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Livestock Science

Livestock Science 157 (2013) 520–526

1871-14http://d

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journal homepage: www.elsevier.com/locate/livsci

Effect of vitamin E or propolis supplementation on semenquality, oxidative status and hemato-biochemical changesof rabbit bucks during hot season

N.M. Hashema,n, A. Abd El-Hady b, O. Hassan b

a Animal Production Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21526, Egyptb Poultry Production Department, Faculty of Agriculture (El-Shatby), Alexandria University, Alexandria 21526, Egypt

a r t i c l e i n f o

Article history:Received 14 March 2013Received in revised form2 September 2013Accepted 6 September 2013

Keywords:Rabbit bucksSemen qualityBlood parametersOxidative stressVitamin EPropolis

13/$ - see front matter & 2013 Elsevier B.V. Ax.doi.org/10.1016/j.livsci.2013.09.003

esponding author. Tel.: þ20 01288719758;ail address: hashemnesreen@yahoo.com (N.M

a b s t r a c t

This study was designed to determine the effects of supplementation of vitamin E orpropolis to bucks’ diets on semen quality, oxidative status and hemato-biochemicalchanges during summer season. Thirty adult male New Zealand white rabbits wererandomly allocated into three equal groups, 10 bucks each. Bucks received a maintenancediet supplemented by vitamin E (150 mg/kg diet, E-group) or propolis (150 mg/kg diet,P-group) or not supplemented (control, C-group) for ten consecutive weeks. Semen andblood samples were collected to analyze semen quality traits and hemato-biochemicalparameters, respectively. Ambient temperature and relative humidity were recorded dailyand used to estimate the temperature humidity index (THI). Results indicated that bucksin the E- and P-groups had better libido (lower reaction time, Po0.05) and higher spermconcentration and viability than the control group (Po0.01). These enhancements wereparallel to an increase in blood plasma testosterone concentrations in the vitamin E andpropolis-treated groups (Po0.01). Additionally, concentrations of seminal plasma totalprotein and initial fructose were significantly increased in the semen of both treated-groups. For blood components, either vitamin E or propolis supplementation enhancedhematopoiesis including the number of red blood cells, hematocrite value and hemoglo-bin concentration (Po0.01). Also, concentrations of blood plasma glucose and highdensity lipoprotein (HDL) were increased, while those of blood plasma cholesterol andtriglycerides were decreased (Po0.01) in both treated-groups. Supplementation withvitamin E or propolis enhanced (Po0.01) oxidative status of the blood plasma, whereaspropolis-treated bucks had the highest value of total antioxidant capacity followed byvitamin E treated-bucks. Also, malondialdehyde activity was lower (Po0.05) in thepropolis-treated bucks compared with the control, while bucks of the E-group wereintermediate. In summary, inclusion of vitamin E or propolis in male rabbits' diets duringthe hot season could be used effectively to mitigate negative impacts of elevatedtemperature on semen quality, oxidative status and hemato-biochemical features.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Under subtropical conditions, ambient temperature is themajor constraining factor controlling animal productivity. In

ll rights reserved.

fax: þ20 35922780.. Hashem).

Egypt, the summer and early autumn seasons (from Mayto September) are not within the thermal neutral zone(Attia et al., 2011). The comfort zone temperature is around21 1C and their productive and reproductive performancescould be impaired, which in turn prevents continuation ofthe breeding season for about 5 months (García-Tomás et al.,2008). Under such environmental conditions, severalphysiological and reproductive disorders are caused by

N.M. Hashem et al. / Livestock Science 157 (2013) 520–526 521

disturbances in blood metabolites, oxidative status, enzy-matic reactions and hormonal secretions (Alvariño, 2000;Marai et al., 2002). Rising free radicals, reactive oxygenspecies (ROS), seems to be one of the most harmful effectsof elevated temperature that initiates peroxidation of poly-unsaturated fatty acids (PUFA). These are important forsperm plasma membrane integrity and subsequent fertility(Altan et al., 2003; Sahin et al., 2001). In the testes, lowconcentrations of ROS are normally produced during testi-cular spermatogenesis and steroidogenesis, and they arenecessary for different sperm functions such as capacitation,hyperactivation, acrosome integrity and sperm–oocyte fusion(Mathur and D'Cruz, 2011). Overproduction of ROS canimpair sperm integrity causing male infertility (Paul et al.,2009). Since cooling of the housing buildings is expensive, anumber of alternative methods are used to modulate theharmful effects of high environmental temperature; focusingmostly on dietary manipulation. Vitamin E is believed to bethe primary component of the antioxidant system of thespermatozoa, and is one of the major membrane protectorsagainst ROS and lipid peroxidation (Surai et al., 1998).Additionally, vitamin E has been shown to increase totalsperm output and sperm concentration in male rabbits(Castellini et al., 2003).

