5±0.

85T

211

1.09

ab±4

.23

159.

21±1

3.39

48.1

2a ±9.

9025

4.73

±21.

5814

3.65

±17.

9114

.30±

0.84

T3

106.

61ab

c ±4.

5812

2.94

±11.

2716

.33b ±

7.51

196.

71±1

8.03

90.0

9±14

.13

17.9

5±1.

43T

411

7.47

a ±4.

7614

0.97

±7.2

923

.49b ±

3.35

225.

54±1

1.67

108.

07±7

.45

16.7

3±0.

32S.

Em

.±4.

3510

.49

7.44

16.7

913

.52

0.94

C.D

. at 5

%12

.85

NS

21.9

5N

SN

SN

SC

.V. %

9.89

18.6

460

.77

18.6

429

.39

14.4

1

Mea

ns in

a c

olum

n w

ith d

iffer

ent s

uper

scrip

ts d

iffer

sign

ifica

ntly

(P<0

.05)

, NS:

Non

-sig

nific

ant.

RO

FC=R

etur

n ov

er fe

ed c

ost.

Tabl

e 2.

Net

retu

rn d

urin

g ex

perim

enta

l fee

ding

.

Sr. N

o.Pa

rtic

ular

T1

T2

T3

T4

1To

tal F

eed

cost

(Rs./

Hea

d)17

464.

7220

218.

3819

403.

0221

379.

542

Rea

lizab

le R

ecei

pt fr

om sa

le o

f milk

(Rs./

Hea

d) 3

2 R

s.37

442.

8646

360.

8635

801.

2241

048.

283

Rea

lizab

le R

ecei

pt fr

om sa

le o

f milk

(Rs./

Hea

d) 2

0 R

s.23

401.

5628

976.

2222

375.

0825

656.

544

Ret

urn

over

feed

cos

t (R

s./ H

ead)

32

Rs.

1977

8.14

2614

2.48

1639

8.2

1966

8.74

5R

etur

n ov

er fe

ed c

ost (

Rs./

Hea

d) 2

0 R

s.59

36.8

487

97.8

429

72.0

642

77.0

06

Diff

eren

ce in

RO

FC o

ver c

ontro

l – 3

2 R

s.-

+616

4.34

-357

9.94

-309

.40

7D

iffer

ence

in R

OFC

ove

r con

trol –

20

Rs.

-+2

821

-296

4.78

-165

9.89

8Sa

ving

in fe

ed c

ost d

ue to

impr

oved

serv

ice

perio

d ov

er c

ontro

l (R

s./ H

ead)

-15

88.4

111

05.6

1-9

46.2

89

Net

Def

eren

ce R

s. 32

-77

52.7

5-2

474.

32-1

255.

6810

Net

Def

eren

ce R

s. 20

-44

09.4

1-1

859.

16-2

606.

17


197

was 168.51±12.6 and 197.04±11.29 in T1 and T2 groups, respectively and the treatment group differed (P<0.05) from each other. Accordingly, the daily return over feed cost (Rs. per head) was 80.88±7.27 and 101±6.50 in T1 and T2 groups, respectively. The same was significantly higher (P<0.05) in T2 as compared to T1 group. The present findings are in consonance with the above scientific reports.

CONCLUSION

In present experiment, higher level of bypass fat at 20 g and 30 g did not seem to have any positive effect on daily realizable receipt and return over feed cost and cost of feeding per liter of milk production. Supplementation of 10 g bypass fat per liter per day was found to be effective in terms of realizable receipt and ROFC in lactating Jaffrabadi buffaloes.

REFERENCES

Anonymous. 2002. EnertiaPFA calcium salts of palm fatty acids (PFA), Rumen Bypass Fat. The Official Answer Guide. ADM Animal Health and Nutrition, 1000 N. 30th Quincy, IL 62301, 877-236-2460.

Association of Official Analytical Chemists, AOAC. 1995. Official Methods of Analysis, 16th ed. Association of Official Analytical Chemists, Washington, DC, USA.

Bhoraniya, V.P. 2009. Influence of supplementing bypass fat during early lactation on milk production in buffaloes. M.V.Sc. Thesis, Anand Agricultural University, Anand.

Elmeddah, Y., M. Doreau and B. Michalet-

Doreau. 1991. Interaction of lipid supply and carbohydrate in the diet of sheep with digestibility and ruminal digestion. J. Agric. Sci., 116: 437-445.

Garg, M.R., P.L. Sherasia and B.M. Bhanderi. 2007. Effect of feeding different levels of bypass fat/protein supplement on milk yield and milk composition in crossbred cows. In Proceedings of International Tropical Animal Nutrition Conference. National Dairy Research Institute, Karnal, India.

Garg, M.R., P.L. Sherasia, B.M. Bhanderi, S.K. Gulati and T.W. Scott. 2002. Effect of feeding rumen protected nutrients on milk production in crossbred cows. Indian J. Anim. Nutr., 19: 191-198.

Goff, J.P. and R.L. Horst. 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci., 80(1): 260-268.

Indian Council of Agriculture Research, ICAR. 1998. Nutrient Requirement of Livestock and Poultry. Second Revised Edition. Indian Council of Agriculture Research, Krishi Anusandhan Bhawan, PUSA New Delhi, India.

National Research Council, NRC. 2001. Nutrient Requirements of Dairy Cattle, 7th ed. National Research Council, National Academy Press, Washington, DC.

Rice, V.A., F.N. Andrew, E.J. Warnick and J.E. Legates. 1970. Breeding and Improvement of Farm Animals, 6th ed. Tata Mc Graw Hill Publishing Co., Bombay, India.

Shankhpal, S.S., R.S. Gupta, S. Parnerkar and A.J. Dharni. 2009. Effect of supplementation of bypass fat on milk production and nutrient utilization in lactating cows. In Proceedings of Animal Nutrition Association World


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Conference, New Delhi, India. Snedecor, G.W. and W.G. Cocharan. 1994.

Statistical Methods, 8th ed. Affiliated East-West Press Private Limited, New Delhi, India.

Talpatra, S.K., S.C. Ray and K.C. Sen. 1940. The analysis of mineral constituents in Biological materials. Indian J. Vet. Sci., 10: 243-258.

Vidhate, P.G., R.D. Kokane and S.T. Hande. 2006. Economic Impact of feeding By-Pass Fat in Crossbred Cows. Journal of Bombay Veterinary College, 14(1-2): 68-72.


199

Original Article

ABSTRACT

Buffalo mange is a contagious skin disease caused by a variety of parasitic mites burrowing in or living on the skin. A female Murrah buffalo was presented with a history of inappetence, sudden decrease in milk yield, bilateral lameness of forelimbs with local hair loss and pruritis. Skin scrapings examination revealed presence of both Sarcoptes and Psoroptes mites. A part of the skin scrapings inoculated on various culture media and stained by Gram’s staining showed gram positive cocci bacteria in clumps. Further identification of bacteria by various biochemical tests confirmed the presence of Staphylococcus aureus as a secondary invader. Antibiotic sensitivity test was performed using eight different commonly used antibiotics. Haematology revealed reduced haemoglobin, PCV and TEC values, leucocytosis, neutrophilia and eosinophilia. The buffalo was treated with 1% Ivermectin at 200 µg/kg body weight, subcutaneously once a week for three weeks, Enrofloxacin at 5 mg/kg body weight, intramuscularly once a day for five days and Meloxicam at 0.5 mg/Kg body weight, once a day, intramuscularly for 5 days. Deltamethrin was also applied to the surrounding environment twice at a two week interval. The buffalo showed significant improvement after the treatment.

Keywords: mange, buffalo, Sarcoptes, Psoroptes, Staphylococcus aureus

INTRODUCTION

Mange is a widespread contagious skin disease and appears to be one of the most important skin diseases of buffalo in tropical and subtropical countries (Jabeen et al., 1998). Water buffaloes (Bubalus bubalis) are infected mainly with Sarcoptes, Psoroptes, and Chorioptes species of mange mites (Afzal et al., 1995).

Sarcoptes scabiei, a burrowing mite causes scabies in humans or sarcoptic mange in a number of animals through host-adapted variants. It generally affects sparsely haired parts of the body. Sarcoptes scabiei var. bubalis is the cause of sarcoptic mange in buffaloes. The disease is sometimes characterized by presence of skin lesions much more severe than other forms of mange and may involve the entire body surface of bovine in a period as short as 6 weeks (Radostits et al., 2007). Psoroptes mites are superficial skin parasites which generally live on the skin of parts of the body well covered with hairs. Infestation may be chronic or even subclinical and localized, often in the ear of the host, or it may be acute and more generalized over the entire body, when it is

CONCURRENT SARCOPTIC AND PSOROPTIC MANGE COMPLICATED WITH STAPHYLOCOCCUS AUREUS IN A MURRAH BUFFALO (BUBALUS BUBALIS)

R.L. Rakesh1,*, K. Mahendran2, K. Karthik3, Priyanka2, A.G. Bhanuprakash2 and V.K. Gupta2

1Division of Parasitology, *E-mail: [email protected],2Division of Medicine, 3Division of Bacteriology and Mycology, Indian Veterinary Research Institute, Izatnagar, Uttar Pradesh, India



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described as psoroptic mange (Bates, 1999). P. ovis and P. natalensis have been reported to cause psoroptic mange in buffaloes (Gill et al., 1989; Sreedevi et al., 2010). Chorioptes bovis is another non-burrowing mite very occasionally reported to cause chronic mange in buffaloes (El-Khodery et al., 2009).

Buffaloes infested with mange usually suffer from intense pruritus and react by vigorously scratching, biting and rubbing against objects, which can cause injuries that can be infected with secondary bacteria leading to serious complications. Thus buffalo mange can severely affect the profitable buffalo production due to dermatitis, hide damage, decreased milk and meat production, reduced performance and sometimes even mortalities in severe complicated cases (Tikaram and Ruprah, 1986). Diagnosis of mange in domestic animals is based on clinical manifestations and the demonstration of mites or their developmental stages in host skin scrapings (Kettle, 1995).

The present paper describes the clinical manifestations and successful therapeutic management of concurrent sarcoptic and psoroptic mange complicated with secondary invasion by Staphylococcus aureus causing cellulitis and resulting in lameness of the affected limbs in a Murrah buffalo (Bubalus bubalis).


History and Clinical Examination: A female Murrah buffalo aged 4 years, housed in the cattle and buffalo farm of Indian Veterinary Research Institute, Izatnagar, Bareilly (India) was presented with a history of inappetence, sudden decrease in milk yield, swollen forelimbs, pruritis

and lameness. Clinical manifestations included marked swelling of the forelimbs especially the right limb with alopecia, crusts and scab formation. In some areas, the affected dermis exuded serum and was covered with granulation tissue (Figure 1). In vital parameters, rectal temperature was increased and heart rate, respiration rate and pulse rate were normal. The animal was dull, weak, unable to stand for long time and gait was altered.

Parasitological examination: Deep skin scrapings from the periphery of the lesions were collected in a test tube containing 3 ml of 10% potassium hydroxide solution. After gentle heating for about 3 minutes, the test tube was centrifuged at 3,000 rpm for 5 minutes. A drop of the sediment was put on a clean glass slide and examined under microscope for the presence of mites. The mites found were identified by morphological characteristics of the species (Soulsby, 1982; Kettle, 1995). The skin scrapings were examined every week for 4 weeks to evaluate efficacy of treatment.

Microbiological examination: A part of the skin scrapings collected aseptically into a sterile container was inoculated on nutrient agar, blood agar and Sabouraud dextrose agar (SDA) separately and incubated at standard conditions. From the pure culture isolated on blood agar which was having hemolysis pattern with golden yellow colonies, it was sub-cultured on to mannitol salt agar. Bacterial colony from the culture was also stained by Gram’s staining. Further, for specific identification of bacterial isolates, biochemical tests like catalase test, coagulase test, DNAse test and oxidase tests were performed (Cowan and Steel, 1965; Schleifer and Kloos, 1975; Thaker et al., 2013). Isolates were also subjected to in-vitro antibiotic susceptibility test (ABST) using eight different commonly used antibiotics: Ampicillin, Amoxycillin, Tetracyclin,


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Gentamicin, Enroflaxacin, Streptomycin, Amikacin and Ciprofloxacin (Himedia, India) (Bauer et al., 1966).

Haematological examination: Blood samples were collected by jugular venipuncture. About 5 ml of blood sample was collected in a clean glass vial using EDTA as anticoagulant for haematological examination. In another vial, 5 ml of blood was collected for harvesting serum sample. A complete haematological examination was carried out as per standard techniques (Jain, 1986). Total protein, albumin and globulin concentration in the serum was determined by using standard biochemical procedures. The blood and serum examination were repeated after 28 days to determine improvement after treatment.

Treatment: The buffalo was treated with 1% Ivermectin at 200 µg/kg body weight, subcutaneously once a week for three weeks, Enrofloxacin at 5 mg/kg body weight, intramuscularly once a day for five days and Meloxicam at 0.5 mg/Kg body weight, once a day, intramuscularly for 5 days. Adjunct to the drugs, Deltamethrin was applied to the surrounding environment twice at a two week interval. Follow up for observation of response to therapy was carried out regularly.


Based on the history and clinical signs, it was diagnosed as a case of buffalo mange. Both Sarcoptes and Psoroptes mites were identified on parasitological examination of the skin scrapings. The smaller mites were roughly circular and had a finely striated cuticle. All their legs were short and the third and fourth pairs did not project beyond the margin of the body. The tarsi of the first, second

and fourth pair of legs in the male and the first and second pair of legs in the female ended in suckers whereas the remaining pairs ended in bristles. The pedicels which bore these suckers or bristles were unsegmented. Their anus was located terminally. These features indicated them as Sarcoptes scabiei var. bubalis (Figure 2). The larger mites were oval; all their legs were long and projected beyond the margin of the body. Their pedicels were long, segmented and bore suckers on the first, second, and third pairs of legs in the male and on the first, second, and fourth pairs of legs in the female. Based on these morphological features they were identified as Psoroptis natalensis (Figure 3). Thus, the buffalo was confirmed to be suffering from both sarcoptic and psoroptic mange (Randhawa et al., 1997; Ramprabhu et al., 2001).

Microbiological examination revealed that there were similar golden yellow colonies on blood and nutrient agar but no growth on SDA. Colonies on blood agar showed hemolysis. Suspecting for Staphylococcus, single colonies were plated on Mannitol salt agar which showed yellow colonies indicating it as S. aureus (Figure 4). Mannitol salt agar is selective for Staphylococcus and S. aureus produce yellow colonies. Further confirmation was done with biochemical tests which showed catalase positive and oxidase negative. There was DNAse and coagulase production. These tests confirm that the isolate was a pathogenic Staphylococcus aureus which can produce disease. ABST performed showed highest zone of inhibition around discs containing Ampicillin followed by Enrofloxacin.

On Haematology, reduction in PCV, Hb, TEC and lymphocytes and increase in TLC, neutrophils and eosinophils were noticed. This finding is in agreement with many workers (Shanthkumar and Suryanarayana, 1995; Dimri et al., 2007; Vishe et al., 2012). Reduced PCV,


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Figure 1. Photograph showing swollen forelimbs, alopecia and moist exudation

Figure 2. Photograph showing Sarcoptes scabiei var. bubalis mite

Figure 3. Photograph showing Psoroptis natalensis mites (male and female in copulation)

Figure 4. Photograph showing S.aureus colonies on Mannitol Salt Agar


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Table 1. Haematological parameters before and after treatment.

Blood parameter Pre-Treatment Post-TreatmentPacked Cell Volume (%) 26 34Haemoglobin (g/dl) 9.0 11.0Total Erythrocyte Count (106/mm3) 5.32 6.54Total Leukocyte Count (103/mm3) 12.98 9.68Differential Leukocyte Count:

a) Lymphocytes (%) 50 64b) Monocytes (%) 5 4c) Neutrophils (%) 34 25d) Eosinophils (%) 10 6e) Basophils (%) 1 1

Total Protein (g/dl) 6.5 8.0Albumin (g/dl) 2.5 3.3Globulin (g/dl) 3.9 4.6Albumin Globulin Ratio (A:G) 0.64 0.72

TEC and Hb values were probably due to the decreased erythroid elements in blood (Vishe et al., 2012). Lymphopenia was probably due to immunosupression. Leukocytosis and neutrophilia were due to secondary bacterial infection. Eosinophilia noticed in present case might be linked to antigen-antibody interactions in tissues rich in mast cells, such as skin and as well as to protracted host-parasite reaction (Ramprabhu et al., 2001). Additionally, the affected buffalo showed hypoproteinaemia and decrease in A:G ratio. This correlates with the observations of Randhawa et al. (1997) and Ramprabhu et al. (2001). Decrease in albumin represents decreased albumin synthesis. A possible cause for this could be the increased rate of acute phase proteins due to skin inflammation. The low level of protein could also be due to loss of serum proteins from the affected dermis (Ramprabhu et al., 2001).

