Livestock Science 139 (2011) 271–276

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Nutrient utilization and milk yield response of early lactating Nili-Ravibuffaloes fed on urea–molasses treated wheat straw fermented withcattle manure

Zia-ul-Hassan, M. Aasif Shahzad⁎, M. Nisa, M. SarwarInstitute of Animal Nutrition and Feed Technology, University of Agriculture, Faisalabad-38040, Pakistan

a r t i c l e i n f o

⁎ Corresponding author at: Institute of Animal Nutriogy, University of Agriculture, Faisalabad, Pakistan. Tel.: +

E-mail address: aasifshah9@hotmail.com (M. Aasif

1871-1413/$ – see front matter © 2011 Elsevier B.V.doi:10.1016/j.livsci.2011.01.008

a b s t r a c t

Article history:Received 21 September 2010Accepted 14 January 2011

This study was aimed to examine the influence of urea–molasses treated wheat straw (WS)fermented with cattle manure (CM) with 4% urea and 4% molasses incubated for 40 days onits chemical composition and varying substitution levels of fermented wheat straw (FWS)with concentrate on nutrients intake and their digestibilities, milk yield and its composition inNili-Ravi buffaloes. Twenty early lactating buffaloes were randomly divided into four groups ina randomized complete block design. Four iso-nitrogen and iso-caloric diets were formulated.There was no replacement of concentrate with FWS in control (FWS 0) diet while FWS 15, FWS25 and FWS 35 dies contained 15%, 25% and 35% replacement of concentrate with FWS,respectively. Nutrients intake remained unaltered (PN0.05) by lactating buffaloes fed dietscontaining varying levels of FWS. However, neutral detergent fiber and acid detergent fiberdigestibilities increased (Pb0.05) in buffaloes fed FWS20 and FWS30 diets than those fedFWS10 and FWS0 diets. Apparent digestibilities of DM, organic matter and CP remainedunchanged (PN0.05) in buffaloes fed different levels of FWS. Similar trend was noticed for milkyield (4% fat corrected milk). The percent milk fat, CP, total solids and solids not fat and theiryields were non-significant (PN0.05) in buffaloes fed varying amounts of FWS. The blood andmilk urea nitrogen (BUN and MUN) didn't differ among Nili-Ravi buffaloes fed diets containingvarying levels of FWS. This study implied that urea–molasses treated WS fermented with CMreplaced 30% dietary concentrate without affecting the milk yield and its quality by earlylactating buffaloes.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Fermented wheat strawCattle manureMilk compositionBuffaloes

1. Introduction

In developing countries, nutritional requirements of live-stock are usually met through crop residues characterized byhigh fiber, low nitrogen and energy. Effective and economicalsources of nitrogen (N) are needed as supplements for betterusage of crop residues in dairy diets (Sarwar et al., 2002).Oilseeds in spite of effective supplements are impractical due tohigh cost. Moreover, ever increasing human population hasconstantly been decreasing the supply of concentrate feed for

tion and Feed Technol-92 41 9201088.Shahzad).

All rights reserved.

ruminant production, due to strong completion with cashcrops. Efficient utilization of existing feedstuff and strongstruggle to explorenew feed resources are in graveneedof timefor better livestock productivity. Enhancement of the nutritivevalue of dry roughages with physical, chemical or biologicaltreatments has been the focus in the last decades (Nisa et al.,2004). However, the involvement of chemicals and healthprecautions has always been a constant constraint in theirextensive use (Khan et al., 1992).

Animal wastes represent a largely unused and almostneglected source of N which is available in sufficient quantitieswhich may be used to satisfy most of the supplementaryprotein needs for livestock production. In Pakistan, about40 million heads of cattle and buffalo produce 400 million ton/

272 Z.- Hassan et al. / Livestock Science 139 (2011) 271–276

annum, about 28 kg/head/day (Sarwar et al., 2002; Haq andMasoud, 1966). Cattle manure (CM) can be used for feedingeither in dry form, chemically treated fresh manure or ensiledwith crop residuesorwithother feed ingredients. Fermentationof urea plusmolasses treatedwheat straw (WS)with CMmightbe an important tool to improve the nutritive value of wheatstraw.

