DEVELOPMENT OF SOYA MILK EXTENDER FOR SEMEN CRYOPRESERVATION OF KARAN FRIES (CROSSBREED CATTLE)

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1 CryoLetters 34 (1), (2013) CryoLetters, DEVELOPMENT OF SOYA MILK EXTENDER FOR SEMEN CRYOPRESERVATION OF KARAN FRIES (CROSSBREED CATTLE) VK Singh, AK Singh, R Kumr nd SK Atrej* Reproductive Biochemistry Lortory, Animl Biochemistry Division, Ntionl Diry Reserch Institute, Hryn, Indi. *Corresponding uthor emil: sktrej@rediffmil.com Astrct Egg yolk sed semen extenders re used widely, with the potentil risk of xenoiotic contmintion. This study ws designed to develop soy milk sed extender to sustitute egg yolk sed extender for ovine semen cryopreservtion. In the first experiment soy milk ws prepred from fresh soy en (Glycine mx). Concentrtion of soy milk in tris sed extender ws stndrdized sed on qulity prmeters of spermtozo during liquid preservtion t 5 C up to 72 h nd compred with egg yolk tris (EYT) extender. Sperm in soy milk tris (SMT) extender with 25% soy milk showed no significnt (P > 0.05) differences in ll the qulity prmeters like motility, viility, memrne nd crosome, s compred to sperm in EYT extender up to 72h in liquid dilution. In the second experiment the Krn Fries semen ws cryopreserved in SMT extender with 25% soy milk (selected from the first experiment) using different concentrtion of glycerol, s cryoprotectnt, rnging from 6-7% with difference of 0.2% to stndrdize optimum concentrtion sed on post thw motility of spermtozo. Glycerol t finl concentrtion of 6.4% ws found to e the est mong ll. Further, semen smples were split nd cryopreserved in newly developed SMT extender contining 6.4% glycerol nd compred with conventionl EYT extender for post thw sperm qulity prmeters nd degree of cryocpcittion. There were no significnt (P > 0.05) differences etween sperm in EYT extender nd SMT extender for post thw motility, viility, memrne, crosome nd cryocpcittion. In conclusion, the newly developed SMT extender mintined comprle semen qulity s compred to EYT extender hence it cn e used to sustitute egg yolk extender for cttle (Krn Fries) semen cryopreservtion. Keywords: Krn Fries; Cttle; Soy milk extender; Spermtozo; Cryopreservtion. INTRODUCTION South Asin nd Pcific (SAP) countries hold pproximtely 18% of the glol cttle popultion nd lck of plnned reeding progrmmes re highlighted in most of these countries (12). Vrious ttempts hve een mde to improve the milk production of ntive zeu cttle through selection nd cross-reeding. The cross reed cttle ( Krn Fries, cross etween Holstein Friesin nd Thrprkr) is result of such n ttempt (27). Artificil 52

2 insemintion (AI) is technique tht cn chieve rpid dissemintion of genetic mteril from few geneticlly proven sires to lrge numer of femles (52). Tody, cryopreserved semen is mostly used for AI glolly. However, cryopreservtion of sperm ecme possile only fter the discovery of glycerol s cryoprotectnt (35). Polge used the egg yolk-citrte extender (45) long with the glycerol for cryopreservtion. Soon fter tht, Tris-uffered egg yolk-glycerol ws developed nd provided excellent protection during semen cryopreservtion (10, 13). Due to the qulities of Tris-uffered egg yolk-glycerol medium, tody it is the most commonly used semen extender for cryopreservtion of ull sperm s well s sperm from mny other species (22). Although, tody egg yolk-sed extenders re lrgely used for semen cryopreservtion (48, 53), it hs got some serious demerits. Egg yolk sed extenders possess risks of xenoiotic contmintion (2, 7) nd presence of steroid hormones (24). It is lso reported tht egg yolk contin sustnces (e.g. high-density lipoproteins nd minerls), which