After treatment, the skin scrapings examination showed progressive decrease in the

number of mites found, but a few live mites were still present on day 7 prompting further two doses of Ivermectin. Meanwhile, antibiotic treatment with Enrofloxacin was continued daily for 5 days, as the bacteria were found highly susceptible to it in ABST. This brought about rapid clinical improvement. By day 21, no more live mites were observed in the skin scrapings. By day 28, the lesions had almost completely healed, hair had grown, and the skin became glossy and regained normal colour and texture. The haematological examination carried out on day 28 revealed remarkable improvements in all the parameters tested, almost returning to their normal physiological range. Similar results were obtained by other workers (El-Khodery et al., 2009; Kotb and Abdel-Rady, 2011), who also found treatment of animal’s environment with Deltamethrin as an adjunct to administration of Ivermectin to be the best protocol for eradication and prevention of re-infestation with mange mites in buffaloes.


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ACKNOWLEDGEMENT

The authors are thankful to the Director, Indian Veterinary Research Institute, Izatnagar, for the facilities provided.

REFERENCES

Afzal, M., A. Hussain, M.S. Mian, A. Muneer and A.R. Rizwi. 1995. Incidence of ectoparasites and its chemotherapy. J. Anim. Health Prod., 5: 146-149.

Bates, P. 1999. Inter- and intra-specific variation within the genus Psoroptes (Acari: Psoroptidae). Vet. Parasitol., 83: 201-217.

Bauer, A.W., W.M.M. Kirby, J.C. Sherris and M. Tarek. 1966. Antibiotic susceptibility testing by a standardized single disk method. Am. J. Clin. Patho., 45(4): 493-496.

Cowan, S.T. and K.J. Steel. 1965. Manual for the Identification of Medical Bacteria, 1st ed. Cambridge University Press, London. 217p.

Dimri, U., R. Ranjan, S.K. Singh, M.C. Sharma, D. Swarup, P. Dwivedi, A.K. Sharma and M. Kataria. 2007. Clinico-pathological and haemato-biochemical changes in buffaloes with sarcoptic mange. Indian J. Vet. Pathol., 31: 160-162.

El-Khodery, S.A., M. Ishii, S.A. Osman and M.H. Al-Gaabary. 2009. Comparative therapeutic effect of moxidectin, doramectin and ivermectin on psoroptes mites infestation in buffalo (Bubalus bubalis). Trop. Anim. Health Prod., 41(7): 1505-1511.

Gill, B.S., J. Singh, B.S. Gill, A. Singh, S.S. Khehra, A. Rai and O. Hussain. 1989. Efficacy of ivermectin against mange and gastrointestinal nematodes of buffalo

(Bubalus bubalis). Vet. Parasitol., 31: 141-147.

Jabeen, F., N. Ahmed, M.A. Chaudhry and I. Javed. 1998. Epidemiology and treatment of sarcoptic mange in buffalo calves around Lahore. Pak. V. J., 18(1): 39-42.

Jain, N.C. 1986. Schalm’s Veterinary Haematology, 4th ed. Lea and Febiger, Philadelphia. 1221p.

Kettle, D.S. 1995. Medical and Veterinary Entomology, 2nd ed. Cab International, Wallingford. 725p.

Kotb, S. and A. Abdel-Rady. 2011. Epidemiological studies of Egyptian buffaloes mange with special reference to efficacy of different therapeutic trials for treatment of mange. Ass. Univ. Bull. Environ. Res., 14(1): 9-22.

Radostits, O.M., C.C. Gay, K.W. Hinchcliff and P.D. Constable. 2007. Veterinary Medicine: a Textbook of the Diseases of Cattle, Horses, Sheep, Pigs and Goats, 10th ed. Saunders Elsevier, Philadelphia. 2156p.

Ramprabhu, R.A., M. Subramanian, A.P. Nambi, S. Prathaban and P. Dhanapalan. 2001. Concurrent sarcoptic and psoroptic mange infestation in Bubalus bubalis. Vet. Arhiv., 71: 53-56.

Randhawa, C.S., R.S. Brar, D.R. Sharma and S.S. Randhawa. 1997. Biochemical responses in mixed chronic psoroptic and sarcoptic mange of buffaloes (Bubalus bubalis). Trop. Anim. Health Prod., 4: 253-254.

Schleifer, K.H. and W.E. Kloos. 1975. Isolation and characterization of staphylococci from human skin. I. Amended descriptions of Staphylococcus epidermidis and Staphylococcus saprophyticus and descriptions of three new species: Staphylococcus cohnii, Staphylococcus haemolyticus, and Staphylococcus xylosus.


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Int. J. Syst. Bacteriol., 25: 50-61.Shanthkumar, G. and C. Suryanarayana. 1995.

Clinicobiochemical and therapeutic studies on mange in buffalo calves. Indian Vet. J., 72: 77-79.

Soulsby, E.J.L. 1982. Helminths, Arthropods and Protozoa of Domesticated Animals, 7th ed. Bailliere Tindall, London. 809p.

Sreedevi, C., G.S.N. Murthy and N.L. Rani. 2010. Therapeutic management of Psoroptes natalensis in buffaloes (Bubalus bubalis). Buffalo Bull., 29(4): 304-307.

Thaker, H.C., M.N. Brahmbhatt and J.B. Nayak. 2013. Isolation and identification of Staphylococcus aureus from milk and milk products and their drug resistance patterns in Anand, Gujarat. Vet. World, 6(1): 10-13.

Tikaram, S.M. and N.S. Ruprah. 1986. Incidence of sarcoptic mange in buffaloes in India. Trop. Anim. Health Prod., 18: 86-90.

Vishe, H.P., K. Pawar, H.K. Gupta and G.S. Rao. 2012. Prevalence and hemato-biochemical studies in parasitic and non parasitic dermatological disorders in Surti buffalo and buffalo calves. Vet. World, 5(4): 230-235.


207

Original Article

ABSTRACT

Murrah, also known as “black gold of India’ from Haryana is the major source of germ-plasm for quality up-gradation of other low producing buffaloes in India. An “Integrated Murrah Development Scheme” (IMDS) has been implemented in Haryana to conserve the top quality Murrah germplasm. The present study is conducted in Haryana to analyze the various constraints faced by the beneficiaries of IMDS. The responses were taken from 160 beneficiaries from a total of 32 villages from 8 blocks of 4 districts of Haryana. Study revealed that “Concerned officials are not much interested to visit the area and conducting regular meetings with beneficiaries” and “delay from project personnel in sanctioning the funds” were the major administrative constraints in order of severity. Under technical constraints “lack of knowledge about scientific feeding, breeding, health-care and management practices of buffaloes” followed by “lack of awareness about the IMDS” were the most severe. “Concerned staff was not taking much interest in imparting awareness training camps”

followed by “Practical demonstration facilities are inadequate under IMDS” were important infrastructural and operational constraints. “Delay in getting incentive money for the owner of animal under scheme” and “preference for jobs rather than dairy-based self-employments” were the economic and socio-psychological constraints, respectively. Difficulty in maintaining records due to illiteracy and lack of awareness, lack of decision-making ability were other miscellaneous constraints faced by beneficiaries of IMDS. To run this scheme in sustainable manner for conservation of Murrah breed, there is dire need to remove of these constraints on priority.

Keywords: constraints, administrative, technical, infrastructural and operational, economic, socio-psychological, IMDS

INTRODUCTION

Haryana state is the major source of germ-plasm and breeding stock for up-gradation of their

ANALYSIS OF CONSTRAINTS FACED BY BENEFICIARIES OF INTEGRATED MURRAH DEVELOPMENT SCHEME (IMDS) IN HARYANA

Y.S. Jadoun1,*, S.K. Jha2, Pragya Bhadauria3 and Rajiv Baliram Kale4

1Department of Veterinary and Animal Husbandry Extension Education, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, *E-mail: [email protected] of Dairy Extension, National Dairy Research Institute, Karnal, Haryana, India3Indian Council of Agricultural Research-Agricultural Technology Application Research Institutes, Ludhiana, Punjab, India4Agricultural Extension, Indian Council of Agricultural Research-Agricultural Technology Application Research Institutes, Jodhpur, Rajasthan, India


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low producing buffalo population in other states of India. But these buffaloes of Haryana having major share of Murrah breed are alarmingly declining. The 18th Livestock Census of India (2007) revealed that there were 59, 53,000 buffaloes in Haryana, against a figure of 60, 35,000 as reflected in the earlier livestock census. Moreover, about 100,000 high-yielding buffaloes per annum in their prime age of production have been sent to slaughter houses in metros and other cities without leaving any progeny behind. In its home tract, a genetic drain in the recent years has been a cause of concern.

This situation has left the Murrah population in a quagmire of genetic stagnation. The fast genetic improvement of Murrah is not only the top priority for the state but also a national concern. The top quality Murrah germplasm presently available in the state needs to be identified through performance-recording to preserve and multiply. For the conservation of Murrah germplasm, the Department of Animal Husbandry and Dairying, Government of Haryana, in collaboration with Department of Animal Husbandry and Dairying, Government of India started an “Integrated Murrah Development Scheme” (IMDS) in the year 2002 to 2003 (Department of A.H. and Dairying, Government of Haryana).With the assumption that Integrated Murrah Development Scheme (IMDS) for rescuing this germplasm would add to germ-pool for future breeding. But the success of any scheme is depends on smooth functioning of scheme without any constraints to the beneficiaries. Hence, it was needed to take feedback of the beneficiaries regarding constraint for getting full benefits from the scheme. For the purpose of this study, the term ‘constraints’ was operationalized as, all those factors, which hinder the process of effective implementation of any dairy

developments programme or scheme, as faced by beneficiaries. The analysis of these constraints is very essential to overcome them for running the scheme sustainably. Keeping this in view the present study was conducted to analyze various administrative, technical, infrastructural and operational, economic, socio-psychological and miscellaneous constraints faced by beneficiaries of IMDS.


The present study was carried out in Haryana state purposively as it is ‘home tract’ of Murrah buffaloes and IMDS has been specifically implemented in the state. Haryana state comprises 21 district divided into the four divisions. Four districts, namely Kurukshetra, Mahendragarh, Bhiwani and Jhajjar were selected, purposively, from each administrative division (i.e., Ambala, Gurgaon, Hisar, Rohtak), thereby covering whole Haryana state, as based on maximum number of Murrah buffaloes under Integrated Murrah Development Scheme (IMDS) in their respective division. Out of four districts, two blocks were selected from each district. Further, four villages were selected, purposively, from each block. Thus, a total of 32 villages from 8 blocks of 4 districts of Haryana state were the locale of study. In order to find out the constraint faced by beneficiaries of IMDS, five beneficiary Murrah owners from each village were selected, thereby making the sample size of 160 respondents for the study. To analyze various constraints faced by beneficiaries of IMDS, an interview schedule was developed under sub-heads namely, administrative, technical, infrastructural and operational, economic, socio-psychological and miscellaneous constraints. The


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data were collected by face to face interview using pre-tested structured schedule. These constraints were ranked on the basis of mean score as obtained by Garrett’s ranking technique.

As per Garrett’s ranking technique, the respondents were asked to enumerate and assign ranks to different constraints, which were used for prioritization of constraints. The orders of merit as given by the respondents were converted into ranks, by using the following formula:

Percent position =

Where, Rij = Rank given for ith problem by jth

individual.Nj = Number of problems ranked by the

jth individual.

The percent position of each rank was then converted into scores, by referring to the table, as given by Garrett. The scores of individual respondents for a particular problem were added and divided by the total number of respondents. The mean scores for all the constraints were arranged in descending order and thus, ranks were assigned to prioritize the constraints.


The constraints as faced by the beneficiary dairy farmers of IMDS in accessing the facilities provided under scheme have been categorized and discussed under following sub- heads.

Administrative constraintsAmong the administrative constraints,

the data presented in Table 1, clearly revealed

that “concerned officials are not much interested to visit the area and conducting regular meetings with beneficiaries” (mean score: 90.45) was ranked first by the beneficiaries, which is supported by the findings of Singh (2006) and Tiwari et al. (2003). The next constraint, in the order of seriousness was found to be: “delay from project personnel in sanctioning the funds” (mean score: 88.36).

“Lack of proper linkages/channel for the owners of buffaloes under scheme for marketing of their animals and its milk & milk products” (mean score: 85.30) followed by “Concerned officials are not communicating the information properly, regarding available facilities under project” (mean score: 60.05) were the other constraints faced by the beneficiaries in order of severity. These constraints may be sort out among A.H. officials, beneficiaries and policy-makers through regular interactions.

Technical constraintsThe data presented in Table 2. revealed

that “lack of knowledge about scientific feeding, breeding, health-care and management practices of buffaloes” (mean score: 92.67) was faced as the most serious constraint, it might be due to lack of training facilities regarding buffalo management practices under IMDS. The second rank was given to “lack of awareness about the IMDS” (mean score: 84.34), which may be due to lack of awareness camps regarding facilities provided under IMDS.

The other technical constraints such as, “Repeat breeding” (mean score 79.29) was ranked third by the beneficiaries, which is in line with the findings of Sharma et al. (2010), it might be because of the lack of trained/ skilled staff and A.I. was not practiced in time by the A.H. personnel. “Less qualified staff working at A.I. centers (mean score 62.04) was ranked fourth by the beneficiaries.

100 ( R ij – 0.50) ( N j)


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“Veterinary doctor/ A.H. officials do not visit the area regularly (mean score 59.54)” was ranked fifth by the beneficiaries, which is in agreement with the findings of Singh et al. (2004) and Tiwari et al. (2003). These constraints may be sorted out by providing regular health check-up and awareness camps by A.H. officials, at regular intervals and recruitment of skilled A.H. personnel as per the requirement of the project.

Infrastructural and operational constraintsIt was observed from the Table 3

“concerned staff was not taking much interest in imparting awareness training camps” (mean score: 91.17) about IMDS which was reported by majority of beneficiaries as major infrastructural and operational constraint. “Practical demonstration facilities are inadequate under IMDS” (mean score: 89.07) ranked second and “lack of A.V. aids for educating the beneficiaries in training programme” (mean score: 82.46) was ranked as the third most important constraints by the beneficiaries. These findings are in line with the findings of Nachimuthu (2002).

The other infrastructural and operational constraints such as, “Illiteracy and poor knowledge of beneficiaries create problem in better understanding about A.H. schemes/programmes” (mean score: 78.04), Similar findings were reported by Anand (2009); Bhamare (2006). “Lack of trained, field-oriented and experienced Veterinary personnel” (mean score: 65.27) was the other infrastructural and operational constraint faced by the beneficiaries.

Economic constraintsAmong the economic constraints, delay in

getting incentive money for the owner of animal under scheme (mean score: 92.45) was faced as

most serious constraint and ranked first, insufficient MSP was given for their male calves purchased by A.H. Officials (mean score: 88.07) was ranked second by the beneficiaries.

Other economic constraints such as “non-availability of credit/loans under the scheme” (mean score: 84.74), followed by “non-availability of A.I. facilities in time for animal under scheme” (mean score: 82.08), “Insurance facilities provided under the scheme is not sufficient” (mean score: 80.83), “high cost of emergency veterinary services” (mean score: 79.44) and “high cost of veterinary medicines” (mean score: 72.78) were in order of seriousness.

Socio-psychological constraintsFrom the Table 5, it is inferred that

various socio-psychological constraints were faced by the beneficiaries, wherein it was found that the “preference for jobs rather than dairy-based self-employments” (mean score: 84.87) was faced as the most serious constraint, and was ranked first. Another constraint “Lack of decision-making ability among beneficiaries” (mean score: 80.43) and “lack of risk-bearing capacity among beneficiaries” (mean score: 72.65) ranked 2nd and 3rd, respectively.

Other constraints such as, “lack of democratic awareness and harmony among dairy farmers about the scheme” (mean score: 69.23 ), followed by “least participation of beneficiaries in various organizations” (mean score: 65.45), “lack of cooperation among beneficiaries” (mean score: 54.43), “lack of faith among beneficiaries in dairy development programme as well as in concerned officials as a measure for improving their economy” (mean score: 07.18) and “resourceful people of the society discourage the BPL farmers to join the scheme” (mean score: 05.14) were ranked in order


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Table 1. Administrative constraints faced by the beneficiaries.

Sr. No. Constraints Mean score Rank

1 Concerned officials are not much interested to visit the area and conducting regular meetings with beneficiaries 90.45 I

2 Concerned officials are not communicating the information, properly, regarding available facilities under project 60.05 IV

3 Delay from project personnel in sanctioning the funds 88.36 II

4There is no proper linkages/channel for the owners of buffaloes under scheme for marketing of their animals and its milk and milk products

85.30 III

Table 2. Technical constraints faced by the beneficiaries.

Sr. No. Constraints Mean score Rank1 Lack of awareness about the IMDS 84.34 II2 Less qualified staff working at A.I. centers 62.04 IV3 Repeat breeding 79.29 III

4Lack of knowledge about scientific feeding, breeding, health-care and management practices of buffaloes

92.67 I

5 Veterinary doctor/ A.H. officials do not visit the area regularly 59.54 V

Table 3. Infrastructural and operational constraints faced by the beneficiaries.