There is an international interest concerning application ofnatural medical sources in animal production fields. Propolisis a natural resinous substance collected by bees from parts ofplants, buds and exudates (Zhou et al., 2008). About 300components, mainly phenolic compounds, have been identi-fied. Most of these isolated compounds belong to three maingroups, flavonoids, phenolic acids and esters (Simoes et al.,2004). Propolis has several therapeutic properties includingantioxidant, antimicrobial, anti-parasitic, antiviral, anti-inflammatory and anti-tumoral properties (Kumazawa et al.,2004; Paulino et al., 2008). Therefore, this study was designed

Table 1Chemical composition of propolis detected by gas chromatography/mass spectr

Compounds

Trimethylsilyl 3-Phenyl-2-PropenoateDodecanoic Acid, Trimethylsilyl Ester6,7-Dihydroxycoumarin Di-TmsTetradecanoic Acid, Trimethylsilyl EsterHexadecanoic Acid, Ethyl EsterCinnamic Acid, 3,4-Dimethoxy-, Trimethylsilyl EsterHexadecanoic Acid, Trimethylsilyl EsterFerulic AcidSilane,[1-(5-Ethenyltetrahydro-5-Methyl-2-Furanyl)-1-Methylethoxy]TrimethyOctadecanoic Acid, Trimethylsilyl Ester3-Methyl-3-Butenyl Isoferulate-Tms-Derivative2-Propen-1-One, 1-(2,6-Dihydroxy-4-Methoxyphenyl)-3-Phenyl-, (E)1,2,4-Tris(Tert Butyldimethylsiloxy)Naphthalene1h-Imidazole-4-Carboxamide, 5-Amino-, Tetrakis(Trimethylsilyl) Deriv.3-(Hydroxymethyl)-1-Phenyl-1-Heptadecyn-3-Ol2′,4′,6′-Tris(Trimethylsilyloxy)Chalcone4-Quinolinamine, N,3-Diphenyl-2-(Phenylmethyl)Bis-O-Trimethylsilyl-Palmitinic Acid-Glycerin-(1)-MonoesterBenzoic Acid, 2-[(Trimethylsilyl)Amino]-3-[(Trimethylsilyl)Oxy]-, Methyl Ester1h-Pyrrole, 5-(3-Methoxyphenyl)-2,3-Diphenyl2-Methyl-1,6- Bis[(Trimethylsilyl)Oxy] Anthra-9,10-Quinone3,5,7-Tris(Trimethylsilyloxy)FlavoneMeso-Dimethyl-R-3,C-6-Bis[(TertButyldimethylsilyl)Oxy]–T-4,5-Epoxycyclohex

to test the efficiency of propolis comparable to vitamin E inmitigating heat stress effects on semen characteristics, oxida-tive status and hemato-biochemical profile in adult malerabbits.

2. Materials and methods

The present study was conducted in the AgriculturalExperimental Station (311 20′N, 301E), Faculty of Agricul-ture, Alexandria University, Alexandria, Egypt. The proce-dures imposed on the animals were carried out meetingthe Directive 2010/63/EU of the European Parliament andof the Council of 22 September 2010 on the protection ofanimals used for scientific purposes.