inhiit cellulr respirtion, ffect metolic ctivities, nd decrese postthw sperm motility (23, 31, 54). To circumvent these prolems numer of non niml origin sed extenders hve een developed nd they re commercilly ville tody. These extenders like AndroMed (2), Biociphos plus (4-5, 17, 28) nd Bioxcell (3, 18-19, 48) hve een evluted for the preservtion of ovine, ovine, cprine nd ufflo semen nd found to mintin etter semen qulity thn egg yolk sed extenders. These commercil extenders re expensive nd their vilility is limited. Unfortuntely, out 90% of the contriution of the livestock sector is from smll holders in Asi. Hence, higher cost of commercil extenders nd their limited vilility mkes them unpprochle for different emerging or smll-scle semen nks in developing countries. Therefore, the mjor ojective of this study ws to develop n economicl plnt sed soy milk semen extender nd compre its performnce in vitro with estlished egg yolk Tris extender. MATERIALS AND METHODS Experimentl design Two experiments were conducted to develop soy milk extender for cttle semen cryopreservtion. In the first experiment, soy milk ws prepred from soy en nd percentge of soy milk in semen extender ws optimized y liquid preservtion of semen t 5 C, sed on stndrd sperm qulity prmeters. In the second experiment, the percent of soy milk in the extender ws selected from first experiment nd used for cryopreservtion of Krn Fries semen. For this glycerol percentge in the finl SMT extender ws optimized sed on post thw motility of spermtozo. Finlly, Krn Fries semen ws cryopreserved in SMT extender with optimized glycerol percentge nd post thw in vitro ssessment of qulity prmeters ws done. Conventionl EYT extender ws tken s control for ll the experiments. Egg yolk tris (EYT) extender Estlished Egg yolk tris (EYT) semen extender contining 20% Egg yolk (v/v) diluted in TCG uffer (274 mm Tris, 87 mm Citric cid, 43 mm Glucose, 10,00,000 IU Benzyl penicillin per litre, 7,50,000 IU Streptomycin per litre) nd ws prepred s descried y Ahmed nd Foote (1). Additionlly, 7.0% Glycerol (v/v), s cryoprotectnt, ws supplemented in the semen extender for freezing. Soy milk tris (SMT) extender Soy milk used in the semen extender ws prepred ccording to Nelson et l., (29) given for soy milk everge preprtion with modifictions. In rief, fifty grms of soy ens were weighed nd ked in microwve oven until turned golden rown. Bked ens were soked overnight in 0.5% NHCO 3 solution. Soked ens were grounded in high-speed 53

3 homogenizer nd filtered through muslin cloth. Finl volume of stock soy milk ws djusted to 300 ml. Soy milk ws utoclved nd stored t 4 C. Soy milk tris (SMT) extender contining grdient of soy milk concentrtions (10%, 15%, 20%, 25% nd 30%), supplemented with TCG (274 mm Tris, 87 mm Citric cid, 43 mm Glucose, 10,00,000 IU Benzyl penicillin per litre, 7,50,000 IU Streptomycin per litre) uffer ph 7.0 ±0.2 were used in the experiment. Estlished Egg yolk tris (EYT) semen extender contining 20% Egg yolk diluted in TCG uffer (1) ws tken s control for ll the experiments. Semen collection nd liquid preservtion Cross reed cttle (Krn Fries) ulls (3-5 yers of ge) housed t Artificil Breeding Reserch Centre, Ntionl Diry Reserch Institute, Krnl, Indi, under uniform nutritionl conditions were used in this study. All the experiments were crried out on ten ejcultes from ech of five rndomly chosen ulls. Semen ws collected two times week using rtificil vgin (IMV, L Aigle Cedex, Frnce). Following collection, semen smples were immeditely evluted on the sis of ejculted volume, colour, mss motility nd individul motility. Ejculted smples hving milky white colour, score of 3 nd