1 Concerned staff not taking much interest in imparting awareness training camps about IMDS 91.17 I

2 Lack of trained, field-oriented and experienced veterinary personnel 65.27 V

3 Practical demonstration facilities are inadequate under IMDS 89.07 II

4 Illiteracy and poor knowledge of beneficiaries create problem in better understanding about A.H. schemes/programmes 78.04 IV

5 Lack of A.V. aids for educating the beneficiaries in training pro-gramme 82.46 III


212

Table 4. Economic constraints faced by the beneficiaries.

Sr. No. Constraints Mean score Rank1 Insurance facilities provided under the scheme is not sufficient 80.83 V2 High cost of emergency veterinary services 79.44 VI3 High cost of veterinary medicines 72.78 VII4 Non-availability of credit/loans under the scheme 84.74 III

5 Non-availability of A.I. facilities in time for animal under scheme 82.08 IV

6 Delay in getting incentive money for owner of animal under scheme 92.45 I

7 Insufficient ‘Minimum support price’ (MSP) was given for their male calves while being purchased by A.H. Officials 88.07 II

Table 5. Socio-psychological constraints faced by the beneficiaries.


1 Resourceful people of the society discourage the BPL farmers to join the scheme 05.14 VIII

2 Lack of cooperation among beneficiaries 54.43 VI3 Least participation of beneficiaries in various organizations 65.45 V4 Lack of faith in modern veterinary schemes/programmes 00.00 ----

5 Lack of democratic awareness and harmony among dairy farmers about the scheme 69.23 IV

6Lack of faith among beneficiaries in dairy development programme as well as in concerned officials as a measure for improving their economy

07.18 VII

7 Lack of decision-making ability among beneficiaries 80.43 II8 Lack of risk-bearing capacity among beneficiaries 72.65 III9 Preference for jobs rather than dairy-based self-employment 84.87 I

Table 6. Miscellaneous constraints faced by the beneficiaries.


1 Poor knowledge of beneficiaries regarding facilities provided under scheme 58.44 V

2 Concerned personnel do not provide proper information about purchasing of dairy equipments/inputs 69.93 II

3 Difficulty in maintaining records due to illiteracy and lack of awareness about importance of records 71.74 I

4 Inadequate medical facilities for sick animals 59.08 IV

5 Inadequate contact of beneficiaries with developmental officials 67.69 III


213

of severity, respectively, by the beneficiaries.

Miscellaneous constraintsApart from the above-mentioned constraints

some of the important miscellaneous constraints as mentioned in the Table 6 that were “difficulty in maintaining records due to illiteracy and lack of awareness about importance of records” (mean score: 71.74); was raked first, which is followed by “concerned personnel do not provides proper information about purchasing of dairy equipments/inputs” (mean score: 69.93); “Inadequate contact of beneficiaries with developmental officials” (mean score: 67.69); “Inadequate medical facilities for sick animals” (mean score: 59.08); and “poor knowledge of beneficiaries regarding facilities provided under scheme” (mean score: 58.44), in the same order, on the basis of their mean score.

CONCLUSION

There were various constraints found regarding the utilization of facilities provided under the IMDS by beneficiary dairy farmers. To run this scheme in smooth and sustainable manner for conservation and improvement in Murrah breed in terms of quality as well as quantity, there is dire need to remove of these constraints on priority with the considerable focus on the implementation of such scheme. The attention of policy makers is needed for more interaction among the farmers and officials, promotion of extension activities for awareness and capacity building of farmers, timely implementation of scheme activities and resource.

REFERENCES

Anand, P. 2009. Impact of Self-Help Groups on growth of dairy farming in Haryana. Ph.D. Thesis, National Dairy Research Institute, Deemed University, Karnal, Haryana, India.

Bhamare, Y.A. 2006. Monetary utilization pattern of women in Self-Help Groups, Parbhani. M.Sc. Thesis, Marathwada Agricultural University, Parbhani, Maharashtra, India.

Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture. Livestock Census. Department of Animal Husbandry, Dairying and Fisheries, Ministry of Agriculture, Government of India, India.

Nachimuthu, K. 2002. Socio-economic and technological impact of animal husbandry programs in Pondichery. Ph.D. Thesis, National Dairy Research Institute, Karnal, India.

Singh, U. 2006. Multidimensional impact of women dairy cooperative societies on beneficiaries in Haryana, Ph.D. Thesis, National Dairy Research Institute, Deemed University, Karnal, Haryana, India.

Sharma, K., S.P. Singh and Gautam. 2010. Constraints perceived by dairy farmers in adoption of recommended buffalo husbandry practices, Indian J. Dairy Sci., 63(3): 225-232.

Tiwari, R.K., J.P. Bisen and P.N. Sharma. 2003. A study on constraints and suggestions regarding adoption of improved animal husbandry practices in Chhattisgarh Plains, Indian Res. J. Ext. Edu., 3(1): 22-29.


215

ABSTRACT

Different workers have reported better semen quality in buffaloes during hot and humid summer than in colder months, whereas contrary observations were also reported by many workers. Therefore, an initial study was attempted to find out the seasonal effect on semen characteristics of the swamp buffaloes of north eastern India. Data of semen characteristics pertaining to four bulls of four years were used for the study. The semen was collected by artificial vagina and the fresh semen was evaluated as per standard methods. The season was divided into Pre-monsoon (March to May), Monsoon (June to September), Post-monsoon (October to November), and winter (December to January) on the basis of meteorological data of the area. The volume of the semen was significantly (P<0.05) higher during Monsoon in comparison to other seasons. Mass activity, initial motility, per cent livability and sperm concentration did not differ significantly among the seasons. In conclusion, there is no significant effect of season on mass activity, initial motility, percent live sperm and concentration in swamp buffalo semen of north east India except the semen volume.

Keywords: semen, season, swamp buffalo, India

INTRODUCTION

India has a rich genetic resource of buffalo comprising of 13 registered riverine buffalo breeds along with many lesser known populations of significant regional importance. The riverine buffalo (Bubalusbubalis) is found throughout the length and breadth of the country, while the swamp buffalo (Bubalus bubalis carabanesis) is restricted to Assam and other Northeastern states of the country (Kalita et al., 2010). These animals are particularly suitable for ploughing paddy fields and providing draught power for varied agricultural activities. They are hardy animals and capable of readily using low-quality feedstuff and well suited to swampy, hot and humid tropical climate of the region. These buffaloes play important role in the socio-economic conditions of the rural population of the region. They are unique germplasm and need considerable attention for conservation. An initial study was made on the seminal attributes of these animals for using in artificial insemination programmes (Das et al., 2007). Buffalo bulls breed round the year but conflicting reports have been published about the semen quality and volume during various seasons. Different workers had studied the influence of season on semen quality in different species of animals (Javed et al., 2000;

SEASONAL VARIATION IN THE CHARACTERISTICS OF THE SWAMP BUFFALO SEMEN OF NORTHEAST INDIA

G.C.Das1, P.K.Das2, S. Deori3,*, H. Mazumdar1, B.N. Bhattacharyya1 and Arundhati Phookan1

1College of Veterinary Science, Khanapara, Guwahati, India2College of Veterinary Sciences and Animal Husbandry, R.K. Nagar, Agartala, India3Indian Council of Agricultural Research, National Research Centre on Yak, Dirang, Arunachal Pradesh, India, *E-mail: [email protected]

Original Article


216

Koonjaenak et al., 2007; Safaa et al., 2008; Tiwari et al., 2012). However, to the best of our knowledge, there is no literature available regarding seasonal effect on the fresh semen quality of these buffaloes. Therefore, an initial study was attempted to find out the seasonal effect on fresh semen characteristics of the swamp buffaloes of north-eastern region of India.

MATERIALS AND METHOD

Semen was collected from four swamp buffalo bulls (aged 3 to 4 years) maintained in the buffalo farm of the Network Project on Swamp Buffalo, College of Veterinary Science, Khanapara, Guwahati, Assam, India, using artificial vagina following twice a week schedule. They were managed uniformly under intensive system of management, fed concentrate mixture as per the body weight and green fodders ad libitum. Immediately after collection semen samples were taken into the laboratory and kept at 37ºC in a water bath. Ejaculates were evaluated for volume (ml), mass activity (0 to 5 scale), initial motility (%), livability (%) and the sperm concentration.

Semen volume (ml) was determined by reading the volume in the pre-warmed graduated glass collection tube. The mass activity of fresh semen was evaluated (100 × magnification) on the basis of a scale from 0 to 5 (0 = all spermatozoa are motionless, 5 = 90% or more of the spermatozoa are rapidly moving waves). Initial sperm motility was evaluated as a percentage, using a drop of semen diluted in Tris buffer at 37oC, and observed on a pre-warmed slide at a 100x magnification under microscope. Per cent livability was estimated by staining with eosin and nigrosin stain and counting 200 cells under microscope

at 400x magnification. The sperm concentration (million/ml) was calculated by diluting the semen (1:400) using a Sahli pipette in a formol citrate solution and measured by counting in an Improved Neubauer chamber at a 400x magnification under a microscope.

Semen characteristics data of four year involving four seasons were taken into account for the study. The season was classified into pre-monsoon (March to May), monsoon (June to September), post-monsoon (October to November), and winter (December to January) on the basis of meteorological data of the area.

The data obtained were subjected to compute analysis of variance (ANOVA). Duncan’s Multiple Range Test was performed to identify significant difference among the seasons as per Snedecor and Cochran (1980).


The mean values (±S.E.) for various characteristics of fresh semen of swamp buffaloes of northeast India viz., semen volume, mass activity, initial motility, present livability and sperm concentration are presented in Table 1. The ejaculate volume in the present study varied from 1.08±0.06 to 1.78±0.21 ml among different seasons with an overall average of 1.28±0.17 ml. Das et al. (2006) recorded a similar value of 1.90±0.16 ml ejaculate volume in swamp buffaloes of Assam. However, in contrast to the present finding Koonjaenak et al. (2007) reported a higher volume of 3.2 to 3.8 ml in swamp buffaloes of Thailand. This difference may be due to variation in genetics, reproductive health status of bulls, age of bulls, frequency of collection, nutrition and management. The reported ejaculate volume in the


217

swamp buffaloes of India is much lower than the values of other riverine buffalo breeds (Javed et al., 2000; Maurya et al., 2003; Bhakat et al., 2011). In the present investigation the semen volume was significantly higher (P<0.05) during monsoon than in other seasons. Javed et al. (2000) also recorded significant effect of season on the ejaculate volume of Nili-Ravi buffalo bull. However, Alavi-Shooushtari and Babazedeh-Habashi, (2006) found semen volume had no significant variation in different seasons in Azarbaijani buffalo. The mass activity in the present investigation varied from 3.51±0.12 to 3.78±0.06 with an overall mean of 3.63±0.08 in a 0 to 5 scale. Similar to present findings Das et al. (2006) reported mass activity of 3.12±0.13 in swamp buffaloes of Assam. Javed et al. (2000) recorded overall mass activity score of 2.65±1.03 in Nili-Ravi buffalo semen. They further reported mass activity score was higher (P<0.05) in dry summer and spring. However, in our study the mass activity varied during different seasons but it was statistically not significant. The initial sperm motility ranged from 77.50±0.92 to 79.66±0.74% with an overall

average of 78.94±0.49%. Koonjaenak et al. (2007) reported initial sperm motility ranged from 72.8 to 75.2% in Thai swamp buffaloes, which is almost consistent with the present findings. They further concluded that the season has no influence on the initial sperm motility. In the present investigation also the initial motility in swamp buffalo semen did not differ significantly between the seasons. Javed et al. (2000) recorded much lower values of overall sperm motility (56.89±0.65%) in Nili- Ravi buffaloes with significantly (P<0.05) lower values in winter than humid summer and autumn. Percentage of live sperm varied from 89.36±0.85 to 90.21±0.80 with an overall mean of 89.91±0.19 in the present study. The present values are higher than the findings of Das et al. (2006) for swamp buffaloes and Alavi-Shooushtari and Babazedeh-Habashi (2006) for Azarbaijani buffaloes. Although, the live sperm percentage varied between the seasons but statistically not significant. Non- significantly highest value was recorded during post-monsoon and lowest during monsoon season. In contrast to our findings, Pant and Mukherjee (1972) reported that the percentage

Table 1. Semen characteristics (Mean±S.E.) of swamp buffalo of north east India during different seasons.

Semen characteristics

SeasonsOverall

Pre-monsoon Monsoon Post-monsoon WinterVolume

(ml) 1.15a±0.05 1.78b±0.21 1.12a±0.04 1.08a±0.06 1.28±0.17

Mass activity (0-5 scale) 3.47±0.07 3.51±0.12 3.75±0.07 3.78±0.06 3.63±0.08

Initial motility (%) 79.35±0.72 77.50±0.92 79.26±0.67 79.66±0.74 78.94±0.49

Livability (%) 89.97±0.77 89.36±0.85 90.21±0.80 90.10±0.71 89.91±0.19

Concentration (million/ml) 1016.13±28.25 1113.33±53.97 998.53±53.53 1100.34±56.49 1057.08±29.07

Means with different superscript in a row differ significantly (P<0.05).


218

of live spermatozoa decreased with increase in air temperature and humidity in Murrah buffaloes.

The sperm concentration (million/ml) varied from 998.53±53.53 to 1113.33±53.97 with an overall average of 1057.08±29.07. Present value is in consistent with the values of Koonjaenak et al. (2007) for Thai swamp buffaloes (1.1 to 1.2 billion/ml). Das et al. (2006) reported a lower concentration of 968.88±104.87 million/ml in swamp buffaloes of Assam in comparison to the present study. Although, highest value was recorded in monsoon and the lowest in post-monsoon season, however, the results are not significant. Koonjaenak et al. (2007) also found that season has no influence on the sperm concentration in the semen of Thai swamp buffaloes. In contrast to our study, Javed et al. (2000) reported influence of season in the sperm concentration of Nili- Ravi bulls during autumn and spring.

In conclusion, present study revealed that there is no significant effect of season on mass activity, initial motility, percent livability and sperm concentration of swamp buffalo semen reared in the north eastern India except the semen volume. The semen volume found to be significantly higher (P<0.05) during monsoon season.

REFERENCES

Alavi-Shooushtari, S.M. and B. Babazedeh-Habashi. 2006. Seasonal variation in the characteristics of the Azarbaijani buffalo (Bubalus bubalis) semen. Iran. J. Vet. Res., 7: 49-54.

Bhakat, M., T.K. Mohanty, V.S. Raina, A.K. Gupta and H.M. Khan. 2011. Frozen semen production performance of Murrah buffalo bulls. Buffalo Bull., 30: 157-162.

Das, G.C., S. Deori and B.K. Das. 2006. Certain seminal attributes of the swamp buffaloes of Assam. In Proceedings of National Symposium on Buffalo for Rural Upliftment, Mumbai, India.

Das, G.C., S. Deori, B.K. Das and R.N. Goswami. 2007. Seminal characteristics of the swamp buffaloes of Assam. Indian Vet. J., 84: 1052-1053.

Javed, M.T., A. Khan and R. Kausar. 2000. Effect of age and season on some semen parameters of Nili-Ravi buffalo (Bubalus bubalis) bulls. Vet. Arhiv, 70: 83-94.

Kalita, R., A. Dandapat, K.B.D. Choudhury, G.C. Das and R.N. Goswami. 2010. Conformation traits of swamp buffalo of Assam at different age groups. Indian J. Anim. Res., 44: 300-302.

Koonjaenak, S., V. Chanatinart, S. Aiumlamai, T. Pinyopumimintr and H. Rodriguez-Martinez. 2007. Seasonal variation in semen quality of swamp buffalo bulls (Bubalus bubalis) in Thailand. Asian J. Androl., 9: 92-101.

Maurya, V.P., R.K. Tuli and R.L. Goyal. 2003. Effect of buffer composition, sephadex grade and column size on filtration based quality improvement of semen from Murrah buffalo bull. Asian Austral. J. Anim. Sci., 16: 165-171.

Pant, K.P. and D.P. Mukherjee. 1972. The effect of seasons on the sperm dimensions of buffalo bulls. J. Reprod. Fertil., 29: 425-429.

Safaa, H.M., M.E. Emarahand and N.F.A. Saleh. 2008. Seasonal effects on semen quality in black Baladi and White New Zealand rabbit bucks. World Rabbit Sci., 16: 13-20.

Snedecor, G.W. and W.G. Cochram. 1980. Statistical Methods, 7th ed. The Iowa State


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University Press, Ames, Iowa, USA. Tiwari, R., G.K. Mishra, R.B. Singh, S.U. Rehman,

K.S. Rathora, S.K. Saxena and M.U. Siddiqui. 2012. Seasonal variations in the quality and freezability of Red Sindhi bull semen. Indian J. Anim. Sci., 82: 1344-1346.