2.1. Animals and their management

Thirty, 12 month old, male, New Zealand White rabbitswith a mean body weight of 3.3570.06 kg were used in thepresent study. Bucks were individually kept in wire cages(40�50�35 cm3) in a naturally ventilated house. A period of16 h of light was provided which included 12 h of natural daylight and 4 h of supplementary electric light. Clean fresh waterwas available for rabbits at all times. Bucks were randomlyallocated into three equal groups: (1) control (C-group), bucksreceived the maintenance diet; (2) vitamin E (E-group), bucksreceived the maintenance diet supplemented with 150mg ofvitamin E/kg diet; and (3) propolis (P-group), bucks receivedthe maintenance diet supplemented with 150mg of propolis/kg diet. Both additives were mixed with diet componentsduring manufacturing of the pellets. Treatments were appliedfor 10 weeks. Bucks were individually fed 200 g/d of themaintenance diet (18% barley, 25% wheat bran, 6% yellowcorn, 18% soy bean, 28% alfalfa hay, 3% molasses, 1% CaCo3, 1%NaCl; 17.8% CP and 2650 K cal/Kg DE) (NRC, 1994). Vitamin E

ometry.

Retentiontime (min)

Area of eachcomponent (%)

17.72 3.7120.11 0.6921.95 0.5124.28 3.5827.09 1.7827.90 2.5628.11 24.4228.84 2.68

l-, Cis 29.16 1.0631.59 2.6032.66 2.9634.44 0.8435.36 1.5735.91 1.9436.22 13.7536.33 1.7336.63 11.0337.23 2.6537.39 0.9639.15 1.6139.58 5.9539.74 10.15

-1-Ene-1,2-Dicarboxylate 41.30 1.27

N.M. Hashem et al. / Livestock Science 157 (2013) 520–526522

was obtained from Epico Co., Jordan, while propolis sampleswere collected in March 2011 from experimental apiary atAlexandria Governorate, Northwestern Egypt, dry subtropicalclimate (311 11′ 53″ N, 291 55′ 9″ E). The chemical analysis ofpropolis (Table 1) was carried out with GC–MS spectro-photometry according to the method of Fernández et al.(2008). A Shimadzu Model GC 2010 Series gas chromatographcoupled with a Shimadzu series mass-selective detectorquadruple mass spectrometer model GCMS-QP 2010 wasused. Propolis had high percentages of long chain fatty acids,mainly palmitic acid (24.42%), flavonoids as 3-(Hydroxy-methyl)-1-Phenyl-1-Heptadecyn-3-Ol (13.75%) and flavonesas 3,5,7-Tris (Trimethylsilyloxy) (10.15%).

2.2. Data collection

2.2.1. Temperature humidity index calculationThe means of ambient temperature and relative humidity

were recorded daily during August to mid October using anautomatic hydrothermograph-computerized system (Datalogger-Log 100/110, Germany) located in the rabbitry. Themaximum and minimum values of ambient temperatureand relative humidity of each day were used to estimate thedaily mean of each parameter. The temperature–humidityindex (THI) was calculated using the equation modifiedby Marai et al. (2002) THI¼db 1C�[(0.31–0.31(RH)) (db1C�14.4)], where db 1C¼dry bulb temperature and RH¼relative humidity percentage/100. The mean values ofambient temperature, relative humidity and THI were30.6570.7 1C, 78.570.2% and 29.3570.3, respectively. Themean values of THI obtained in this study were classified assevere heat stress (28.9 to o30.0) according to the classifi-cation of Attia et al. (2011).

2.2.2. Semen collection and evaluationSemen samples were collected every 17 days (days 17, 34,

51 and 68) of the experimental period. Semen samples werecollected using an artificial vagina and a teaser doe. Reactiontime for every buck was calculated using a stopwatch as thetime interval from mounting a doe until complete ejacula-tion. The volume of each ejaculate was recorded afterremoval of the gel mass. Sperm concentration was deter-mined after semen dilution (1:100) with physiological salineby the method of Smith andMayer (1955). Assessment of thepercentage of live and dead spermatozoa was performedusing an eosin–nigrosine blue staining mixture (Blom, 1950).The percentage of motile sperm (progressive motility) wasestimated by visual examination under 40�magnificationsusing a phase-contrast microscope with heated stage. Theinitial fructose concentration in semen was determinedimmediately after collection using commercial kits (Diagnos-tics Biochem, Canada Inc.).