more mss ctivity nd more thn 80% progressive motility ws used in ll the experiments. For semen preservtion t 5 C ech ejculte ws split into six liquots. One liquot ws diluted in egg yolk tris (EYT) extender while other 5 were extended in grdient soy-milk tris (SMT) extender with 10, 15, 20, 25 nd 30 percent soy milk respectively. Sperm concentrtions in semen smples were determined y hemocytometer method (37) nd finl concentrtion of sperms ml 1 ws chieved y diluting either with EYT or SMT extender. Extended semen smples were cooled grdully to 5 C y trnsferring into refrigerted condition nd stored for 72 h. Semen qulity prmeter like percent motility, viility, memrne nd crosome were evluted t 0, 24, 48 nd 72 h. Semen qulity ssessment Individul motility. For motility ssessment, 5 µl of diluted semen ws trnsferred on grese-free glss slide on 37 C heting plte nd cover slip ws pplied. Sperm showing flgellr movement were oserved in t lest five rndomly chosen microscopic fields. A totl of 200 cells were counted per slide t 200x mgnifiction, nd two slides were evluted per ejculte. The men of oservtions ws tken s the finl motility score (39, 51). Viility. For viility evlution, 10 µl of diluted semen ws mixed with 5 µl of 5% eosin B nd 5 µl of 10% nigrosin on slide (49) nd incuted for 2 min t 37 C. The eosinnigrosin stined sperm were smered on grese-free slide nd llowed to dry for min. A totl of 200 vile (white) nd non-vile (red) sperm were counted in rndomly chosen fields under microscope (BX-51, Olympus, Jpn) t 400x mgnifictions. A minimum of two slides were exmined per ejculte (6) nd percent viility ws clculted. Memrne. The hypoosmotic swelling test (HOST) ws used to evlute memrne (40). In rief, 100 µl of diluted semen ws dded to 900 µl of 100 mosm hypo-osmotic solution (49.95 mm fructose, mm trisodium citrte) nd incuted for 1 hour t 37 C. After incution, liquot of semen suspensions ws plced on clen, dry nd grese-free glss slides nd covered with cover-slip, nd exmined under 400x mgnifiction using phse-contrst microscope. A minimum of 200 spermtozo were counted per slide for different types of swelling ptterns. Similrly, 200 spermtozo were counted in 300 mosm isotonicsolution (99.96 mm fructose, mm trisodium citrte). The sperm with coiled tils, incuted in isotonic solution, were considered s morphologiclly norml. Hence, the percentge of morphologicl norml spermtozo recorded in isotonic solution ws sutrcted from the percentge of coiled til spermtozo incuted in hypoosmotic solution. Acrosome. Acrosome ws ssessed ccording to Hncock (21). A thin smer of diluted semen ws ir-dried nd fixed t room temperture efore trnsferring into 54

4 freshly prepred giems solution for 90 min. Acrosoml of 200 spermtozo were recorded from minimum five different fields under 1000x mgnifiction (46). Tle 1. Motility, memrne (HOST), viility nd crosome of Krn Fries spermtozo diluted in control, soy 10%, soy 15%, soy 20%, soy 25% nd soy 30% during storge t 5 C for 72 h. Vlues re the mens±sem of ten ejcultes from ech of five rndomly chosen Krn Fries ulls. Different letters indicte significnt (P < 0.05) difference. Vlues with different smll letters re significntly different within the row, nd vlues with different cpitl letters re significntly different with in the column Time % EYT SMT 10% SMT 15% SMT 20% SMT 25% SMT 30% 0 h Motility 71.0±3.6 A 54.0±4.5 A 53.0±4.6 A 57.0±5.6 A 69.0±2.9 A 52.0±4.8 A Viility 81.6±1.2 A 74.2±3.8 c A 73.0±3.0 c A 77.0±3.1 c A 79.2±3.1 A 72.8±3.4 c A Mem. Acrosome 58.6±5.4 A 45.6± 7.2 A 46.0±6.8 A 47.8±7.5 A 50.2±6.9 A 41.2±6.9 A 96.8±0.3 A 95.2±0.9 A 95.4±1.8 A 96.6±1.0 A 97.4±0.8 A 96.6±0.6 A 24 h Motility 59.0±1.8 B 