221

ABSTRACT

A total of forty ejaculates were collected from eight buffalo bulls by artificial vagina method. Each ejaculate was splitted into two parts, one part was used for in vitro capacitation by TALP medium, and the rest was frozen in Tris-egg yolk-citrate-glycerol extender. The results revealed that the live acrosome reacted and total acrosome reacted frozen thawed spermatozoa differed significantly (P<0.01) from in vitro capacitated spermatozoa at different hours of incubation. The incidence of hyperactivated motility, total HOST-reacted spermatozoa, sperm membrane protein and cholesterol level of frozen thawed spermatozoa also differed significantly (P<0.01) from in vitro capacitated spermatozoa during different hours of incubation except at 0 h for hyperactivated motility, 6 h for total HOST-reacted spermatozoa, 1 h for sperm membrane protein level and at 2 h for cholesterol level. The study suggests that cryopreservation induces capacitation-like changes of the swamp buffalo spermatozoa in respect of acrosomal status, plasma membrane integrity, membrane protein and cholesterol levels.

Keywords: capacitation, cryopreservaion, spermatozoa, swamp buffalo

INTRODUCTION

Artificial insemination (AI) is useful for the improvement of milk productivity in buffaloes by the propagation of animals with high genetic potential (Ciptadi et al., 2012). The best preservation technique to date of post-thaw survival is restricted to about 50% of the sperm population (Watson, 1995). The final cryopreservation goal of semen is not only to maintain the initial motility but also to maintain the necessary metabolism to produce viable sperm to survive in the female reproductive tract at the time of fertilization. The freezing and thawing process provokes morphological or biochemical cryogenic damage resulting in sperm dysfunction and changes in cell’s membrane. The exposure of spermatozoa to low temperatures shortens their capacitation time, changing the membrane lipid architecture, membrane permeability and the reducing efficiency of enzymes extruding calcium ions. These changes resemble capacitation, and are likely to reduce long-term sperm viability and alter their motility. The term “cryocapacitation” have been introduced to emphasize the fact that cryopreservation procedures induce capacitation-like changes in spermatozoa (Watson, 1995; Cormier and Bailey, 2003). These cooling-related capacitation-like changes in spermatozoa, may affect the fertility of

Original Article

CRYOPRESERVATION INDUCES CAPACITATION-LIKE CHANGES OF THE SWAMP BUFFALO SPERMATOZOA

D.J. Talukdar1, K. Ahmed1, S. Sinha1, S. Deori2,*, G.C. Das1 and Papori Talukdar3

1College of Veterinary Science, Khanapara, Guwahati, Assam, India2National Research Centre on Yak, Dirang, Arunachal Pradesh, India, *E-mail: [email protected] Dairy Research Institute, Karnal, Haryana, India


222

cryopreserved semen, by rendering the cells less stable in the female reproductive tract, after artificial insemination and therefore relatively short-lived. Such changes cannot easily be distinguished from true capacitation. Keeping in view, the study had been planned with the objective to study the functional characteristics of in vitro capacitated and frozen-thawed spermatozoa of swamp buffalo bull.


A total of 40 semen samples were collected by artificial vagina method from each eight swamp buffalo bulls aged five to eight years maintained at College of Veterinary Science, Guwahati, Assam, India. Each ejaculate was evaluated for volume, mass activity, and initial motility immediately after collection. Samples having volume 1.0 ml or more, mass activity 3 or more and initial sperm motility 70% or more were divided into two parts, one part was used for in vitro capacitation by TALP medium (NaCl-92.9 mM; KCl-4 mM; NaHCO3-25.9 mM; CaCl2 .2H2O-10 mM; MgCl2.6 H2O-0.5 mM; sodium lactate-7.6 mM; sodium pyruvate-1.3 mM; HEPES-20 mM; glucose-0.25%; heparin-200 μg/ml, BSA-0.6%, Penicillin G-40 IU/ml, Streptomycin sulphate-50 µg/ml and Deionised triple distilled water up to 1000 ml) at concentration of 6×109 spermatozoa/ml (Rogers and Yanangimachi, 1975) at 37oC with 5% CO2 for 6 h and the rest was frozen with Tris-egg yolk-citrate extender [Tris (hydroxy methyl) amino methane-2.422 g; citric acid monohydrate-1.36 g; fructose-1.0 g; penicillin G sodium-1000 IU/ml; streptomycin sulphate-100 mg/ml; double distilled water up to 100ml] with 20% egg yolk and 6.4% glycerol to yield approximately 60 million motile

sperm/ml. The extended semen was equilibrated for 4 h at 5oC before filling in 0.25 ml French mini straws. After filling and sealing, the semen straws were placed in a rack at 4 cm above liquid nitrogen in the vapour phase for 8 min and finally plunged into liquid nitrogen container (-196oC) and stored. The frozen semen straws were thawed at 37oC for 30 seconds. Each sample of in vitro capacitated sperm was evaluated at one hour interval starting from 0 h and all the samples of frozen thawed and in vitro capacitated were evaluated for hyperactivated motility (Marquez and Susan, 2004), total HOST-reacted sperm (Jeyendran et al., 1984), acrosomal status by using FITC labelled Pisum sativum agglutinin (Kaul et al., 2001) and live intact acrosome using Eosin-Nigrosin-Giemsa staining technique (Tamuli and Watson, 1994), sperm membrane protein (Cheema et al., 2011) and cholesterol level (Srivastava et al., 2013) by using quality kit (Siemens Ltd., 589, Sayajipura, Ajwa Road, Vadodara-390 019, Gujarat, India) in a Systronics Spectrophotometer 106.The statistical analysis of the data was done using SAS Enterprise Guide 4.2 version.


The results of analysis of variance (ANOVA) are presented in Table 1. ANOVA revealed highly significant effect of source (in vitro capacitated vs frozen thawed spermatozoa).

Before fertilizing of the oocyte, mammalian spermatozoa undergo the sequence of membrane alterations associated with accumulation of calcium ion and the increase of tyrosine phosphorylation resulting in sperm hyperactivation (Hewitt and England, 1998; Petrunkina et al., 2003), which is characterized by high-amplitude and asymmetrical


223

Tabl

e 1.

Fuc

tiona

l int

egrit

y of

in v

itro

capa

cita

ted

and

froz

en th

awed

swam

p bu

ffalo

sper

mat

ozoa

(mea

n*±

se).

Stag

e Pa

ram

eter

sIn

vitr

o ca

paci

tatio

n (h

ours

)Fr

ozen

Tha

wed

01

23

45

6

Hyp

erac

tivat

ed

Mot

ility

(%)

14.0

0a ± 0

.80

35.7

5b ±1.

1252

.87c ±

1.38

60.2

5d ±1.

0574

.50e ±

1.78

47.5

0f ±1.

1141

.25g ±

1.30

16.0

0a ±0.

76

Tota

l HO

ST re

acte

d Sp

erm

(%)

82.5

5a ± 1

.00

78.0

2b ±1.

2677

.22b ±

1.12

75.5

5bc±1

.21

71.7

0cd±1

.49

69.2

5d ±1.

6161

.57e ±

2.20

62.2

0e ±1.

31

Live

acr

osom

e re

acte

d sp

erm

(%)

(Eos

in-N

igro

sin-

Gie

msa

stai

n)

8.62

a ±0.

3345

.57b ±

2.97

52.4

2cd±2

.49

50.9

2bd±2

.39

56.9

2c ±1.

8854

.27cd

±1.8

349

.37bd

±2.5

927

.28e ±

0.81

Tota

l acr

osom

e re

acte

d sp

erm

(%)

(FIT

C-P

SA)

7.90

a ±0.

5943

.50b ±

2.46

53.2

0c ±1.

4468

.00d ±

1.87

69.6

5d ±1.

3968

.47d ±

1.42

75.1

2e ±1.

2438

.72f ±

1.79

Sper

m m

embr

ane

Prot

ein

(mg/

109

sper

m)

5.13

a ±0.

123.

92b ±

0.13

3.25

c ±0.

122.

64d ±

0.11

2.55

d ±0.

131.

82e ±

0.06

1.45

f ±0.

083.

95b ±

0.10

Cho

lest

erol

(µg/

108

sper

m)

21.9

5a ±0.

4419

.08b ±

0.51

14.6

5c ±0.

5112

.94d ±

0.47

11.0

7e ±0.

598.

45f ±

0.54

5.64

g ±0.

4614

.74c ±

0.60

*40

obse

rvat

ions

.M

eans

bea

ring

diffe

rent

supe

rscr

ipts

in a

row

diff

er si

gnifi

cant

ly.


224

flagellar beating that assists sperm in penetrating the oocyte zona pellucida (Marquez and Susan, 2004). Hyperactivation has been considered part of the capacitation process because sperm have been observed to hyperactivate while undergoing capacitation. During capacitation, several sperm proteins become phosphorylated on tyrosine residues and this phosphorylation has been demonstrated to be regulated by a cAMP pathway through activation of protein kinase A (PKA). Some of the proteins that become tyrosine phosphorylated during capacitation have been localized to the flagellum, and therefore it has been proposed that they are involved in hyperactivation (Si and Okuno, 1999). The incidence of hyperactivated motility of frozen thawed spermatozoa in the present study differed significantly (P<0.01) with hyperactivated motility of in vitro capacitated spermatozoa at different hours of incubation and the incidence was slightly higher than 0 h and lower than 1 h which might be due to cryopreservation as cryopreserved spermatozoa have poor calcium efflux mechanisms and are less efficient in extruding calcium ions resulting in rapid accumulation of cytosolic calcium ion (Bailey et al., 1994) and when these intracellular ion concentrations reach threshold levels, capacitation followed by acrosome reaction is triggered without the activation of zona pellucida receptors in the sperm plasma membrane or the associated signal transduction system (Bailey et al., 2000).

The determination of the acrosome status in cryopreserved sperm is of the fundamental importance as cryopreservation directly damages sperm membrane, which could be followed by a loss of the acrosomal matrix contents. The incidence of total live acrosome reacted spermatozoa in the present observation differed significantly (P<0.01) between in vitro capacitated spermatozoa at

different hours of incubation and frozen thawed spermatozoa. The incidence of live acrosome reacted spermatozoa in frozen thawed spermatozoa was higher than 0 h and lower than 1 h of incubation, which might be due to partial capacitation of buffalo spermatozoa occurs during freezing and thawing followed by equilibration (Watson, 1995).There is substantial evidence that cryopreservation promotes the premature capacitation of spermatozoa (Bailey et al., 2000; Watson, 2000) and this cryocapacitation is frequently cited as one of the factors associated with the reduced longevity of cryopreserved spermatozoa in the female reproductive tract by a loss of the acrosomal matrix contents. Detection of the viability and stages of acrosomal exocytosis, either spontaneous or induced, was carried out using fluorescent probes. FITC-Pisum satiuum lectin (FITC-PSA) was used to assess acrosomal status by staining glycoproteins in the acrosome of permeabilised spermatozoa. Fluorescein-conjugated plant lectins like fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC- PSA) from the edible pea has been used as a selective acrosomal staining of the spermatozoa of human (Cross et al., 1986), stallion (Casey et al., 1993) and monkey (Cross et al., 1989). As PSA binds to the acrosomal contents, the progress of the acrosome reaction is indicated by the intensity and distribution of fluorescence over the acrosomal region. The acrosome reaction in buffalo spermatozoa commences just anterior to the equatorial segment and proceeds in an arborizing fashion towards the apical ridge (Watson and Plummer, 1986). Therefore, as the reaction progresses, more acrosomal contents will be lost and, therefore, less fluorescence will be seen. Staining only in the equatorial segment is also characteristic of a cell that has only recently completed its acrosome


225

reaction whereas cells devoid of staining in this region have fully completed the acrosome reaction some time previously (Tesarik et al., 1993). The total acrosome reacted (PSA-ve) spermatozoa in the present observation differed significantly (P<0.01) between frozen thawed spermatozoa and in vitro capacitated spermatozoa at different hours of incubation. The incidence in frozen thawed spermatozoa was slightly higher than in 0 h and lower than in 1 h of incubation. This might be due to cryocapacitation which could be followed by a loss of the acrosomal matrix contents (Cross et al., 1986).

The plasma membrane integrity of sperm is of crucial importance for optimal sperm function and only a sperm with an intact plasma membrane can undergo a series of complex changes in the female reproductive tract and can acquire the ability to fertilize an oocyte (Yanagimachi, 1994). The mean percentage of total HOST reacted spermatozoa of frozen thawed semen in the present study differed significantly (P<0.01) with in vitro capacitated spermatozoa at different hours of incubation except 6 h where the values was in close proximity. This might be due to cryopreservation results partial capacitation that induced membrane phase changes, which are thought to result in lateral phase separation of membrane components and increased membrane permeability for solutes (Hammerstedt et al., 1990). The disruption of plasma membrane integrity caused by disarrangement of lipids within the membrane during cryocapacitation may induce further cellular damage and consequently lead to irreversible damage to its integrity (Jeyendran et al., 1984; Watson, 1995).

Capacitation is a post-testicular developmental and maturational process of mammalian spermatozoa occurring during their transit through the female reproductive tract with

modification of sperm surface proteins, added or removed and an array of proteins have been shown to undergo tyrosine phosphorylation in different species (Luconi et al., 1996; Galantino et al., 1997). During fertilization, mammalian sperm membrane proteins are also involved in the penetration of cumulus matrix, recognition of zona pellucida and fusion with the oocyte plasma membrane (Myles and Primakoff, 1997). In the present observations, there was leakage of proteins from the frozen thawed as well as capacitated spermatozoa but the leakage was significantly more in the latter as compared to the former. However, the leakage of proteins in the frozen thawed spermatozoa may be because of acrosomal damage and in capacitated, because of acrosomal damage as well as due to capacitation and acrosome reaction. As the mean sperm membrane protein level of spermatozoa in frozen thawed semen differed significantly (P<0.01) from in vitro capacitated spermatozoa at different hours of incubation except 1 h where the values was in close proximity. The similarity of sperm membrane protein level in frozen thawed and in vitro capacitated sperm at 1 h of incubation at 37oC in TALP medium in the present study indicated the initiation of capacitation of spermatozoa which corroborated the findings of earlier workers (Watson, 1995; Cromier and Bailey, 2003; Kadirvel et al., 2009) who reported that the cryopreserved sperm are in a partially capacitated state. The alteration in the sperm membrane proteins might be due to sublethal damage which was occurred during cryopreservation leading to loss of sperm surface proteins (Lessard et al., 2000), segregation of membrane proteins (De Leeuw et al., 1990), inactivation of membrane-bound enzymes and decreased lateral protein diffusion within the membrane (Watson, 1995).The molecular mechanisms of capacitation are not


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completely elucidated; however, recent studies have demonstrated involvement of numerous structural and biochemical modifications in spermatozoa, such as changes in membrane composition and membrane fluidity (Harrison et al., 1996; Green and Watson, 2001), increased intracellular calcium (Visconti et al., 1998), activation of ion channels (Florman et al., 1998) and generation of reactive oxygen species (de Lamirande et al., 1997). Various reports suggested an active participation of the sperm plasma membrane in the process of capacitation, mainly through the loss of cholesterol (Cross, 1998; Visconti et al., 1999). The cholesterol efflux during in vitro capacitation increases the disorder of phospholipid packing, and results in increased bilayer permeability (Cross, 1998). Similar to physiological and in vitro capacitation, a significant reduction of cholesterol content after cryopreservation was observed in the present study. Our results are in agreement with Cerolini et al. (2001) and Kadirvel et al. (2009) who observed decreased free cholesterol content and increased phospholipids and triglycerol content after freezing-thawing of boar semen. Furthermore, sperm membranes are known to release phospholipids into surrounding medium during cold shock (Darin-Bennett et al., 1973). The mechanism of loss of cholesterol during cryopreservation is not completely understood. However, most of the cholesterol loss is due to slow diffusion from cell and a net transfer of cholesterol from rat and bovine sperm to the medium has already been demonstrated (Ehrenwald et al., 1988). Release of cholesterol from the membrane plane, a probable consequence of peroxidative damage to membrane lipids, could lead to the premature capacitation of cryopreserved bull spermatozoa (Cormier et al., 1997). The loss of phospholipids in cryopreserved samples occurs at a more rapid rate

when compared to fresh samples and follows the pattern expected for lipid peroxidation (Alvarez and Storey, 1992). The present study observed that the mean cholesterol level of spermatozoa in frozen thawed semen differed significantly (P<0.01) from in vitro capacitated spermatozoa at different hours of incubation except 2 h where the values was in close proximity. Therefore, cholesterol efflux may represent an integral part of the intrinsic regulatory property of sperm to undergo capacitation-like changes during cryopreservation. The reduction in cholesterol content which was similar in post thawed spermatozoa and in vitro capacitated spermatozoa after 2 h of incubation indicated the partial capacitation status of post thawed spermatozoa.

CONCLUSION

In conclusion, this study showed that cryopreservation induces capacitation-like changes of the Swamp buffalo spermatozoa in respect of acrosomal status, plasma membrane integrity, membrane protein and cholesterol levels.

ACKNOWLEDGEMENTS

The author expresses gratefulness to the Director of Post Graduate Studies, Assam Agricultural University, and to Dr. R.N. Goswami, the Dean, Faculty of Veterinary Science, Assam Agricultural University, Khanapara for providing necessary facilities needed for the study.