However, scientific information regarding effect of urea–molasses treated wheat straw ensiled with manure and itsfeeding value for lactating Nili-Ravi buffaloes is limited.Therefore, the present study was conducted to evaluate theeffect of urea–molasses treated wheat straw ensiled withmanure on DM and CP content and the influence of varyingsubstitution levels of FWSwith concentrate on nutrients intakeand digestibility, milk yield and its composition in Nili-Ravibuffaloes.

2. Materials and methods

2.1. Preparationof fermentedwheat strawand laboratory analysis

Wheat straw was thoroughly mixed with CM in the ratioof 70:30. Urea (4%) and molasses (4%) were mixed in knownquantity of water, which was later on adjusted to maintain50% moisture in treated WS. This solution was uniformlysprinkled on mixture of WS and CM. Then the mixture of WS,CM, urea and molasses was thoroughly mixed, packed incemented pits (15′×8′×6′) and material in these pits wasallowed to ferment for 40 days. Pits were covered with 4inches thick layer of rice straw, followed by plastic filmcovering, which was plastered with a blend ofWS andmud toavoid any cracking on drying (Nisa et al., 2004). It wasassumed that plastic film and mud plastering providedanaerobic conditions. The plastic film was removed after40 days and a sample of FWSwas withdrawn. After taking thesample from the pit, the plastic film was put back to keep thepit sealed. The sample of FWS was analyzed for DM and CP.The net energy for lactation (NEL) was calculated by theequations derived by Conrad et al. (1984).

After completion of fermentation period, the DM of FWSwas analyzed by drying it at 105 °C in a forced draft oven untila constant weight was attained (AOAC, 1990). Percent N(total-N and TCA insoluble-N) and crude protein wereestimated by Kjeldhal method (AOAC, 1990). Crude proteinwas calculated by multiplying N% with factor 6.25 (AOAC,1990). The NDF and ADF were also analyzed according to theprocedure described by Van Soest et al. (1991).

2.2. Animals, diets and data collection

Twenty early lactating (20±5 days) Nili-Ravi buffaloeswere randomly divided in four groups using completelyrandomized design (CRD). Four iso-nitrogenous and iso-caloricrations having 0% (FWS0), 10% (FWS10), 20% (FWS20) and 30%(FWS30) FWS were formulated (NRC, 2001) (Table 2).

The experiment lasted for 120 days. First 15 days was givenfor dietary adaptation and remaining 105 days was given forsample collection. Feed intake and milk production wererecorded daily. Buffaloes were milked twice daily, andindividual milk yields were recorded. Milk samples werecollected at two consecutive milking (pm and am) fortnightly

with 12-h interval and preserved in 2-bromo-2-nitro-propane-1-3-diol and kept refrigerated (6 °C) until analysis (Johnson etal., 2002).

Milk samples were analyzed for fat, total solids (TS), solidsnot fat (SNF), CP (AOAC, 1990) and milk urea-N (MUN) bycolorimetric assay (Broderick, 1986). Blood samples werealso collected from jugular vein at 3, 6, 9 and 12 h post-feeding fortnightly and transferred immediately to laboratoryin icebox for blood urea-N (BUN) analysis (Broderick andKang, 1980).

During the lastweek, a digestibility trialwas conducted. Theacid insoluble ash was used as a digestibility marker (VanKeulen and Young, 1977). Fecal grab samples were taken twicedaily such that a sample was obtained for every 3-h interval of24-h period (Khan et al., 2004). Feed offered and orts weresampleddaily and compositedby animal for analysis. Diets, ortsand fecal samples were analyzed for DM, N (AOAC, 1990), NDF(Van Soest et al., 1991) and ADF (Goering andVan Soest, 1970).

2.3. Laboratory analysis

Dry matter was analyzed by drying it at 135 °C until aconstant weight was reached (method 930.15; AOAC, 1990).Protein-N of FWS was analyzed using an acidified extract(20 g of fresh sample in 200 ml of 0.01 NHCl, agitated at 21 °Cfor 22 h) and deproteinized with trichloracetic acid (TCA;Novozamsky et al., 1974). Nitrogen fractions (Total-N, TCAinsoluble-N) of WS, CM and FWS were done by the Kjeldahlmethod (955.04; AOAC, 1990). Crude protein was calculatedby multiplying %N with factor 6.25 (method 984.13; AOAC,1990). Non-protein-N and TP contents of other than FWSwere analyzed following methods of AOAC (1990), 991.21and 991.23, respectively.