Seminal plasma was obtained by centrifugation ofsemen samples at 700� g for 20 min, at 4 1C, and thenstored at �20 1C. The concentrations of seminal plasmatotal protein and albumin were measured using commer-cial kits (Reactivos GPL, Barcelona, Spain). Seminal plasmaglobulin concentration was obtained by subtracting thevalues of albumin from the corresponding values of totalprotein. Activities of seminal plasma aspartate transami-nase (AST; EC 2.6.1.1) and alanine transaminase (ALT; EC

2.6.1.2) were determined using kits obtained from Reacti-vos GPL, Barcelona, Spain.

2.2.3. Blood collection and hemato-biochemical analysesBlood samples were collected with heparinized tubes

from the ear vein of each buck on days 17, 34, 51 and 68 ofthe nutritional treatment. A part of each sample was used toassess the hematological parameters including red bloodcells count (RBCs), numbers of platelets, white blood cells(WBCs) and its differential count according to Feldman et al.(2000). The percentage of packed corpuscular volume (PCV),mean corpuscular volume (MCV), mean corpuscular hemo-globin (MCH), and mean corpuscular hemoglobin concentra-tion (MCHC) were also calculated according to Feldman et al.(2000). Plasma was separated from blood by centrifugationat 700� g for 20 min and stored at �20 1C. Blood plasmawas analyzed for concentrations of total protein, albumin,glucose, cholesterol, triglycerides, low density lipoprotein(LDL) and high density lipoprotein (HDL) using colorimetricmethod by commercial kits obtained from Reactivos GPL,Barcelona, Spain. Blood plasma globulin was estimated bysubtracting albumin values from the corresponding values oftotal protein. Total antioxidant capacity (TAC) and activity ofmalondialdehyde (MDA) in blood plasma were also analyzed(Reactivos GPL, Barcelona, Spain). Blood plasma testosteroneconcentration was measured using solid-phase enzymeimmunoassay (ELISA) kits obtained from Diagnostics Bio-chem, Canada Inc. The lower limit of assay detection was0.02 ng/ml. The intra- and inter-assay coefficients of varia-tion (CV) were 9.6% and 6.1%, respectively.

2.3. Statistical analysis

Data including hematological and plasma biochemicalparameters and semen characteristics were analyzed usingMIXED procedure for repeated measurements of SAS(2001). In the statistical model, treatment, time of sam-pling and their interactions were included as fixed effects,while buck effect was introduced as a random effect.Percentages of live and dead sperm were subjected tosquare root transformation to approximate a normal dis-tribution (SAS, 2001). Differences among treatment groupswere tested by the Duncan's new multiple range test. Allthe results were expressed as the mean (7SEM). Thestatistical significance was accepted at Po0.05.

3. Results

3.1. Effects on sexual activity and semen characteristics

Effects of supplementing vitamin E or propolis on reac-tion time of bucks, semen characteristics and seminal plasmabiochemical parameters are presented in Table 2. Propolis-treated bucks had higher sexual activity reflected by lowerreaction time (Po0.05) compared with bucks in the controlgroup, while vitamin E-treated bucks were intermediate.Concentrations of sperm were higher (Po0.01) in bothtreatment groups compared with the control group. VitaminE supplementation decreased significantly percentage ofdead sperm, while propolis supplementation resulted in anintermediate value compared with the C-group and E-group.

Table 3Effect of vitamin E or propolis supplementation compared with controlon hematological parameters measured on days 17, 34, 51 and 68(means7SEM) in male New Zealand White rabbits.

Parameter1 Treatment

Control Vitamin E Propolis SEM

Hb, g/dl 10.6B 11.2A 11.5A 0.2RBC, �106/ml 5.7B 6.1A 6.3A 0.1PCV, % 35.9B 37.1A 37.7A 0.7Platelets, �103/ml 277 282 288 5.3MCV, mm3 63.2a 60.9ab 59.9b 0.9MCH, pg 18.6 18.5 18.1 0.3MCHC, g/dl 29.3 28.8 28.3 0.3WBC, �103/ml 6.4B 6.9AB 7.0A 0.1Lymphocytes, % 34.3 35.1 35.5 0.6Nutrophiles, % 58.5 56.9 57.2 0.6Monocytes, % 4.2 4.4 4.1 0.2

ab Means with unlike superscripts within rows differ (Po0.05).AB Means with unlike superscripts within rows differ (Po0.01).