36.0±4.8 B 38.0±6.4 B 42.0±5.6 B 55.0±3.8 B 45.0±3.1 A Viility 70.8±4.4 B 48.8±6.7 d B 50.4±6.5 c B 61.6±4.0 B 70.4±1.7 A 58.4±4.1 cd B Mem. Acrosome 45.2±5.2 AB 29.4±6.4 AB 27.2±6.7 B 32.0±8.1 c AB 44.6±5.8 c AB 35.2± ±1.5 A 90.2±1.8 AB 90.0±2.0 AB 91.8±1.5 c BC 92.6±0.4 c B 93.4±0.4 c B c AB 48 h Motility 39.0±4.0 C 21.0±3.6 C 23.0±3.0 C 26.0±3.6 C 38.7±2.3 C 33.0±3.0 B Viility 62.6±2.7 B 36.0±8.0 BC 39.6±7.1 BC 44.2±6.4 C 58.8±3.8 B 45.4±5.617 BC Mem. Acrosome 33.8±5.7 B 18.2±5.6 BC 16.6± 5.6 BC 20.8± 6.3 BC 28.4±6.9 c BC 22.8±5.0 c B 90.8±0.7 B 87.0±1.6 BC 87.2± 2.1 BC 87.6± 1.4 CD 92.0±0.6 B 93.0±0.6 B 72 h Motility 30.0± 2.7 C 13.0±1.2 C 15± 2.2 c C 19.0±1.8 cd C 30.0±1.5 C 23.0±2.0 d B Viility 45.4±3.4 C 24.8±1.2 C 29.2± 0.7 c C 31.6±2.9 c C 49.6±3.3 B 37.6±2.8 d C Mem. Acrosome 24.0±4.8 B 8.0±1.6 C 7.6± 0.2 d C 12.4±1.4 c C 20.0±2.0 C 14.6±0.67 c B 88.0±0.8 B 85.0±2.0 C 85.4± 1.8 C 85.8±1.9 D 89.4±0.8 C 91.0±0.6 c C Optimiztion of glycerol for cryopreservtion To optimize glycerol concentrtion in SMT extender with 25% soy milk (selected from the experiment one), grdient of glycerol rnging from 6.0% - 7.0% with difference of 0.2% ws used in the cryopreservtion of Krn Fries semen. Bsed on post thw motility of spermtozo, the optimum percentge of glycerol in SMT extender ws decided. 55

5 Semen cryopreservtion Semen collection nd evlution were done in similr mnner s it ws done in experiment one. Semen hving more thn 3 mss ctivity nd 80 percentge individul motility were tken for cryopreservtion. Glycerol percentges used for cryopreservtion of crossred ull semen with EYT nd SMT extenders were 7.0 nd 6.4% respectively. Ech ejculte ws split into three liquots, one liquot ws used for fresh semen qulity prmeter nlysis nd two were used for cryopreservtion in EYT nd SMT extenders respectively. Smples were diluted to finl concentrtion of sperms ml 1, filled nd seled into medium-sized (0.25 ml) French strws. Seled strws were then equilirted t 4 C for 4 h. After equilirtion, the strws were kept in nitrogen vpour, 5 cm ove liquid nitrogen, for 10 min nd then plunged into liquid nitrogen for storge. Frozen strws were thwed t 37 C for 30 s in wter th nd used for the study of semen qulity prmeter. Semen qulity prmeter ssessment Sperm culture medium. Modified Tyrode s Hepes-uffered medium (sp-talph) ws used for wshing of spermtozo (ph , osmolrity: mosmol / kg). The medi were prepred s descried y Glntino-Homer et l., (16). Assessment of sperm cryocpcittion. Immeditely fter thwing, semen smples in EYT nd SMT extender were collected seprtely into 15 ml polypropylene tues nd centrifuged t 275 g for 6 min ech. The extended seminl plsm ws discrded nd pellet ws sujected to two wshes with 3ml of sp-talph nd centrifugtion t 275 g for 5min ech. The loose sperm pellet ws resuspended with 3ml of sp-talp nd sujected to finl centrifugtion step to remove the sp-talph medi. The pellet ws resuspended with sp- TALP nd the sperm concentrtion ws determined y hemocytometer nd djusted to 100 x 10 6 cells ml -1 with sp-talp. To study the extent of cryocpcittion, wshed spermtozo were incuted with 100µg/ml Lyso-Phosphtidyl Choline (LPC, known inducer of crosome rection in cpcitted cells only) for 15 min t 38.5 C in cell culture incutor with 5% CO 2 nd 85% reltive humidity in ir (33). The smples were then sujected to doule stining procedure using trypn lue nd giems stins to differentite etween crosome-rected (live/ded) nd non-rected (live/ded) cells (47). The percentge of live spermtozo undergoing crosome rection counted under right field microscope (BX-51, Olympus, Jpn) t 400x mgnifiction. Two hundred cells were counted per smple from rndomly