Acid detergent fiber was determined using acetyl-trimethylammonium bromide detergent in 0.5 M sulfuric acid (Goeringand Van Soest, 1970). Neutral detergent fiber was determinedusing sodium sulfite and amylase [Van Soest et al., 1991(method A for NDF)].

Total-N in milk was estimated by Kjeldhal method(920.105; AOAC, 1990). Milk CP contents were calculated bymultiplying %Nwith 6.38. Milk TS were determined by heatingmilk sample on steam bath (10–15 minutes), followed byheating at 98–100 °C for 3 h in a hot air oven (925.23; AOAC,1990). Milk fat was determined by using fat extraction tubesfollowing the method of Roese Gottlieb (905.02; AOAC, 1990).Solids not fat were calculated by the difference of TS and milkfat. Fat correctedmilk (FCM;4% fat)was calculated as describedby Tyrrell and Reid (1965) using equation; 4% FCM=milk (kg/day)×(44.01×milk fat%+163.56)/339.60. Milk samples weredeproteinizedwith sulfosalicylic acid and stored at−20 °C andanalyzed for urea (Broderick, 1986).

For BUN, plasma was collected by centrifugation at 1500 ×gfor 40 minutes and stored at −4 °C until it was analyzed forurea. Urea-N in the plasma was determined following conver-sion of urea to ammonia (NH3) with urease. Thawed plasma(0.25 ml) was brought to room temperature (20 °C) andincubated for 10 minutes with 1.5 ml of a urease solution(4.03 U/ml, Sigma Chemical Co). The reaction was terminatedby vortexing with 0.15 ml of 65% (wt/vol) TCA and incubatingon ice for 30 minutes. After centrifugation at 21,000 ×g for

Table 2Ingredients and chemical composition of diets (DM basis).

273Z.- Hassan et al. / Livestock Science 139 (2011) 271–276

10 minutes, urea in the supernatantwas determined (Broderickand Kang, 1980).

Ingredients, % Diets a

FWS0 FWS10 FWS20 FWS30

Wheat straw 30.00 30.00 30.00 30.00Fermented wheat straw b 0.00 10.00 20.00 30.00Sunflower meal 25.00 25.00 25.00 25.00Wheat bran 21.00 21.50 9.90 2.89Rice polish 4.70 0.00 0.00 0.00Pop corns 3.90 3.90 3.90 3.90Molasses 12.00 5.00 4.99 0.00

2.4. Statistical analysis

Data collected regarding nutrients intake, their digestibil-ities, milk yield and its composition were analyzed statisticallyin randomized completely block design using analysis ofvariance technique. Means were separated for significance byusing Duncan's multiple range test (Steel and Torrie, 1984).

Corn oil 0.00 1.50 3.00 5.05Dicalcium phosphate 2.00 2.00 2.00 2.00Urea 1.40 1.10 1.21 1.16

Chemical composition (% of DM)Dry matter 83.52 82.92 79.02 74.18Organic matter 73.55 72.30 67.03 60.06Crude protein 16.15 15.88 15.85 16.16True protein 10.48 11.87 13.84 14.16Neutral detergent fiber 52.47 54.14 57.32 58.09Acid detergent fiber 29.41 32.24 32.79 34.88NEL, Mcal/kg 1.54 1.50 1.49 1.48

a The diets FWS0, FWS10, FWS20 and FWS30 contain 0%, 10%, 20% and 30%DM from FWS as a replacement of concentrate.

b

3. Results

3.1. Chemical composition of FWS

Dry matter content decreased in FWS after 40 days ofincubation. The CP of 4% urea and 60% molasses treated WSfermented with CM increased compared to unfermented WS(Table 1). The TP content of FWS also increased from 0.23% to9.21% compared to WS. The NDF and ADF contents decreasedin WS fermented with CM.