1 Values represented in the table are means of four samples.

Table 4Effect of vitamin E or propolis supplementation compared with controlon plasma biochemical parameters, total antioxidant capacity (TAC),malondialdehyde activity (MDA) and plasma testosterone concentrationmeasured on days 17, 34, 51 and 68 (means7SEM) in male New ZealandWhite rabbits.

Parameter1 Treatment

Control Vitamin E Propolis SEM

Total protein, g/dl 6.2 6.6 6.8 0.2Albumin, g/dl 4.2 4.2 4.5 0.2Globulin, g/dl 1.9 2.3 2.3 0.2Alb/Glo. ratio 2.6 2.0 2.7 0.3Glucose, mg/dl 134.4B 143.8A 151A 2.5Cholesterol , mg/dl 114.8A 104.8B 101.9B 3.3Triglycerides, mg/dl 172.4A 141.9B 140.4B 3.6LDL, mg/dl 21.6a 18.6b 20.4ab 0.9HDL, mg/dl 42.8B 49.5A 51.8A 2.7TAC, mM/L 1.1C 1.5B 1.8A 0.1

N.M. Hashem et al. / Livestock Science 157 (2013) 520–526 523

Values of seminal plasma total protein were higher in bothtreated-groups than that of the C-group (Po0.01). Concen-trations of seminal plasma albuminwere increased (Po0.01)in the P-group compared with the other two groups, whileseminal plasma globulin concentrations recorded the highestvalue in the P-group followed by E-group and C-group(Po0.01). Bucks-treated with vitamin E or propolis hadhigher (Po0.01) values of seminal plasma initial fructosecompared with the control bucks. On the contrary, seminalplasma AST and ALT activities did not differ significantlyamong buck groups.

3.2. Effects on hematological parameters

There were marked effects on several hematologicalparameters attributable to the supplementation of dietwith vitamin E or propolis (Table 3). The concentrations ofhemoglobin, number of RBC and PCV were higher in thebucks treated with vitamin E or propolis (Po0.01). Thenumber of WBC and MCV value were increased signifi-cantly in the P-group compared to the C-group, while bothparameters scored intermediate values in the E-group.No effects of treatments on differential count of whiteblood cells and number of platelets were observed.

3.3. Effects on blood plasma biochemical parametersand testosterone concentration

Supplementation of vitamin E or propolis increasedthe concentrations of plasma glucose compared with thecontrol group (Table 4). The concentrations of cholesteroland triglycerides were lower in both treated-groups com-pared with the control group (Po0.01), while HDL showedopposite trend (Po0.01). The concentrations of LDL weresignificantly lower in the E-group than in the C-groupbut intermediate in the P-group. Additionally, propolistreatment resulted in higher concentrations (Po0.01) oftotal antioxidant capacity and plasma testosterone

Table 2Effect of vitamin E or propolis supplementation compared with controlon reaction time, semen characteristics and seminal biochemical para-meters measured on days 17, 34, 51 and 68 (means7SEM) in male NewZealand White rabbits.

Parameter1 Treatment

Control Vitamin E Propolis SEM

Reaction time, s 4.9a 3.8ab 2.9b 0.3Ejaculate volume, ml 0.54 0.59 0.69 0.1Progressive motility, % 75.4 83.0 80.1 2.9Sperm concentration,�106/ml 118.3B 175.7A 187.7A 5.6Live sperm, % 90.9b 93.9a 92.6ab 1.0Dead sperm, % 9.1a 6.1b 7.4ab 1.0Total protein, g/dl 2.50C 2.90B 3.30A 0.1Albumin, g/dl 1.30B 1.46B 1.68A 0.1Globulin, g/dl 1.20b 1.45ab 1.60a 0.8Alb/Glo. ratio 1.20 1.09 1.11 0.1Initial fructose, mg/dl 26.9C 30.7B 34.0A 1.5AST, IU/l 78.2 67.6 67.2 3.4ALT, IU/l 93.1 86.5 78.8 7.1

ab Means with unlike superscripts within rows differ (Po0.05).ABC Means with unlike superscripts within rows differ (Po0.01).

1 Values represented in the table are means of four samples.

MDA, IU/L 149.1a 132.9b 127.9b 3.7Testosterone, ng/ml 1.7C 2.3B 2.5A 0.1A

ab Means with unlike superscripts within rows differ (Po0.05).ABC Means with unlike superscripts within rows differ (Po0.01).