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REFERENCES

Alvarez, J.G. and B.T. Storey. 1992. Evidence for increased lipid peroxidation damage and loss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm during cryopreservation. J. Androl., 13: 232-241.

Bailey, J.L., J.F. Bilodeau and N. Cormier. 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl., 21: 1-7.

Bailey, J.L., L. Robertson and M.M. Buhr. 1994. Relationships among in vivo fertility, computer-analysed motility and in vitro Ca2+ flux in bovine spermatozoa. Can. J. Anim. Sci., 74: 53-58.

Casey, P.J., R.B. Hillman, K.R. Robertson, A.I. Yudin, I.K. Liu and E.Z. Drobnis. 1993. Validation of an acrosomal stain for equine sperm that differentiates between living and dead sperm. J. Androl., 14: 289-297.

Cerolini, S., A. Maldjan, F. Pizzi and T.M. Ghozzi. 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction, 121: 395-401.

Cheema, R., A.K. Bansal, G.C. Bilaspuri and V.K. Gandotra. 2011. Correlation between the proteins and protein profile(s) of different regions of epididymis and their contents in goat buck. Anim. Sci. Pap. Rep., 29: 75-84.

Ciptadi, G.M., A. Nasich, N. Budiarto and V.M.A. Nurgiartiningsih. 2012. The estrus synchronization response following PGF2α treatment in Indonesian Madura cattle with different body condition scores. Pak. Vet. J., 32: 624-626.

Cormier, N. and J.L. Bailey. 2003. A differential mechanism is involved during heparin-and

cryopreservation-induced capacitation. Biol. Reprod., 69: 177-185.

Cormier, N., M.A. Sirard and J.L. Bailey. 1997. Premature capacitation of bovine spermatozoa is initiated by cryopreservation. J. Androl., 18: 461-468.

Cross, N.L. 1998. Role of cholesterol in sperm capacitation. Biol. Reprod., 59: 7-11.

Cross, N.L., P. Morales, M. Fukuda and E. Behboodi. 1989. Determining acrosomal status of the cynomolgus monkey (Macaca jbscicularis) sperm by fluorescence microscopy. Am. J. Primatol., 17: 157-163.

Cross, N.L., P. Morales, J.W. Overstreet and F.W. Hanson. 1986. Two simple methods for detecting acrosome-reacted human sperm. Gamete Res., 15: 213-226.

Darin-Bennett, A., A. Poulos and I.G. White. 1973. The effects of cold shock and freeze-thawing on release of phospholipids by ram, bull and boar spermatozoa. Aust. J. Biol. Sci., 26: 1409-1420.

De Lamirande, E., H. Jiang, A. Zini, H. Kodama and C. Gagnon. 1997. Reactive oxygen species and sperm physiology. Rev. Reprod., 2: 48-54.

De Leeuw, F.E., H.C. Chen, B. Colenbrander and A.J. Verkleij. 1990. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology, 27: 171-183.

Ehrenwald, E., J.E. Parks and R.H. Foote. 1988. Cholesterol efflux from bovine sperm. I. Induction of the acrosome reaction with lysophosphatidyl choline after reducing sperm cholesterol. Gamete Res., 20: 145-157.

Florman, H.M., C. Arnoult, I.G. Kazam, C. Li and M.B. O’Toole. 1998. A perspective on the


228

control of mammalian fertilization by egg activated ion channels in sperm: a tale of two channels. Biol. Reprod., 59: 12-16.

Galantino-Homer, H.L., P.E. Visconti and G.S. Kopf. 1997. Regulation of protein tyrosine phosphorylation during bovine sperm capacitation by cyclic adenosine 3’5’-monophosphate-dependent pathway. Biol. Reprod., 56: 707-719.

Green, C.E. and P.F. Watson. 2001. Comparison of the capacitation-like state of cooled boar spermatozoa with true capacitation. Reproduction, 122: 889-898.

Hammerstedt, R.H., J.K. Graham and J.P. Nolan. 1990. Cryopreservation of mammalian sperm: what we ask them to survive. J. Androl., 11: 73-88.

Harrison, R.A.P., P.J.C. Ashworth and N.G.A. Miller. 1996. Bicarbonate/CO2, an effector of capacitation, induces a rapid and reversible change in the lipid architecture of boar sperm plasma membranes. Mol. Reprod. Dev., 45: 378-391.

Hewitt, D.A. and G.C. England. 1998. An investigation of capacitation and the acrosome reaction in dog spermatozoa using a dual fluorescent staining technique. Anim. Reprod. Sci., 51: 321-332.

Jeyendran, R.S., H.H. Van der Ven, M. Perez-Pelaez, B.G. Carbo and L.J.D. Zaneveld. 1984. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J. Reprod. Fertil., 70: 219-228.

Kadirvel, G., S. Kumar, A. Kumaresan and P. Kathiravan. 2009. Capacitation status of fresh and frozen-thawed buffalo spermatozoa in relation to cholesterol

level, membrane fluidity and intracellular calcium. Anim. Reprod. Sci., 116: 244-253.

Kaul, G., G.S. Sharma, B. Singh and K.K. Gandhi. 2001. Capacitation and acrosomal reaction in buffalo bull spermatozoa assessed by chlortetracycline and Pisum sativum agglutinin fluorescence assay. Theriogenology, 55: 1457-1468.

Lessard, C., S. Parent, P. Leclerc, J.L. Bailey and R. Sullivan. 2000. Cryopreservation alters the level of the bull sperm surface protein P25b. J. Androl., 21: 700-707.

Luconi, M., C. Krausz, G. Forti and E. Baldi. 1996. Extracellular calcium negatively modulates tyrosine phosphorylation and tyrosine kinase activity during capacitation of human spermatozoa. Biol. Reprod., 55: 207-216.

Marquez, B. and S.S. Susan. 2004. Different signaling pathways in bovine sperm regulate capacitation and hyperactivation. Biol. Reprod., 70: 1626-1633.

Myles, D.G. and P. Primakoff. 1997. Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg. Biol. Reprod., 56: 320-327.

Petrunkina, A.M., K. Simon, A.R. Gunzel-Apel and E. Topfer-Petersen. 2003. Regulation of capacitation of canine spermatozoa during co-culture with heterologous oviductal epithelial cells. Reprod. Domest. Anim., 38: 455-463.

Rogers, B.J. and R. Yanagimachi. 1975. Retardation of guinea-pig sperm acrosome reaction by glucose: The possible importance of pyruvate and lactate metabolism in capacitation and the acrosome reaction. Biol. Reprod., 13: 568-575.


229

Si, Y. and M. Okuno. 1999. Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. Biol. Reprod., 61: 240-246.

Srivastava, N., S.K. Srivastava, S.K. Ghosh, K. Amit, P. Perumal and A. Jerome. 2013. Acrosome membrane integrity and cryocapacitation are related to cholesterol content of bull spermatozoa. Asian Pac. J. Reprod., 2: 126-131.

Tamuli, M.K. and P.F. Watson. 1994. Use of a simple staining technique to distinguish acrosomal changes in the live sperm sub-population. Anim. Reprod. Sci., 35: 247-250.

Tesarik, M.D., C. Mendoza and A. Carreras. 1993. Fast acrosome reaction measure: a highly sensitive method for evaluating stimulus-induced acrosome reaction. Fertil. Steril., 59: 424-430.

Visconti, P.E., H. Galantino-Homer, G.D. Moore, J.L. Bailey, X.P. Ning, M. Fornes and G.S. Kopf. 1998. The molecular basis of sperm capacitation. J. Androl., 19: 242-248.

Visconti, P.E., X. Ning, M.W. Fornes, J.G. Alvarez, P. Stein, S.A. Connors and G.S. Kopf. 1999. Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. Dev. Biol., 214: 429-443.

Watson, P.F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and assessment of their post-thawing function. Reprod. Fertil. Dev., 7: 871-891.

Watson, P.F. 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod.

Sci., 60-61: 481-492.Watson, P.F. and J.M. Plummer. 1986. Relationship

between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa. J. Exp. Zool., 238: 113-l18.

Yanagimachi, R. 1994. Mammalian fertilization, p. 189-317. In The Physiology of Reproduction. Raven Press, New York.


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ABSTRACT

The ruminal parameters, in vivo, in vitro, in sacco digestibility, and water extractable dry matter (DM), were measured in buffaloes fed rice straw and elephant grass with supplementation of fresh leaves of Sesbania grandiflora. The design was a 2x2 factorial arrangement with three replications. The first factor of the experiment was feed (rice straw or elephant grass); the second factor was supplementation of 4 kg/day of fresh leaves of Sesbania grandiflora. The buffaloes received the forage at 80% of their requirement and the sesbania leaves were supplemented once in the morning. Ruminal ammonia concentration, bacteria population and volatile fatty acids were significantly higher for the diets with Sesbania grandiflora supplementation. Supplementation also significantly improved in vivo DM and NDF digestibility but had no effect on in vitro measurement of digestibility nor on “a”, “b” and “c” parameters in the in sacco test. However, the effective DM degradability value as measured by the in sacco method was significantly increased by supplementation compared to the control. In vivo DM digestibility was more closely correlated with water extractable DM values than with any of the in vitro analyses. It is concluded that farmers could supply a supplement of Sesbania grandiflora leaves to buffaloes fed low quality forages to improve

their rumen function and production.

Keywords: ruminant, legume leaves, rumen microbes, feed digestion, supplement, rumen environment, water extractable DM

INTRODUCTION

Due to the demand for land for crop production in Vietnam, the grassland areas for ruminants has been reduced. This is one of the factors that has contributed to the reduction of the buffalo population reduction in the Mekong delta (Nguyen, 1988). However, buffalo meat has an important role for people in this region. Recently, buffalo production has been favourably considered by farmers as a means of raising their income (Nguyen, 2012), but low quality of buffalo feeds, which are mainly crop residues, has dominated in their diets and has limited the buffalo performance. Therefore some supplements to diets were used to improve cattle and buffaloes performance such as cotton seed cake, soybean extraction meal, coco nut cake, multi nutrient cake, etc (Nguyen and Nguyen, 2015). Beside them, plant protein sources as alternative sources of supplements in buffalo diets have been abundant and available in this region such as Sesbania, Leuceana and duckweeds. Estimation of feed digestibility by the in vivo

Original Article

A RESPONSE OF IN VITRO, IN SACCO AND IN VIVO DIGESTIBILITY AND RUMEN PARAMETERS OF SWAMP BUFFALOES SUPPLEMENTED

SESBANIA GRANDIFLORA LEAVES

Nguyen Van Thu* and Nguyen Thi Kim Dong

Department of Animal Sciences, College of Agriculture and Applied Biology, Can Tho University, Vietnam, *E-mail: [email protected]


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technique in ruminants has been useful; however, the high cost and time required have been limiting factors to its common use. Alternatively, in vitro and in sacco digestibility methods have been used effectively due to low cost and many more feed samples can be evaluated (Lopez et al., 2000). Therefore, this study aimed to investigate whether the rumen environment and feed digestibility could be improved by supplementation of buffalo diets with leaves of Sebania grandiflora, using in vitro and in sacco digestibility procedures.


The study was carried out at the experimental farms of Cantho University. It was a 2x2 factorial design experiment with 3 replications. The first factor was feed (rice straw or elephant grass) and the second factor was supplementation (with and without leaves of Sesbania grandiflora). The experimental animals were swamp male buffaloes (420±25 kg live weightfitted with rumen cannulae. The experimental period was 3 weeks including one week for diet adaptation. The fresh Sesbania grandiflora leave was supplemented once at a level of 4 kg per day in the morning. The experimental animals were fed at 7.00 am and 2.00 pm.

Samples of rumen contents were collected at 3 h post-feeding to measure rumen pH, ammonia N (NH3-N), protozoa and bacteria populations. Rumen pH was measured by pH meter and NH3-N was analyzed by the micro Kjeldahl method. For counting protozoa the preparation of rumen content samples followed the procedure of Dehority (1984) and a 0.2 mm deep chamber under 100 x magnification was used. Total bacteria populations were counted in a Neubauer chamber

under 1200 x magnification after the preparation of rumen content samples following the procedure of Warner (1962). Total VFA were measured by steam distillation following the procedure described by Barnett and Reid (1957).

Feeds and refusals were collected daily and pooled weekly for analysis of DM to calculate feed intake. Feeds and samples for rumen incubation were analyzed for DM, organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL) and ash following the procedure of AOAC (1990) and Van Soest et al. (1991). Animals were weighed on two consecutive days at the beginning of the experiment and at the end of each period to calculate the live weight change.

Feed samples were dried and ground to pass a 1mm sieve for the rumen incubations. In sacco incubation was made at 12, 24, 48, 72 and 96 h in duplicate to measure feed degradability following the method of Ørskov et al. (1980). Their values were also fitted to the non-linear model DMD = a+b (1-e-ct) following Ørskov and McDonald (1979). DMD is the dry matter disappeared after time (t), “a” is the intercept of the degradation curve at time zero, “b” is the fraction which degrades with time at rate “c” and “a+b” represent potential degradability. The effective dry matter digestibility (ED) was calculated following Ørskov and McDonald (1979) by ED = a+bc[1-e-

(c+k)t]/(c+k), where k = 0,0246 in the case of buffalos (Bartocci et al., 1997) and “a”, “b” and “c” values fit to the DMD = a+b (1-e-ct) as above.Feed samples were also used for measuring OM degradability in vitro at 12, 24, 48, 72 and 96 h by using rumen fluid as described by Goering and Van Soest (1970). In vivo DM, OM and NDF digestibility were determined by faecal collection for 7 days (Mc Donald, 1998). The water extractable DM


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(WEDM) was determined in duplicate for three representative samples of each of the leaves following the procedure described by Ly and Preston (1997). The samples (1 g) were put in bags (50 x 150 mm) made from nylon filter cloth with a pore size of 45 to 55 microns and thereafter washed at random in one, two, three or four consecutive cycles of 30 minutes each. The volume of water used in every cycle was in the ratio of 3 litres per bag. After washing, the dry matter in the residue was estimated by microwave radiation to constant weight.

Data were analyzed by the General Linear Model using the software of Minitab (1998) Comparisons between feeds, and supplementation, were made by the Tukey test.


Feed composition

Rice straw and elephant grass were low in crude protein but a high fiber content, while Sesbania leaves were high in crude protein but low fiber. Thus supplementing Sesbania leaves to the rice straw and elephant grass improved the crude protein content of the diets. Rice straw had a higher lignin content compared to elephant grass.

Rumen parameter, VFA production, bacteria and protozoa populations Ruminal pH was not effected by supplementation (Table 2) and was in the range suitable for the growth and activities of bacteria (Maeng, 1998). Ammonia concentration in the rumen was higher when the buffaloes were fed elephant grass, rather than rice straw, and when the diets were supplemented with Sesbania leaves. The rumen

ammonia concentration reflected the crude protein levels in the diets and was associated with higher numbers of bacteria. VFA concentration was higher on the supplemented diet; however there was no difference between the grass and rice straw diets. These results are similar to the findings of Bitende and Ledin (1996); Kaitho et al. (1998).

Water extractable DM values and in vivo digestibility

Loss of DM after washing and digestibility coefficients were higher for the elephant grass diet compared with the rice straw diet and for the diets supplemented with sesbania leaves (Table 3). There was a close relationship (R2 = 0.91) between the DM loss by washing and the DM digestibility.

The coefficients in the in sacco model of digestibility mostly favoured the grass versus the rice straw and sesbania leaf supplementation versus no supplementation (Tables 4 and 5). However the relationships between digestibility coefficients derived from this model and in vivo digestibility were much lower than when the water extractable DM was the predictor.Nguyen Van Thu and Preston (1999) found that supplement of Sesbania grandiflora leaves to a basal diet of rice straw brought about improvements in the rumen environment and the feed intake of swamp buffaloes. Similarly, Thongsuk et al. (2011) in their results also concluded that a way of using local protein rich trees for a suitable and worthwhile method improved the quality of buffalo feeding systems in the tropics.

CONCLUSIONS AND IMPLICATIONS

Sesbania grandiflora leaf supplementation improved rumen function parameters and


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Table 1. Chemical composition of the experimental feeds (as % of DM, except for DM which is on fresh basis).

DM OM Ash CP CF EE NDF ADF LigninRice straw 79,9 83,8 16,2 4,43 32,0 2,07 69,6 41,4 13,1Elephant grass 11,5 86,3 13,7 9,86 32,1 3,76 71,5 38,3 9,09Sesbania leaves 22,4 90,5 9,50 21,8 - - 37,3 22,5 -Rice straw + Sesbania leaves 65,8 84,2 15,8 8,82 33,6 4,08 56,1 37,2 10,8E. grass + Sesbania leaves 12,6 85,6 14,4 12,1 31,4 4,52 60,3 36,6 10,3

DM = dry matter, OM = organic matter, CP = crude protein, NDF = Neutral detergent fiber, ADF = acid detergent fiber, CF = crude fiber and EE = Ether extract.