4% urea plus 4% molasses treated WS ensiled with CM (70:30 DM) for40 days.

Table 3Dry matter, organic matter, crude protein, neutral detergent fiber and aciddetergent fiber intakes and their digestibilities by Nili-Ravi buffaloes fed dietcontaining different levels of fermented wheat straw.

Items Diets a

FWS0 FWS10 FWS20 FWS30 SE

Dry matter (DM)

3.2. Lactation trial

Nutrient intakes of diets containing different levels of FWSare given in Table 3. Organic matter (OM), DM, CP, NDF andADF intakes by lactating buffaloes fed diets containingvarying levels of FWS remained unaltered (PN0.05). TheNDF and ADF digestibilities in lactating buffaloes weresignificantly higher (Pb0.05) with FWS20 and FWS30 dietsthan those of FWS10 and FWS0 diets. However, the non-significant (PN0.05) differences in apparent DM, OM and CPdigestibilities have been observed in lactating buffaloes feddifferent levels of FWS (Table 3).

Milk yield (4% FCM) by buffaloes fed diets containingdifferent levels of FWSwas similar (Table 4). The percentmilkfat, CP, TS and SNF and their yields were non-significantly(PN0.05) in buffaloes fed varying amounts of FWS. The BUNand MUN in Nili-Ravi buffaloes fed diets containing varyinglevels of FWS were similar (Table 4). The graphical presen-tation of BUN trend with different diets is shown in Fig. 1.

Table 1Chemical composition of wheat straw, molasses, cattle manure and fermentedwheat straw (DM basis).

Items, % Wheatstraw

Molasses Cattlemanure

FWS a

Dry matter 93.58 77.24 19.74 59.40Crude protein 2.34 2.77 7.98 15.10True protein 0.23 – 3.01 9.21Non-protein nitrogen 0.34 – 0.80 1.00Neutral detergent fiber 84.04 – 52.14 48.82Acid detergent fiber 55.62 – 32.16 29.53NEL, Mcal/kg 1.02 1.18 1.42 1.47

a Fermented wheat straw.

4. Discussion

4.1. Chemical composition of FWS

Decrease in DM of FWS might be attributed to anaerobicmicrobial activity to degradeDM for theirmultiplication duringfermentation period (40 days). These results are consistentwith Singh et al. (1985), who reported decreased DM ofroughageswith increased fermentation period. An increased CP

Intake, kg/day 8.83 10.51 9.32 7.95 0.47Apparent DM digestibility, % 60.76 63.26 60.33 59.60 1.97

Organic matter (OM)Intake, kg/day 6.49 7.59 6.34 4.77 0.45Apparent OM digestibility, % 65.14 66.82 64.14 62.73 2.15

Crude protein (CP)Intake, kg/day 1.43 1.68 1.47 1.27 0.07Apparent CP digestibility, % 78.12 79.42 76.63 76.14 1.10

Neutral detergent fiber (NDF)Intake, kg/day 4.63 5.69 5.35 4.62 0.25Apparent NDF digestibility, % 47.34b 48.22b 51.81 a 52.55 a 0.72

Acid detergent fiber (ADF)Intake, kg/day 2.59 3.39 3.06 2.77 0.15Apparent ADF digestibility 45.41b 42.95b 50.23 a 50.61 a 1.07

a The diets FWS0, FWS10, FWS20 and FWS30 contain 0%, 10%, 20% and 30%DM from FWS as a replacement of concentrate.

Table 4Milk yield and composition of Nili-Ravi buffaloes fed diets containingdifferent levels of fermented wheat straw.