1 Values represented in the table are means of four samples.

concentrations compared with those in bucks treated byvitamin E and bucks of the C-group (Table 4). The activityof MDA was higher (Po0.05) in the C-group than in theP-group but had an intermediate value in the E-group.

3.4. Treatment by time effects

Concentrations of sperm were significantly affected bythe treatment by time interaction. Sperm concentration onday 51 was higher (Po0.05) in the propolis-treated bucksthan in the other two groups. The maximum sperm con-centrations were observed (Po0.05) on day 68 of thetreatment when sperm concentration (million/ml) was330715.3 in the E-group and 289714.7 in the P-groupversus 14976.4 in the C-group (Po0.05) (Fig. 1). Therewere no treatment� time interactions for the seminal

b

75105135165195225255285315345

17 34 51 68

Sper

m c

once

ntra

tion

(mill

ion

/ml)

Time (days)

Control Vitamin E Propolisa

b

b

b

a

a

Fig. 1. Treatment� time interaction means (7SEM) for sperm concen-tration. abMeans within times with unlike letters differ (Po0.05).

14

16

18

20

22

24

26

17 34 51 68

Pla

sma

LDL

( mg/

dl)

Time (days)

Control Vitamin E Propolisa

b

b

20

30

40

50

60

70

17 34 51 68

Pla

sma

HD

L (m

g/dl

)

Time (days)

Control Vitamin E Propolis

ab

b

aabb

a aab

bb

Fig. 2. Treatment� time interaction means (7SEM) for blood plasmahigh density lipoprotein (HDL) and low density lipoprotein (LDL).abMeans within times with unlike letters differ (Po0.05).

N.M. Hashem et al. / Livestock Science 157 (2013) 520–526524

plasma biochemical parameters, hematological parameters,and most blood biochemical parameters. However, suchinteractions were significant for concentrations of bloodplasma HDL and LDL (Fig. 2). Bucks supplemented withvitamin E had significantly higher concentrations of HDL ondays 17, 34 and 51 compared with the control bucks, whilethese increases in propolis-treated bucks were on days 51and 68 compared with the control. Vitamin E and propolissupplementated bucks had lower concentrations of LDL onday 17 compared with the control.

4. Discussion

During the summer season, rabbit bucks suffer fromsevere heat stress as reflected by the high values of THI(29.35 on average). However, distinct improvements insexual activity, semen quality and oxidative status of bucks

were observed as a result of diet supplementation withvitamin E or propolis. Yousef et al. (2003) found thatvitamin E supplementation enhanced libido, ejaculatevolume, sperm concentration, semen initial fructose con-centration and decreased lipid peroxidation. Also, vitaminE combined with selenium supplementation during sum-mer season increased serum testosterone concentrationand sperm concentration (El-Maasry et al., 1994). Compar-ison between the two additives showed that propolis-treated bucks had higher libido and initial concentration offructose followed by vitamin E-treated bucks. Theseenhancements in propolis-treated bucks could be asso-ciated with higher concentration of testosterone recordeddue to propolis supplementation, particularly some semi-nal plasma components as well as libido of bucks that aretestosterone-dependent processes. For instance, fructose(the main energy source for sperm cells) production by theaccessory sex glands is dependent on the secretion oftestosterone by Leydig cells (Okab, 2007). The positiveeffects of propolis on semen quality and testosteronelevels observed in the present study are in accordancewith those obtained by Capucho et al. (2012) and Yousefand Salama (2009) when propolis was fed to male rats.

Further improvements in seminal plasma total protein,albumin and globulin were observed due to vitamin E orpropolis supplementation. High protein content in seminalplasma increases its buffering capacity and, in turn,improves the semen quality (Okab, 2007).

Adding vitamin E or propolis to the bucks, diets markedlyincreased sperm concentration, however, statistical analysis ofthe treatment by time interaction showed that increases inthe sperm concentrations due to nutritional supplementationswere only significant on days 51 and 68. This may be due tothe fact that the duration of spermatogenesis process in themale rabbits takes, on average, about 47 days (Boiti et al.,2005). Castellini et al. (2007) reported that the beneficialeffects of vitamin E on sperm concentration require a mini-mum of 4–5 weeks. This time is needed for increasing vitaminE levels in seminal plasma, reaching the site of action and forspermatogenesis.