Table 2. Effect of Sesbania leaves supplementation on Rumen parameters, VFAs production, bacteria and protozoa population of the experimental buffaloes.

Feed(F) Supplementation (S) Significance level

Ele.grass Rice straw Suppl. No suppl. F S F x SpH 6,62 6,84 6,69 6,77 * ns nsAmmonia, mg/100 ml 18,1 8,11 16,7 9,48 *** *** nsBacteria count, x 109/ml 2,83 2,02 2,76 2,09 ** * nsProtozoa count, x 105/ml 9,94 7,43 9,84 7,54 ns ns nsVFAs, mM 113 101 116 98,5 ns * ns

ns non significant difference, *Significant difference (P<0.05). **Significant difference (P<0.01); ***Significant difference (P<0.001).

Table 3. Effect of the supplement on the washing loss values and digestibility in vivo.

Feed (F) Sesbania (S) Significance level

Elephant grass

Rice straw Sesbania No

sesbania F S F x S

Wash loss value at 45 min, % 25,5 17,0 22,8 19,7 *** * nsWash loss value at 90 min, % 28,2 19,7 26,4 21,6 *** * nsDM digestibility, % 66,6 51,1 60,5 57,2 *** * nsNDF digestibility, % 69,4 57,1 66,2 60,3 *** * nsADF digestibility, % 65,6 48,6 58,9 55,3 *** ns ns

ns non significant difference, * Significant at P<0.05, ** Significant at P<0.01; *** Significant at P<0.001.


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Table 4. Effect of sesbania supplementation on coefficients of the in sacco rumen degradation of OM.

Feed (F) Sesbania (S) Significance level

Elephant grass Rice straw Sesbania No sesbania F S F x Sa, % 15,9 5,36 10,7 10,6 ns ns nsb, % 54,7 57,9 54,3 58,2 ns ns nsa+b, % 70,6 63,2 65,0 68,9 * ns nsc, %/h 3,95 2,98 3,00 3,94 ns ns ns

A intercept of degradation curve at time zero, b fraction which degrades with time at rate c and a+b represent potential degradability of y = a+b(1-e-ct). ns non significant difference, * Significant at P<0.05.

Table 5. Effect of sesbania supplementation on DM digestibility in sacco at 48 h.

Feed (F) Supplementation (S) Significance level

Ele.grass Rice straw Suppl. No suppl. F S F x Sa, % 23,0 15,2 19,0 19,2 ** ns Nsb, % 51,7 60,2 59,5 52,4 * ns *a+b, % 74,7 75,4 78,6 71,5 ns ns Nsc, %/h 2,82 1,75 2,86 1,71 * * NsED, % 50,0 38,1 46,6 41,5 *** ** Ns

A intercept of degradation curve at time zero, b fraction which degrades with time at rate c and a+b represent potential degradability of y = a+b(1-e-ct); ED effective digestibility; ns non significant difference, *Significant difference (P<0.05). **Significant difference (P<0.01); ***Significant difference (P<0.001).


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coefficients of digestion in swamp buffaloes fed elephant grass or rice straw as the basal diet. The water extractable DM was a better predictor of in vivo DM digestibility. Farmers could use sesbania leaves to improve performance of their buffaloes due to the supplement being available in their gardens.

ACKNOWLEDGEMENTS

Financial support of this work was provided by SAREC/Sida under the MEKARN project. The author would like to thank the Department of Animal Husbandry, Faculty of Agriculture, Cantho University, Vietnam for use of their facilities. The author also would like to thank Dr. T.R. Preston and Dr. Brian Ogle and Mr. Börje Ericson for their kind help.

REFERENCES

Association of Official Analytical Chemistry, AOAC. 1990. Official Methods of Analysis, 15th (ed.) Washington, DC. 1: 69-90.

Barnett, A.J.G. and R.L. Reid. 1957. Studies on the production of volatile fatty acids from grass by rumen liquor in an artificial rumen. The volatile fatty acid production from grass. J. Agric. Sci. Cam., 48: 315-321.

Bartocci, S., A. Amici, M. Verna, S. Terramoccia and F. Martillotti. 1997. Solid and fluid passage rate in buffalo, cattle and sheep fed diets with different forage to concentrate ratios. Livest. Prod. Sci., 52: 201-208.

Bitende, S.N. and L. Inger. 1996. Effect of doubling the amount of low quality grass hay offered and supplementation with Acacia tortilis

fruits or Sesbania sesban leaves, on intake and digestibility by sheep in Tanzania. Livest. Prod. Sci., 45: 39-48.

Dehority, B.A. 1984. Evaluation of subsampling and fixation procedures used for counting rumen protozoa. Appl. Environ. Microb., 48: 182-185.

Gorering, R.J. and P.J. Van Soest. 1970. Forage fiber analysis (apparatus, reagents and some application). Agriculture Handbook No. 379, United States Department of Agriculture, National Academic, Washington DC. 1-19.

Kaitho, R.J., A. Tegegne, N.N. Umunna, I.V. Nsahlai, S. Tamminga, J. Van Bruchem and J.M. Arts. 1998. Effect of Leucaena and Sesbania supplementation on body growth and scrotal circumference of Ethiopian highland sheep and goats fed teff straw basal diet. Livest. Prod. Sci., 54: 173-181.

López, S., J. Dijkstra and J. France. 2000. Prediction on energy supply in ruminant, with emphasis on forage, p. 63-94. In Givens, D.I., E. Owen, R.F.E. Axford and H.M. Omed. (eds.) Forage Evaluation in Ruminant Nutritive, CAB International, UK.

Ly, J. and T.R. Preston. 1997. An approach to the estimation of washing losses in leaves of tropical trees. Livestock for Rural Development, 9(3).

Maeng, W.J., H.J. Kim, M.B. Chang and H. Park. 1998. Control of rumen fermentation through microbial population change, p. 511-517. In Proceeding Pre-conference Symposia, The 8th World Conference on Animal Production. Seoul. Korea.

McDonald, P., R.A. Edwards, J.F.D. Greehalgh and C.A. Morgan. 1998. Digestibility evaluation of foods, p. 220-237. In Animal Nutrition 5th


237

ed. Longman, Harlow, UK.Minitab. 1998. Minitab Reference Manual Release

12.21. Minitab Inc.Nguyen, C.D. 2012. Effect of the parent live

weight and intensive feeding on growth and meat production of swamp buffaloes. Ph.D. Thesis, National Institute of Animal Sciences. Livestock in Vietnam, Vietnam.

Nguyen, H.D. 1998. A study of economic and technical criteria of raising swamp buffaloes in the South of Vietnam. B.Sc. Thesis, Can Tho University (Vietnamese), Vietnam.

Nguyen, V.T. and T.K.D. Nguyen. 2015. Effect of dietary crude protein levels supplemented by multi-nutrient cakes on feed intake, rumen parameters and nitrogen retention of Lai Sind cattle. Journal of Science, Can Tho University (Vietnamese), 37(1): 11-17.

Nguyen, V.T. and T.R. Preston. 1999. Rumen environment and feed degradability in swamp buffaloes fed different supplements. Livestock Research for Rural Development, 11(3).

Ørskov, E.R. and I. McDonald. 1979. The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agr. Sci., 92: 499-503.

Ørskov, E.R, F.D. De B. Hovell and F. Mould. 1980. The use of nylon bag technique for the evaluation of feedstuffs. Trop. Anim. Health Pro., 5: 195-213.

Thongsuk, J., C. Vongpipatana, S. Usawang and S. Thongruay. 2011. The use of tropical protein-rich leaves as supplements to Thai swamp buffalo receiving a basal diet of rice straw and treated leucaena (Leucaena leucocephala). Trop. Anim. Health Prod., 43(1): 57-67.

Van Soest, P.J., J.B. Robertson and B.A. Lewis. 1991. Symposium: Carbohydrate methodology, metabolism and nutritional implications in dairy cattle: methods for dietary fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci., 74: 3585-3597.

Warner, A.C.I. 1962. Enumeration of rumen micro-organisms. J. Gen. Microbiol., 28: 119-128.


239

ABSTRACT

The study was aimed to assess the effect of varicosity and pregnancy on the blood flow parameters in 22 cranial tibial veins of 17 buffaloes using B-mode and Doppler ultrasonography. Out of these, five buffaloes were suffering from unilateral hind limb varicosity and 12 were clinically healthy non-pregnant (n=6) or in advanced stages of pregnancy (n=6). The study highlights a significant increase in the Doppler blood flow parameters of varicosity affected veins in comparison to clinically healthy cranial tibial veins, with highest percentage change in volume flow/minute. A significant lower vessel diameter and end diastole velocity of the cranial tibial vein was also recorded in healthy advanced pregnant compared to that of healthy non-pregnant buffaloes.

Keywords: ultrasound, varicose, bovine, saphenous, pregnant, cranial tibial

INTRODUCTION

Varicosity is the condition of veins which may develop on the tail (Tyagi and Singh, 2001; Kulkarni et al., 2005), teat and udder (Tyagi and Singh, 2001; Rambabu et al., 2009; Larde et al.,

2013), fore limb, hind limb, vulva, and scrotum in bovine animals (Tyagi and Singh, 2001). Clinically, varicose veins may appear enlarged, twisted and tortuous. The etiopathogeny of varicose veins is not clearly defined but, as per the medical literature, it may be congenital or pregnancy induced and is more commonly reported in females than in males (Barros Junior et al., 2010). Persistent pressure on the walls of the vein, make them stretched and less flexible. This leads to weakening of valves which causes leaking of blood backward, stasis and dilation of vein resulting in varicosity.

Anatomically, the cranial tibial vein (CTV) is joined by dorsal pedal and the dorsal metatarsal vein. Proximally, CTV joins with caudal tibial vein and drains blood into the femoral vein along with the saphenous vein in bovine (Pasquini et al., 2003). Although varicosity of cranial tibial vein and its lower veins has been reported in buffaloes (Tyagi and Singh, 2001), but scanty work to study its pathogenesis or treatment has been done. In humans, Doppler ultrasonography of varicose veins is done to study the pattern of incompetence at superficial and deep venous junctions, incompetent perforators and their distribution to aid in the selection of surgical or non-surgical treatment of the patients (Irodhi et al., 2011). Medical literature correlates limb varicosity with pregnancy (Barros Junior et al., 2010). Limb varicosity in buffalo is

Original Article

VARICOSITY AND PREGNANCY INDUCED BLOOD FLOW CHANGES IN THE CRANIAL TIBIAL VEIN IN BUFFALOES: B-MODE AND DOPPLER ULTRASOUND STUDY

Vandana Sangwan*, Jitender Mohindroo, Ashwani Kumar and Shashi Kant Mahajan

Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab, India, *E-mail: [email protected]



240

sometimes life threatening as the affected vein might gets injured or burst due to high pressure of blood in it. The preliminary work on the objective documentation of varicosity affected cranial tibial vein could be of value in assessing severity of disease by determining the changes in the blood flow patterns as compared to that of normal vein. As per author’s knowledge, there is paucity of literature on the B-mode and Doppler ultrasonography of normal or varicosity affected cranial tibial vein in bovine animals. Considering the above facts, the present study was planned with the following objectives: 1. To evaluate the difference in the Doppler blood flow parameters of varicosity affected cranial tibial vein and the contra lateral healthy vein of the varicosity affected buffaloes. 2. To evaluate the effect of pregnancy on the blood flow parameters of cranial tibial vein. 3. To compare the change in the blood flow parameters of the contra lateral clinically healthy CTV of varicosity affected buffaloes and clinically healthy non-varicosity affected buffaloes.

MATERIALS AND METHOD

Twenty-two CTVs were evaluated using B-mode and Doppler ultrasonography in 17 adult she-buffaloes. The study included: CTV of clinically healthy non-gravid buffaloes (Group 1, n=6), clinically healthy gravid (third trimester) buffaloes (Group 2, n=6), varicosity affected gravid buffaloes (Group 3a, n=5) and normal contra lateral CTV of varicosity affected gravid buffaloes (Group 3b, n=5). The buffaloes in Group 3 were suffering from unilateral varicosity in hind limb (Figure 1). Signalment, history of trauma and clinical examination findings were recorded.

Radiography of the metatarsal region was done in buffaloes having history of injury. In Group 3 buffaloes, lateral aspect of both limbs, from mid tibia to mid metatarsal region was prepared for ultrasonography (Figure 2). Similar site of the right hind limb was prepared for ultrasonography in Group 1 and 2 buffaloes. The buffaloes were restrained in a cattle crate in standing position. The B mode and Doppler ultrasonography was done using a 7 to 12 MHz linear multi-frequency transducer on Wipro Logiq III expert ultrasound machine with in-built function for color flow and pulsed wave Doppler.

The B-mode scanning was aimed to visualize the morphology of the vein and valves. The vein was scanned in the longitudinal view and the color-flow mode was switched-on to visualize the flow of blood within the vessel lumen. Gain and pulse repetition frequency (PRF) adjustments were made to minimize color flow aliasing. The MD cursor was placed at the site where pulse Doppler was desired. When the flow was visualized, the pulsed-wave mode was switched-on. The angle of incidence was kept at 60º and the other parameters such as sample volume and PRF were set, as required. A mean of three values, taken at three different sites in the distal tibial region, were used for statistical analysis. Variables of Doppler study including peak systole (PS) cm / s, end diastole (ED) cm / s, TA Max cm/s, pulsality index (PI), resistive index (RI), acceleration (Acce) cm / s2 and TA Mean cm/s were automatically determined by the machine software upon freezing the frame. The diameter of the vessel from the intima to the intima and distance of the vessel from the transducer was determined electronically using in-built caliper of the machine. The diameter of the vessel was further used by the machine software to calculate volume flow in ml/minute.


241

The results of Doppler ultrasonography in Group 1, 2, 3a and 3b were statistically analyzed for mean and standard deviation (SD) using SPSS 16.0 software. Difference between the values of Doppler parameters of vessels in Group 1 and 2, Group 3a and 3b, Group 1 and 3b, Group 2 and 3b were tested using 2-tailed Student’s t-test at 0.05 and 0.01 level of significance (p). The percent change in the Doppler values of varicosity affected and its contra lateral vein was also calculated.


The buffaloes of Group 3 had a mean ± SD body weight 538±59.75 kg (range 450 to 660 kg) which was comparable to that of buffaloes in Group 1 and 2 (Table 1). The buffaloes in Group 3 were adult, aged between 6 and 10 year and were in third trimester of pregnancy. Feeding, urination and defecation were normal in all the buffaloes. The duration of varicosity ranged from one month to one year. Three buffaloes had a history of injury in the affected limb. Clinical examination revealed involvement of cranial tibial, dorsal pedal and dorsal metatarsal vein, extending up to mid tibial region. The affected limbs were painful on palpation and blood could be felt gushing up and down in the veins.

The skin was hard, dry and keratinized in the ventral portion of the limb, but in between the folds of the bulge, it was moist. In humans also venous hypertension is associated with skin pigmentation, lipodermatosclerosis and ulcerations (Irodhi et al., 2011). The buffaloes showed partial weight bearing at rest and mild lameness from affected limb while walking. The veins were more engorged and tortuous, in the lower limb near hoof, leading to bulging of skin. This bulging

had led to a moist area under the fold, which was infested with maggots in four buffaloes. Maggots had lead to kicking and irritation in the buffaloes, thus injuring the limb further. The varicosity was limited to hind limbs only, but not to same side of the body. Buffaloes of Group 3a had varicosity in left (n=3) or in right hind limb (n=2). Lateral radiograph of the metatarsal region revealed mild periosteal reaction (n=2) or lytic changes and cortical thickening (n=1).

Medical literature supports multiple theories for the occurrence of limb varicosity with pregnancy and its disappearance with dead fetus or delivery (Barros Junior et al., 2010). But, in buffaloes, limb varicosity is usually huge and the animals are always in some stage of pregnancy except for a few weeks in a year. Moreover, the telephonic follow up did not reveal resolution of varicosity, though some improvement in the wounds was noticed after calving. Mechanical compression of the uterus on the iliac veins and inferior vena cava, especially in the last trimester of pregnancy, hormonal theory, increase in pelvic circulation during pregnancy, hereditary predisposition, structural alterations in the wall of vessel and venous valve alterations are some of the possible etiologies for the limb varicosity explained in medical literature. Etiology of varicose veins may be post thrombotic or congenital or acquired arteriovenous fistulae (Barros Junior et al., 2010). However, in the present study, the etiology could not be ascertained as the buffaloes were not kept with the same owner throughout the life and were presented in the advanced stage of varicosity. But, history of trauma in three buffaloes of this study suggests the condition may be secondary or acquired.


242

Tabl

e 1.

Com

paris

on o

f sig

nalm

ent a

nd D

oppl

er u

ltras

onog

raph

y va

riabl

es (M

ean

± SD

) of c

rani

al ti

bial

vei

n in

hea

lthy

non-

grav

id (G

roup

1),

heal

thy

grav

id (G

roup

2),

varic

ose

(Gro

up 3

a) a

nd n

orm

al c

ontra

late

ral v

ein

of v

aric

osity

affe

cted

buf

falo

es (G

roup

3b)

.