Items Treatments a

FWS0 FWS10 FWS20 FWS30 SE

Buffaloes, n 5 5 5 5Milk yieldkg/day 9.16 9.81 9.98 10.18 0.354% FCMb 10.37 11.47 11.67 11.94 0.38

Milk composition, %Fat 4.91 5.11 5.81 5.51 0.16Protein 2.75 3.40 4.77 3.49 0.32Total solids 14.25 14.12 15.10 14.41 0.26Solids not fat 9.94 9.75 10.54 9.94 0.23

Production, kg/dayFat 0.44 0.50 0.58 0.56 0.02Protein 0.28 0.39 0.56 0.42 0.04Total solids 1.47 1.63 1.76 1.72 0.06Solids not fat 1.03 1.12 1.23 1.19 0.05BUN c, mg/dl 22.59 24.33 25.78 27.01 2.16MUNd, mg/dl 21.66 24.29 26.94 28.11 3.99

a The diets FWS0, FWS10, FWS20 and FWS30 contain 0%, 10%, 20% and 30%DM from FWS as a replacement of concentrate.

b 4% FCM calculated as Tyrrell and Reid (1965).c Blood urea nitrogen.d Milk urea nitrogen.

274 Z.- Hassan et al. / Livestock Science 139 (2011) 271–276

content of FWS after 40 days of incubation might be attributedto sufficient availability of time and nutrients for microbialmultiplication. These findings are in linewith that of Khan et al.(1992), who reported increased CP content of sugarcanebaggassewhen ensiledwith CM. They reported that CP contentincreased from 18.4% to 22.2% in sugarcane baggasse whenfermented for 60 days, respectively. Moreover, increase in CPmight be attributed to more availability of NH3-N, as a result ofmoreureahydrolysis. TheNH3-Nafterfixing inFWSmighthaveincreased its CP. These results are in line with that of Lopez-Guisa et al. (1991), who reported that increased CP content ofFWS with increasing molasses level might be due to theavailability of fermentable carbohydrates and suitable growthmedia for microbial multiplication leading to more NH3-Nfixing in FWS (Lopez-Guisa et al., 1991). Energy is usually thelimiting nutrient for growth of anaerobic microbes andprovision of urea and molasses might have increased themicrobial mass that leads to increased CP and TP (Staples et al.,1981). Thus provision of carbon skeleton and energy formicrobial growth in FWS might have synchronized with NH3

released from urea hydrolysis, consequently increasing thebacterial growth, CP and TP. The ammoniation has also beenreported to increase the CP (Heinrichs and Conrad, 1984; Dasset al., 2000). They explained that WS after being ammoniatedincreased the N content by breaking the linkage betweenhemicellulose and lignin of WS.

4.2. Lactation trial

The results of present study are consistent with thosereported previously by Dass et al. (2001), Mehra et al. (2001)and Nisa et al. (2004), who observed no difference in DMintake, when they fed the urea treated WS ensiled withfermentable sugars or organic acids to buffaloes. However,

Parasad et al. (1998) and Wanapat et al. (2000) reportedhigher DM intake by lactating cattle fed urea or NH3 treatedstraw than those of untreated straw. The lack of difference innutrient intakes in present studymay be because all diets hadapproximately similar NDF and ADF concentration, whichprobably have similar passage from the alimentary canal.Sarwar et al. (2004) reported increased intake of urea treatedWS ensiled with fermentable carbohydrates and attributedthis increase to favorable changes in fiber fraction thatoccurred during ensilation. They explained faster digestionrate of urea treated WS ensiled with fermentable carbohy-drate that resulted in higher DM intake compared with ureatreated WS ensiled without fermentable carbohydrates. Non-significant difference in nutrient intake also indicated thatfermentation of urea plus molasses treated WS with CMnullified the gut-filled effects of WS that limits the intakewhen fed to buffaloes (Nisa et al., 2004).

The lack of difference in DM and CP digestibilities inlactating buffaloes fed varying levels of FWS was probablybecause of the similar ruminal rate of disappearance anddigestion of FWS and concentrates. HigherNDFdigestibilities inlactating buffaloes fed FWS diets compared with FWS0 dietmight be because of better ruminal pH and NH3 concentrationas it was noted in our previous studywhen similar substitutionof FWS was made with concentrate in buffalo bulls diets(unpublished data). The WS diet contained 70% DM fromconcentrate that might have lowered the ruminal pH (NRC,2001) and which consequently might have reduced thecellulolytic activity (Khan et al., 2004). This low cellulolyticactivity might have lowered the NDF and ADF digestibilitieswith FWS0 diets. The higher NDF and ADF digestibility withFWS20 and FWS30 diets may be attributed to the betterutilization of NDF. Rates of NDF disappearance and extent ofdigestion followed similar trend as NDF degradability of dietscontaining FWS (unpublished data). A negative relationshipbetween degree of lignification and cell wall digestion inforages was well established (Williams et al., 1984). Anincreased surface area of lingo-cellulose in urea treated WSensiled with fermentable carbohydrates (Sarwar et al., 2004)resulted in increased accessibility to microbial attack (Streeterand Horn, 1982; Lewis et al., 1987). Fermentation of urea plusmolasses treated WS with CM has probably increased thefragility of straw (Zorilla-Rios et al., 1985) and thus itsdigestibility.