Vitamin E or propolis supplementation resulted insignificant increases in hematopoiesis. These improvementsin hematological parameters may enhance blood ability tocarrying oxygen to different tissues and in turn improvingdifferent metabolic and physiological functions. In hotclimates, several biochemical changes in blood plasma areobserved as a part of the heat stress syndrome reflected indecreased concentration of glucose and increased concentra-tion of cholesterol and total lipids (Marai et al., 2002). On thecontrary, bucks treated with vitamin E or propolis had higherconcentrations of plasma glucose indicating the energyavailability for different physiological and biochemical func-tions. Additionally, lipid distribution in the blood plasmashowed different patterns among the experimental groups.Concentrations of cholesterol, triglycerides and LDL weredecreased in both treatment groups, while those of HDLwere increased. HDL has several pivotal biological functionsincluding (1) facilitating transportation of hydrophobic lipidmolecules like cholesterol and triglycerides to move withinthe water-based blood stream; (2) transporting cholesterolinto steroidogenic organs such as the adrenal gland, ovary,

N.M. Hashem et al. / Livestock Science 157 (2013) 520–526 525

and testis; and (3) removing the excess molecules of LDLthrough the liver. These facts may explain the increasedtestosterone concentrations and decreased concentrations oftriglycerides and cholesterol (increased cellular uptake)obtained in the present study in both treatment groups(Bernardo et al., 2000).

Vitamin E had lower concentrations of plasma LDL com-pared with control and propolis-treated bucks which mayindicate that the cell membrane of vitamin E-treated bucks ismore stable and resistant to membrane lipid peroxidationeffects of reactive oxygen species. Vitamin E is considered asthe most important endogenous antioxidant for LDL mole-cules (Gursu et al., 2004). Interestingly, these decreases inplasma LDL were significant at day 17 but not later in theexperimental period. This may be due to the increased heatstress effects through the experiment which may haveincreased the biological requirements from vitamin E. Themarked changes observed in blood plasma biochemical para-meters reflected the ability of vitamin E or propolis to mitigatethe harmful effects of heat stress, facilitating more adaptationof the animal for hot climate and stimulating importantphysiological processes such as incorporation of cholesterolin steroidogenesis, mainly testosterone synthesis, which wasincreased significantly in both treatment groups.

Treatment with vitamin E or propolis reduced theoxidative stress during the summer season, representedby increased total antioxidant capacity and decreasedactivity of the malondialdehyde (MDA; the end productof lipid peroxidation) in the blood. The role of vitamin Eas an antioxidant agent has been reported in severalstudies (Castellini et al., 2003; Yousef et al., 2003). Propolishas been successfully used to reduce thiobarbituricacid-reactive substance levels and induces activation ofantioxidant enzymes such as superoxide dismutase andcatalase against free radicals (Jasprica et al., 2007). Thebiological and antioxidant effects exhibited by propoliscould be related to the effect of the phenolic compounds(flavonol galangin; hydroxycinnamic acids, caffeic acid, p-cumaric acid, ferulic acid and caffeic acid phenethyl ester)of propolis (Lee et al., 2003; Russo et al., 2006). Similarly,in the present study, as denoted from chemical analysis ofpropolis many bioactive components as flavones, flavo-noids and long chain fatty acids were detected. Thesebioactive components are antioxidant agents capable ofscavenging free radicals and thereby providing protectionagainst lipid peroxidation (Paulino et al., 2008; Yousef andSalama, 2009). Thus, propolis has parallel effects onoxidative status of animals to that obtained with vitaminE supplementation, supporting the potential bioactive roleof propolis as a natural antioxidant agent.

5. Conclusion

Dietary manipulation using feed additives such asvitamin E and propolis offers an easily applicable measurefor improving sexual activity and semen quality ofrabbit bucks during hot seasons in the subtropics. Futureresearch should address the possible enhancements inseminal traits and different biological functions that couldbe obtained by dietary inclusion of propolis or vitamin Eunder normal environmental temperature.

Conflict of interest

None of the authors of this manuscript have anyconflict of interest.

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