S.

No.

Vari

able

sG

roup

1(n

=6)

Gro

up 2

(n=6

)G

roup

3b(

n=5)

Gro

up 3

a (n

=5)

% C

hang

e in

Gro

up 3

a co

mpa

red

to 3

bM

ean±

SD

Ran

geM

ean±

SDR

ange

Mea

n±SD

Ran

geM

ean±

SDR

ange

1B

ody

wei

ght (

Kg)

495.

00±5

5.77

(4

50.0

0 to

600

.00)

571.

83±8

4.29

(450

.00

to 6

50.0

0)53

8.00

±59.

75(4

50.0

0 to

660

.00)

538.

00±5

9.75

(450

.00

to 6

60.0

0)N

ot a

pplic

able

2G

esta

tion

perio

d (M

onth

s)N

ot a

pplic

able

6.67

±1.0

3 (6

.00

to 8

.00)

6.

40±1

.34

(5.0

0 to

8.0

0)6.

40±1

.34

(5.0

0 to

8.0

0)N

ot a

pplic

able

3A

ge (Y

ear)

5.50

±1.0

5 (4

.00

to 7

.00)

5.17

±1.1

7 (4

.00

to 7

.00)

7.

80±1

.48

(6.0

0 to

10.

00)

7.80

±1.4

8(6

.00

to 1

0.00

)N

ot a

pplic

able

4Pe

ak S

ysto

le v

eloc

ity (

PS)

cm /

s10

.36±

4.27

(5

.46

to 1

5.91

)8.

04±2

.39

(5.9

0 to

12.

28)

14.7

6±5.

09$

(9.5

5 to

21.

19)

127.

01±4

5.12

**

(52.

69 to

166

.26)

760.

50

5En

d D

iast

ole

velo

city

(ED

) cm

/ s

8.19

±3.3

9 (4

.75

to 1

3.06

)4.

28±1

.68^

(2.3

8 to

6.3

9)

8.45

±4.0

6(4

.07

to 1

2.43

)76

.45±

28.2

9**

(26.

68 to

97.

72)

804.

73

6TA

max

cm

/ s

7.88

±5.0

5 (1

.80

to 1

4.34

)5.

02±2

.01

(2.4

1 to

8.5

7)9.

37±5

.58

(3.3

1 to

14.

59)

91.5

6±32

.59**

(36.

65 to

123

.49)

877.

16

7Pu

lsal

ity In

dex

(PI)

1.97

±2.0

1 (0

.27

to 4

.86)

2.45

±1.2

0 (0

.98

to 4

.34)

1.

55±1

.24

(0.4

3 to

2.9

0)1.

04±0

.63

(0.4

5 to

2.0

2)

-33.

55

* S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 3

a &

3b,

at p

<0.0

5,

**

Sign

ifica

nt d

iffer

ence

bet

wee

n G

roup

3a

& 3

b, a

t p<0

.01.

^ S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 1

& 2

at p

<0.0

5,

$ S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 2

& 3

b at

p<0

.05,

†† Si

gnifi

cant

diff

eren

ce b

etw

een

Gro

up 1

& 3

b at

p<0

.01


243

Tabl

e 1

(con

tinue

d). C

ompa

rison

of s

igna

lmen

t and

Dop

pler

ultr

ason

ogra

phy

varia

bles

(Mea

n ±

SD) o

f cra

nial

tibi

al v

ein

in h

ealth

y no

n-gr

avid

(G

roup

1),

heal

thy

grav

id (G

roup

2),

varic

ose

(Gro

up 3

a) a

nd n

orm

al c

ontra

late

ral v

ein

of v

aric

osity

affe

cted

buf

falo

es (G

roup

3b)

.

S.

No.

Vari

able

sG

roup

1(n

=6)

Gro

up 2

(n=6

)G

roup

3b(

n=5)

Gro

up 3

a (n

=5)

% C

hang

e in

Gro

up 3

a co

mpa

red

to 3

bM

ean±

SD

Ran

geM

ean±

SDR

ange

Mea

n±SD

Ran

geM

ean±

SDR

ange

8R

esis

tive

Inde

x (R

I)0.

79±0

.79

(0.1

7 to

1.8

7)0.

70±0

.25

(0.3

7 to

1.1

1)0.

46±0

.12

(0.3

1 to

0.5

8)0.

52±0

.21

(0.3

1 to

0.8

2)

13.0

4

9A

ccel

erat

ion

cm /

s²30

.81±

33.4

8(6

.78

to 9

8.11

)11

2.99

±135

.84

(22.

28 to

348

.25)

38.6

8±8.

64(2

7.59

to 5

0.04

)92

7.27

±647

.73*

(219

.37

to 1

720.

92)

2297

.29

10TA

mea

n in

cm

/ s

3.89

±2.3

3(0

.83

to 7

.42)

2.57

±0.9

9 (0

.94

to 3

.97)

4.22

±3.4

7(0

.05

to 7

.58)

53.7

1±18

.45**

(23.

02 to

69.

45)

1172

.75

11Vo

lum

e flo

w in

ml /

min

116.

03±8

9.17

(30.

51 to

280

.09)

51.5

7±25

.97

(17.

60 to

93.

99)

62.5

7±61

.64

(0.7

4 to

132

.06)

5489

.47±

4594

.95*

(164

7.45

to 1

1908

.94)

8673

.33

12Ve

ssel

dia

met

er in

cm

0.74

±0.1

1 (0

.62

to 0

.90)

0.59

±0.1

4^

(0.4

1 to

0.7

9)0.

57±0

.062

††

(0.4

7 to

0.6

3)1.

58±0

.56*

(1.0

4 to

2.3

1)17

7.19

13D

ista

nce

of v

esse

l fro

m th

e tra

nsdu

cer i

n cm

0.77

±0.3

8 (0

.16

to 1

.34)

0.68

±0.2

0 (0

.46

to 1

.00)

0.67

±0.1

0(0

.57

to 0

.63)

0.62

±0.1

3(0

.50

to 0

.84)

-7.4

6

* S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 3

a &

3b,

at p

<0.0

5,

**

Sign

ifica

nt d

iffer

ence

bet

wee

n G

roup

3a

& 3

b, a

t p<0

.01.

^ S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 1

& 2

at p

<0.0

5,

$ S

igni

fican

t diff

eren

ce b

etw

een

Gro

up 2

& 3

b at

p<0

.05,

†† Si

gnifi

cant

diff

eren

ce b

etw

een

Gro

up 1

& 3

b at

p<0

.01


244

B-mode ultrasonographyIn standing position, the CTV in Group

3a, was superficial and distended, but, in Group 3b, 2 and 1 it was mildly palpable at the distal end only. The lateral aspect of CTV was scanned for ultrasonography, as the lower limb was very painful. The CTV of Group 3a was in-collapsible than that of other groups. Maximum of three valves were seen in the Group 3a, which were very flaccid and were lying close to the vessel wall with mild fluttering (incompetent valves). The valves were not able to close the vessel lumen completely. The number of valves visualized in Group 3b, 1 and 2 ranged from 1 to 3 with only one valve seen in 12 out of 17 CTVs. The increase in the lumen diameter and the in-collapsible nature of the vein in Group 3a might be the reasons for better visualization of valves compared to that of Group 1, 2 and 3b. Studies on human patients reveal agenesis or hypoplasia of the iliac-femoral valve, which supports the hydrostatic pressure of a blood column from the heart to the inguinal region, may result in unilateral varicosity (Junior et al., 2010). The failure of check valves in the perforating veins, allows high pressure generated in the deep veins by the muscular contractions to be transmitted directly to the unsupported superficial veins (Irodhi et al., 2011).

The mean ± SD vessel diameter of Group 3a buffaloes was 1.58±0.56 cm (range 1.04 to 2.31) which was significantly (p=0.012) higher than that of Group 3b (Figure 3). A 177.19 % change in vessel diameter of CTV in Group 3a than that of Group 3b was recorded (Table 1). The CTV wall was significantly (p=0.008) thicker in Group 3a (0.31±0.029 cm) than that of Group 3b (0.08±0.02 cm) due to keratinization of the wall. Sacculations were seen in the varicose vein while a smooth, uniform diameter was observed in Group 3b, 1 and 2

buffaloes. Chronic stasis of blood leads to dilatation and sacculations in the affected vein resulting in the increase in wall thickness and diameter of the vein (Junior et al., 2010). No echogenic mass or thrombi within the vessel lumen were detected in any of the varicosity affected CTV. Thrombosed tarsal vein in cattle due to metastatic abscessation has also been reported to be incompressible and had an increase in vessel diameter which progressively reduced to normal within 5 month (Kofler et al., 1996; Kofler and Kubber-Heiss, 1997).

The vessel diameter of healthy CTV in advanced pregnant buffalo of Group 3b (p=0.009) and Group 2 (p=0.058) was found to be significantly less than that of non-pregnant buffaloes in Group 1 (Figure 3). This decrease in CTV diameter was in contrast to Boivin et al. (2000) findings in human females where the diameter of competent and incompetent veins increased in third trimester compared to first trimester which returned back to initial values in puerperium. This could be attributed to the difference in the standing posture of humans (bipedal) and bovine animals (quadripedal).

Color Doppler ultrasonographyVenous reflux with a mixture of red and

blue color was visualized in CTV of Group 3a (Figure 4), while minimum or no color flow was observed in CTV of Group 3b, 1 and 2. Venous reflux is the retrograde flow of blood in the veins caused by absent or incompetent valves (Irodhi et al., 2011). Even the spectral flow was missing in certain veins of healthy animals which were not included in the present study. It was noticed that partial weight bearing improved the blood flow in healthy CTV (Figure 5). In human patients also, the Doppler ultrasound examination of the limb veins is preferred in standing position, while supporting weight on the contra lateral extremity (Irodhi et al.,


245

2011). But, in buffaloes it was difficult to make the animal support weight on one hind limb.

Differences in the values of doppler parameters of CTV between various groups

All the velocities, PS (p=0.005), ED (p=0.006), TA max (0.005), Acce (p=0.036) and TA mean (p=0.004) were recorded to be significantly higher in the vein of Group 3a than that of Group 3b (Figure 6). The volume flow/minute in the vein of Group 3a was significantly higher (p=0.035) than in Group 3b which was also highest in percent change (8673.33%) among all the Doppler variables recorded in the present study (Figure 7). Detailed mean ± SD Doppler values with percent change are given in Table 1.

A significant increase in ED (p=0.038)

and vessel diameter (p=0.058) was observed in the CTV of Group 1 than that of Group 2 which might be related delayed emptying of the blood due to pregnancy and less vessel diameter. However, except vessel diameter no significant change was observed in the Group 1 and 3b.

Among pregnant buffaloes (Group 3b and 2), only PS was found to be significantly (p=0.038) higher in Group 3b however, other Doppler variables were comparable.

Along with the tibial, the dorsal pedal and the dorsal metatarsal veins are more affected being more ventrally located. Till now surgical intervention is not reported as a treatment modality in varicosity of cranial tibial vein in buffaloes. Though, varicosity of coccygeal veins can be treated through tail amputation at an early stage

Figure 1. Photograph showing hind limb varicosity in a buffalo.

Figure 2. Photograph showing localization of CTV in buffalo.


246

^

$

0

0.5

1

1.5

2

2.5

vessel diame ter

healthy non-gravid healthy gravid contralateral to varicose gravid varicose gravid

Figure 3. Bar graph showing comparison between the vessel diameter of CTV in buffalo. (^= significance between healthy gravid and non-gravid at P<0.05, $ = significance between varicose and contra lateral healthy CTV at P<0.05) .

Figure 4. Spectral display showing Doppler scan of varicose CTV in a buffalo.


247

^

$$ $

$$$ $

$

0

20

40

60

80

100

120

140

160

180

PS ED TA max TA mean Healthy non-gravid healthy gravid

contralateral varicose (healthy gravid) varicose gravid

Figure 5. Spectral display of healthy CTV (contra lateral to the varicose CTV) in a buffalo.

Figure 6. Bar graph showing comparison between the velocities of CTV in buffalo. {^= significance at 5% between healthy gravid and non-gravid, $$= significance at 1 % level of significance between varicose and healthy CTV (gravid, non-gravid and contra lateral)}.


248

in buffaloes. In medical science, there are various modalities available for the treatment of varicosity. In humans long standing jobs are considered to be an inciting cause for varicosity. Now days in India, buffaloes are not left loose for pasture feeding; but are tied to one post and have very little space for movement. Long standing posture, pregnancy, injury to the limb or any congenital defect may be the reason for varicosity. The study highlights that since, a large volume of blood is stacked in the varicosity affected limb and the animal is in continuous pain and irritation, there is a need to understand the predisposing factors and etio-pathogenesis of the condition and to formulate effective preventive or therapeutic protocol for varicosity of CTV in buffaloes.

CONCLUSION

The study concludes that pregnancy lowered the diameter and ED of CTV in buffaloes. A significant increase in Doppler blood flow parameters occurred in varicosity affected CTVs in comparison to clinically healthy veins in buffaloes.

ACKNOWLEDGMENTS

Authors acknowledge the financial support provided by the University Grant Commission, India and the Professor-cum-Head, Department of Veterinary Surgery and Radiology, GADVASU, Ludhiana for providing facilities to conduct this study. Scrutinizing the manuscript for grammatical and language corrections by Mrs. Balvinder Tuli, Former Head, Department of English, G.N.N

$

0

1000

2000

3000

4000

5000

6000

Volume flow

non-gravid healthy gravid healthy

contralateral varicose (healthy gravid) varicose gravid

Figure 7. Bar graph showing comparison between the volume flow of CTV in buffalo. ($= significance at 5% level of significance between varicose and healthy CTV (gravid, non-gravid and contra lateral).


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College, Doraha, Ludhiana (Punjab), India is also duly acknowledged.

REFERENCES

Boivin, P., A. Cornu-Thenard and Y. Charpak. 2000. Pregnancy-induced changes in lower extremity superficial veins: an ultrasound scan study. J. Vasc. Surg., 32: 570-574.

Irodhi, A., S.N. Keshava, S. Agarwal, I.P. Korah and D. Sadhu. 2011. Ultrasound Doppler evaluation of the pattern of involvement of varicose veins in Indian patients. Indian J. Surgery, 73(2): 125-130.

Junior, N. de B., M.D.C.J. Perez, J.E. de Amorim and F.M. Junior. 2010. Pregnancy and lower limb varicose veins: prevalence and risk factors. J. Vasc. Bras., 9(2): 29-35.

Kofler, J., A. Buchner and A. Sendlhofer. 1996. Application of real time ultrasonography for the detection of tarsal vein thrombosis in cattle. Vet. Rec., 138(2): 34-38.

Kofler, J. and A. Kubber-Heiss. 1997. Long-term ultrasonographic and venographic study of the development of tarsal vein thrombosis in cow. Vet. Rec., 140(26): 676-678.

Kulkarni, M.D., A.S. Kadam, A.V. Khanvikar and O.N. Ladukar. 2005. Vein varicosis in a Pandharpuri buffalo - A case report. Buffalo Bull., 24: 2.

Larde, H., S. Nichols, A. Desrochers, M. Babkine, D. Francos, P.Y. Mulon and Y. Couture. 2013. Milk flow obstruction caused by varicose vein of the teat in dairy cattle. Vet. Surgery, 42: 885-891.

Pasquini, C., T. Spurgeon and S. Pasquini. 2003. Anatomy of Domestic Animals, 10th ed. Sudz Publishing, USA. 435p.

Rambabu, K., M. Sreenu, R.V.S. Kumar and T.S.C. Rao. 2009. Ultrasonography of the udder and teat in buffaloes. Buffalo Bull., 28: 5-10.

Tyagi, R.P.S and J. Singh. 2001. Ruminant Surgery. CBS Publishers, New Delhi. 259p.


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ABASTRACT

In order to evaluate the effect of different antibiotics in extender a study was conducted on 36 ejaculates from six Murrah buffalo bulls maintained at Artificial Breeding Research Centre (ABRC), National Dairy Research Institute (NDRI), Karnal. Each ejaculate wassplit in to three parts and the each part was diluted in Tris egg yolk dilutor with separate combination of antibiotics [Conventionally used Benzyl penicillin (1000 IU/ml) + Streptomycin (1000 ug/ml) (PS), Benzyl penicillin (500 IU/ml) + Streptomycin (1000 ug/ml) + Gentamicin (500 ug/ml) (PSG) and Benzyl penicillin (500 IU/ml) + Streptomycin (1000 ug/ml) + Amikacin (1000 ug/ml) (PSA)]. The microbial load and sperm quality was evaluated in refrigerated (0, 24 and 48 h) and cryopreserved (at -196oC) semen. The results depicted that individual sperm motility, sperm abnormalities, percent intact acrosome, HOST was significantly higher and lowest bacterial load was observed in semen fortified with benzyl penicillin, streptomycin and amikacin combination at refrigerated temperature, but the differences in bacterial load between different treatment combinations were

not significant. The results revealed that benzyl penicillin, streptomycin and amikacin combination had higher efficacy in maintaining the individual sperm motility, percent intact acrosome and HOST, but non-eosinophilic counts and sperm abnormalities were higher in semen treated with amikacin combination. No significant difference in bacterial load was observed in different antibiotic combination treatments in cryopreserved semen, but lowest values of bacterial load were observed in semen treated with benzyl penicillin, streptomycin and amikacin combination. Overall the results suggested that fortification of semen extender with benzyl penicillin, streptomycin and amikacin combination would have positive effect on preservability of semen.