Although the differences in 4% FCM production were non-significant, the tendency for a linear increase by buffaloes feddiets with decreasing nonstructural carbohydrates concen-tration may be attributed to increasing oil in the diets tomaintain NEL. Such findings were previously described bymany workers (Cant et al., 1991; Sarwar et al., 1992a,b).However, the FWS has comparable digestibility and intake tothat of concentrate, which did not cause any significantchange in 4% FCM yield when substituted for concentrates inthe diets of lactating buffaloes. Rakes et al. (1978) alsodemonstrated that milk yields of Holstein cows were notdepressed when screened manure solids provided up to 33%of the fiber in blended complete diets. Arave et al. (1990) feddifferent levels of processed poultry excreta (PPE) to thelactating cows and reported higher 4% FCM yield across allPPE diets. They reported that feeding lactating dairy cows upto 17% poultry excreta had no effect on FCM yield.

FWS0

20.00

22.00

24.00

26.00

28.00

30.00

3.00 6.00 9.00 12.00

Hours

BU

N (

mg/

dl)

FWS10

20.00

22.00

24.00

26.00

28.00

30.00

3.00 6.00 9.00 12.00

Hours

BU

N (

mg/

dl)

FWS20

20.00

22.00

24.00

26.00

28.00

30.00

3.00 6.00 9.00 12.00Hours

BU

N (

mg/

dl)

FWS30

20.00

22.00

24.00

26.00

28.00

30.00

3.00 6.00 9.00 12.00Hours

BU

N (

mg/

dl)

Fig. 1. Effects of different diets1 on the blood urea nitrogen in Nili-Ravi buffaloes.

275Z.- Hassan et al. / Livestock Science 139 (2011) 271–276

The BUN and MUN values observed in Nili-Ravi lactatingbuffaloes during this study were higher than those reportedpreviously (Broderick et al., 1993) for cattle. These higher ureavalues of blood and milk may be due to species difference(Sarwar et al., 1997). Broderick et al. (1993) reported higherurea concentrations in blood and milk in lactating cows whenfed urea treated corn silage compared with control diet. Urea isthe primary form of excretory-N in mammals, and concentra-tions of BUN have long been known to reflect efficiency ofdietary CP utilization by ruminants (Lewis, 1957). Ureaequilibrates rapidly throughout body fluids, including milk.The concentration of MUN, therefore, is thought to reflect theconcentration of BUN (Rook and Thomas, 1985). The MUN andBUN are the useful indices of ruminal NH3 concentrations atsimilar dietary CP percentages in the diet (Oltner andWiktorsson, 1983; Broderick, 1986; Broderick et al., 1990). Inthe present study, all experimental diets have similar concen-tration of CP that might have similar ruminal degradability. Asimilar BUN and MUN in buffaloes fed diets containing varyinglevels of FWS indicated that release and fixation of NH3 inmicrobialmasswere synchronizedwith available energy in the

1 The diets FWS0, FWS10, FWS20 and FWS30 contain 0%, 10%, 20% and30% DM from FWS as a replacement of concentrate.

rumen that led to similar absorption of excess NH3 in the bloodcirculation and thus urea in milk.

5. Conclusion

From this study, it can be concluded that urea plusmolasses treated WS fermented with CM did not affect thenutrient intake, digestibility, BUN, MUN, milk yield and itscomposition when substituted for concentrate up to 30% DMin lactating buffalo ration. However, extensive feeding trialsinvolvingmore number of buffaloes are warranted before anypractical recommendation.

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