Keywords: antibiotic, bacterial load, buffalo semen, preservability, semen quality

INTRODUCTION

AI using frozen semen technology is an important biotechnological tool for the rapid improvement in milk production and germplasm

Original Article

EFFECT OF DIFFERENT ANTIBIOTIC COMBINATIONS IN EXTENDER ON BACTERIAL LOAD AND SEMINAL CHARACTERISTICS OF MURRAH BULLS

G.S. Meena1, M. Bhakat1,*, V.S. Raina1, A.K. Gupta2, T.K. Mohanty1 and R. Bishist3

1Artificial Breeding Research Centre (ABRC), Indian Council of Agricultural Research (ICAR), National Dairy Research Institute (NDRI), Karnal, India, *E-mail: [email protected] Cattle Breeding Division (DCB), Indian Council of Agricultural Research (ICAR), National Dairy Research Institute (NDRI), Karnal, India3Veterinary Science, Dr. Y.S. Parmar University of Horticulture and Forestry, NauniSolan, Himachal Pradesh, India


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improvement. The success of AI technique is associated with effective prolongation of fertile life of spermatozoa obtained from high pedigree bulls under in vitro storage condition. However, there in inherent problem of quality semen production in buffalo due to poor libido, seasonality, low epididymal sperm reserve, low sperm harvest, freezability, contamination of micro-organisms and other foreign particles. Further, unhygienic surroundings, repeated entry of penis into AV, improper handling of semen and inflammatory condition of reproductive system adversely influence the microbial quality of semen. The AI in buffalo is not as popular as in cattle because there is low fertility rate with frozen semen (Shukla and Misra, 2007). Even under best conditions, some microbial contamination can occur as the microbes have ample access to contaminate the semen during collection, processing and preservation stages. Further, the extender used for extension of semen is rich in nutrients most suitable for the growth of microorganisms. Bacteria present in the ejaculates can affect fertilization directly (Morrell, 2006).

Addition of antibiotics in freezing diluents may affect the viability or fertility of cryopreserved bovine spermatozoa by controlling the bacterial load (Morrell, 2006). As it has been proven that bacteria in buffalo semen are resistant to single antibiotics like penicillin (Ahmed et al., 2001). So recently combination of antibiotics for preservation of semen has been advocated (Akhter et al., 2008). Though it is known that antibiotics reduce bacterial load in any subject including semen but scanty information is available on the use of different combinations of antibiotics with specific dose on the preservability of buffalo bull semen. Therefore, the present study was conducted to compare the efficacy of alternate combinations of antibiotics vis-à-vis the antibiotics conventionally used in

semen extender for preservation of semen of Murrah buffalo bulls.


Thirty six ejaculates (initial motility >70%) of six Murrah buffalo bulls maintained at Artificial Breeding Research Centre of National Dairy Research Institute (NDRI) Karnal, were collected twice a week from each bull using sterilized artificial vagina during spring season. Each ejaculate wassplited in to three parts and the each part was diluted in Tris egg yolk dilutor with separate combination of antibiotics. The combinations were Benzyl penicillin (1000 IU/ml) + Streptomycin (1000 ug/ml) (PS) as control as well Benzyl penicillin (500 IU/ml) + Streptomycin (1000 ug/ml) + Gentamicin (500 ug/ml) (PSG) and Benzyl penicillin (500 IU/ml) + Streptomycin (1000 ug/ml) + Amikacin (1000 ug/ml) (PSA) antibiotic combinations. Thereafter, each of three parts was further splitted into two equal parts. Out of these one part of each was placed in refrigerator at 5oC and other part was subjected to freezing for further analysis. Semen was analysed for motility, non-eosinophilic counts, acrosome integrity and plasma membrane integrity by hypo osmotic swelling test. The microbial load and semen quality was evaluated in different hours of refrigeration (0, 24 and 48 h) and frozen (at -196oC) thawed semen. The bacterial count /ml of the sample were estimated by multiplying the dilution factor with the mean number of colonies in media plates (Bacterial load /ml = Bacterial colonies counted on plate X Dilution factor). The percent data on semen characteristics were subjected to arcsine transformation and microbial count was subjected to log transformation for analysis (Snedecor and


253

Cochran, 1994). Data was analyzed by two-way ANOVA with interaction and least squares analysis technique(Harvey, 1975).


Least squares means of individual sperm motility, percent non-eosinophilic counts, HOS reacted spermatozoa and total plate counts in different treatment groups at 0, 24 and 48 h of preservation at refrigerated temperature as well as pre freeze and post thawed semen samples in different treatment groups are presented in Table 1 and 2.

Preservation of semen at 5oCThe overall least squares means of

individual sperm motility (%) estimates in semen preserved at 5oC for 0, 24 and 48 h are presented in Table 1. At 0 h of preservation, individual motility was significantly higher in PSA group. However, at 24 and 48 h of preservation no significant difference was observed in individual motility in different groups.

The of average non-eosinophilic sperm (%) estimates in semen preserved at 5oC for 0, 24 and 48 h are presented in Table 1. After 24 h of preservation the non-eosinophilic sperm percent were significantly higher in PSA compared to PS group; however, there was no significant difference among other treatment combinations. At 0 and 48 h of preservation there was no significant difference in non-eosinophilic sperm percent among different antibiotic combinations. The total sperm abnormalities at 0, 24 and 48 h of preservation are presented in Table 1. After 0, 24 and 48 h of preservation, the total sperm abnormalities were significantly lower in PSA group as compared to

PS and PSG.The percent values of intact acrosome at 0, 24 and 48 h of preservation are presented in Table 1. At 0 and 48 h of preservation there was no significant difference in the percent intact acrosome values. After 24 h of preservation, intact acrosome values were significantly higher in PSA than PSG group. It is clear from the findings that the combination of antibiotics used has no damaging effect on sperm survival. Results of present study are in agreement with those of Bhakat and Raina (2001). The liveability of the spermatozoa was sustained till 48 h of storage with a decrease in motility. The acrosomal damage due to preservation at refrigerated temperature observed in the present study was in conformity with the findings of various workers (Bhakat and Raina, 2001) who also reported increased acrosomal damage with the process of semen preservation at 4oC to 7oC temp. The average HOST (%) estimates in semen preserved at 5oC. After 0 and 24 h of preservation, the HOST percent values were significantly better in PSA than PS and PSG. After 48 h of preservation, there was no significant difference in HOST values in different antibiotic combinations treatment groups. The decrease in quality with increase in storage hours are in consonance with the finding of Azawi and Ismaeel (2012) in ram semen.

The average microbial load (10 X) in semen preserved at 5oC after 0, 24 and 48 h are presented in Table 1. After 0, 24 and 48 h of preservation, no significant difference was observed among all antibiotic combinations. However, the values of microbial load were lower in PSA followed by PSG and PS. The reduction in bacterial count in semen samples following addition of different combinations of antibiotics are in agreement with the studies of Yániz et al. (2010) and Azawi and Ismaeel (2012).


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Table 1. Mean ± SE for semen characteristics (%) and bacterial load in refrigerated semen (up to 48 h) preserved with different antibiotic combinations in extenders.

Time Quality TestsAntibiotics

PS PSG PSA

0 h

IM 73.51a±0.01 74.52a±0.01 76.54b±0.01N-eosin 83.41±0.01 83.70±0.01 84.69±0.02

SPA 8.87a ±0.00 8.48a±0.01 5.80b±0.00IA 89.38±0.01 87.630.01 90.02±0.02

HOST 54.18a±0.01 55.48a±0.01 60.44b±0.01TPC 173.08±1.17 149.12±1.12 127.32±1.10

24 h

IM 55.06±0.01 56.90±0.01 59.70±0.01N-eosin 63.76a±0.01 66.33ab±0.01 68.16b±0.01

SPA 8.50a±0.43 8.67a±1.43 7.17b±0.79IA 74.84ab±0.01 72.24b±0.02 76.66a±0.01

HOST 48.60a±1.68 49.97a±3.71 52.52b±2.48TPC 37.43±0.02 38.30±0.02 40.01±0.02

48 h

IM 46.71a±0.02 47.99b±0.01 49.58b±0.01N-eosin 46.71a±0.02 47.99b±0.01 49. 58b±0.01

SPA 13.16a±0.00 12.15a±0.00 10.20 b±0.00IA 57.54±0.02 55.65±0.01 56.18±0.03

HOST 31.55±0.02 32.39±0.02 34.27±0.02TPC 100.45±1.16 94.18±1.12 72.79±1.09

PS = Penicillin + Streptomycin; PSG = Penicillin + Streptomycin + Gentamicin; PSA = Penicillin + Streptomycin + Amikacin IM: Individual sperm motility (%); N-eosin: Non-eosinophilic count (%); SPA: Total sperm abnormalities (%); HOST: Hypo-osmotic swelling reactivity (%);IA: Intact acrosome (%); TPC: Total plate count (X10) (cfu/ ml).Values with different superscripts within a column differ significantly.


255

Preservation of semen at -196oCIndividual motility values in pre freeze and

post thaw semen are presented in Table 1 and Table 2. In pre freeze semen the percent motility values were significantly higher in PSA than PS and PSG. Similarly, in post thawed semen percent individual motility was significantly better in PSA than PS and PSG groups. The present findings are in agreement with those of Ronald and Prabhakar (2001); Ahmed and Mohan (2002) who reported that the sperm motility treated with Amikacin and Norfloxacin before and after freezing was significantly higher than control.

The percent values of non-eosinophilic sperm in pre freeze and post thaw semen are presented in Table 1 and Table 2. In pre freeze semen no significant difference was observed for live spermatozoa values in different treatment groups, but in post haw semen, percent non-eosinophilic sperm values were significantly higher in PSA and PSG than PS. However, no significant difference was observed between PSA and PSG

groups. The percent total abnormality values in pre freeze and post thaw semen are presented in Table 1 and Table 2. In pre freeze semen, the percent total abnormalities were significantly lower in PSA than PS and PSG. In post thaw semen, the percent total abnormalities were significantly higher in PS as compared to PSG and PSA. The difference between PSA and PSG was not significant.

The percent intact acrosomes in pre freeze and post thaw semen are presented in Table 1 and Table 2. In both pre freeze and post thawed semen, no significant difference was observed in percent intact acrosome values of different treatment groups. However, in both pre freeze and post thawed semen PSA group had highest intact acrosome values. The acrosomal damage due to cryopreservation observed in the present study is in conformity with various workers (Nehring and Stähr, 2001) who reported increased acrosomal damage with the process of semen freezing. HOST percent values in pre freeze and post thaw semen are presented in Table 1 and Table 2. In pre freeze

Table 2. Mean ± SE for semen characteristics (%) and bacterial load in post freeze semen with different antibiotic combinations in extender.

Quality TestsAntibiotics

PS PSG PSAIM 46.28a±0.01 49.60 b±0.01 50.93 b±0.01

N-eosin 54.92a±0.01 57.79b±0.01 58.13b±0.00SPA 19.16a±0.43 14.63b±1.43 14.54b±0.79IA 61.35ab±0.01 63.30b±0.02 65.28a±0.01

HOST 38.20a±0.01 42.83 b±0.01 42.84b±0.01TPC 115.57±1.18 97.95±1.18 76.43±1.18

PS = Penicillin + Streptomycin; PSG = Penicillin + Streptomycin + Gentamicin; PSA = Penicillin + Streptomycin + AmikacinIM: Individual sperm motility (%); N-eosin: Non-eosinophilic count (%); SPA: Total sperm abnormalities (%); HOST: Hypo-osmotic swelling reactivity (%);IA: Intact acrosome (%); TPC: Total plate count (X10) (cfu/ ml).Values with different superscripts within a column differ significantly.


256

semen, HOST percent values were significantly better in PSA than PSG and PS. In post thaw semen, the HOST percent values were significantly better in PSA and PSG than PS. However, no significant difference in HOST percent values was observed between PSG and PSA groups.

The microbial load values (X 10) in pre freeze and post thaw semen are presented in Table 1 and Table 2. In pre freeze and post thaw semen, no significant differences were observed among different antibiotic combinations. However, values of microbial load were lowest in PSA group followed by PSG and PS. Results of present study are in accordance with the earlier studies of Ahmed and Mohan (2002), who reported better efficacy of Amikacin and penicillin in reducing the bacterial counts and improving the motility of semen. Studies are also akin with the report of Ronald and Prabhakar (2001) who revealed that Amikacin, Chloramphenicol and Gentamicin were effective against isolated organisms. Antibiotic addition helps in reduction of microbial population, otherwise there was interference with motility (Diemer et al., 1996) due adherence of microorganism with spermatozoa and impair sperm quality and fertilizing capacity (Griveau et al., 1995; Thibier and Guerin, 2000; Morrell, 2006) due to production of reactive oxygen species and toxins (Fraczek et al., 2007) as well as induce acrosome reaction (El-Mulla et al., 1996).

CONCLUSION

It can be concluded that there is scope of substitution of conventionally used penicillin and streptomycin antibiotics with a combination of benzyl penicillin, streptomycin and amikacin antibiotics in extenders, which have positive effect

on preservability of semen. Antibiotic addition is essential to prevent proliferation of microorganisms in ejaculated spermatozoa.

ACKNOWLEDGEMENT

Authors are thankful to Director, ICAR, NDRI to provide the necessary funds to carry out the research programme.

REFERENCES

Ahmed, K. and G. Mohan. 2002. Effect of antibiotics on the bacterial load and quality of semen of Murrah buffalo bulls at different stages of freezing. Indian J. Anim. Sci., 72(2): 138-139.

Ahmed, K., A.A. Kumar and G. Mohan. 2001. Bacterial flora of preputial washing and semen of Murrah buffalo bulls and their antibiotic sensitivity pattern. Indian Journal of Comparative Microbiology, Immunology and Infectious Diseases, 22(1): 63-64.

Akhter, S., M.S. Ansari., S.M.H. Andrabi., N. Ullah and M. Qayyum. 2008. Effect of antibiotics in extender on bacterial and spermatozoal quality of cooled buffalo (Bubalus bubalis) bull semen. Reprod. Domest. Anim., 43: 272-278.

Azawi, O.I. and M.A. Ismaeel. 2012. Influence of addition of different antibiotics in semen diluent on viable bacterial count and spermatozoal viability of Awassi ram semen, Vet. World, 5(2): 75-79.

Bhakat, C. and V.S. Raina. 2001. Effect of preputial washing and antibiotic treatment on bacterial load and preservability of


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frozen bovine semen. Indian J. Anim. Sci., 71: 1127-1130.

Diemer, T., W. Weidner, H.W. Michelmann, H.G. Schiefer, E. Rovan and F. Mayer. 1996 Influence of Escherichia coli on motility parameters of human spermatozoa in vitro. Int. J. Androl., 19(5): 271-277.

El-Mulla, K.F., F.M. Kohn and M. Dandal. 1996. In vitro effect of Escherichia coli on human sperm acrosome reaction. Arch. Androl., 37: 73-78.

Fraczek, M., A. Szumala-Kakol, P. Jedrzejczak, M. Kamieniczna and M. Kurpisz. 2007. Bacteria trigger oxygen radical release and sperm lipid peroxidation in vitro model of semen inflammation. Fertil. Steril., 88: 1076-85.

Harvey, W.R. 1975. Least Squares Analysis of Data with Unequal Sub-class Numbers. ARS H-4, USDA, Washington, DC.

Morrell, J.M. 2006. Update on semen technologies for animal breeding. Reprod. Domest. Anim., 41: 63-67.

Nehring, H. and B. Stähr. 2001. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract. Achieves of Animal Breeding, 44: 121.

Ronald, B.S.M. and T.G. Prabhakar. 2001. Bacterial analysis of semen and their antibiogram. .Indian J. Anim. Sci., 71: 829-831.

Shukla, M.K. and A.K. Misra. 2007. Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation. Anim. Reprod. Sci., 97: 175-179.

Snedecor, G.W. and W.G. Cochran. 1994. Statistical Methods, 6th ed. Oxford and IBH Publ. Co., New Delhi.

Thibier, M. and B. Guerin. 2000. Hygienic aspects

of storage and use of semen for artificial insemination. Anim. Reprod. Sci., 62: 233-251.

Yániz, J.L., M.A. Marco-Aguado, J.A. Mateos and P. Santolaria. 2010. Bacterial contamination of ram semen, antibiotic sensitivities, and effects on sperm quality during storage at 15oC. Anim. Reprod. Sci., 122: 142-149.

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