chosen five different fields. Sttisticl nlysis Results were expressed s the mens ± stndrd error men (SEM). A difference with vlue P < 0.05 ws considered sttisticlly significnt. Two-wy ANOVA nd Tukey s test were pplied to compre the normlly distriuted dt for motility, memrne, viility nd crosome etween vrious tretment groups in liquid dilution y using the Sttisticl Product nd Service Solutions, Version softwre (SPSS Inc., Chicgo, IL, USA). However, One-wy ANOVA ws pplied to compre results oserved in optimiztion of glycerol nd cryopreservtion. For motility percentge Arc Sine trnsformtion ws crried out to get normlized dt efore nlysis. RESULTS Experiment 1 Stndrd sperm qulity prmeters like percentge (%) motility, viility, memrne (HOST) nd crosome of Krn Fries ull spermtozo diluted in EYT extender (control) nd SMT extender with concentrtion grdient of soy milk (soy 10%, soy 15%, soy 20%, soy 25% nd soy 30%) were ssessed during storge t 5 C for 72 h 56

6 nd results re shown in Tle 1. Upon dilution of semen in different extenders spermtozo showed significntly different (P < 0.05) motility etween egg-yolk tris (EYT) extender nd soy-milk tris (SMT) extender with 10%, 15%, 20% nd 30% upto 72 h. However, SMT extender with 25% soy milk showed no significnt difference (P > 0.05) in motility with EYT extender. Percent viility of liquid preserved spermtozo ws found significntly low (P < 0.05) in SMT extender with 10%, 15% nd 30% soy milk s compred to EYT extender t 0, 24, 48 nd 72 h. However, upto 24 h there ws no significnt difference (P > 0.05) in percent viility of spermtozo in SMT extender with 20% nd 25 % soy milk with EYT extender. Moreover, SMT extender with 25% soy milk only showed non significnt difference (P > 0.05) in percent viility upto 72 h, with EYT extender. There ws no significnt difference (P > 0.05) in memrne of spermtozo liquid preserved in EYT extender nd SMT extender with 25% soy milk upto 72 h. Moreover, no significnt difference (P > 0.05) ws found in crosome immeditely fter dilution in ll the extenders ut fter 24 h of liquid preservtion SMT extender with 10%, 15% nd 20% soy milk showed significnt difference (P < 0.05) in crosome wheres SMT extender with 30% soy milk remin non significnt (P > 0.05) upto 48h. Only SMT extender with 25% soy milk showed non significnt (P > 0.05) crosome upto 72 h with egg yolk extender. Hence, SMT extender with 25% soy milk ws elected for cryopreservtion of Krn Fries ull semen in Experiment 2. Experiment 2 Attempt ws done to cryopreserve spermtozo in selected SMT extender which showed non significnt difference (P > 0.05) in ll qulity prmeters during liquid preservtion with egg yolk extender (Experiment 1). Glycerol, s cryoprotectnt, ws used nd its concentrtion in finl extender ws stndrdized in SMT extender with 25% soy milk nd post thw motility ws ssessed. Results showed tht post thw motility of spermtozo cryopreserved in SMT extender with 6.4% of glycerol ws significntly higher (P < 0.05) thn others (Fig. 1). Cryopreservtion of semen in SMT extender with 25% soy milk nd 6.4% glycerol ws done nd compred with conventionl egg yolk extender for stndrd semen qulity prmeters. There ws no significnt difference (P > 0.05) etween EYT nd SMT extenders for post thw motility, viility, memrne nd crosome (Fig. 2). Degree of cryocpcittion of spermtozo ws lso ssessed in oth the extenders nd found to e sttisticlly non significnt (P > 0.05). However, the percentge of live cpcitted spermtozo ws less in soy milk extender (Fig. 3). DISCUSSION Qulity of preserved spermtozo depends on numer of fctors. However, dilution of semen in suitle extender is one of the importnt fctors ffecting sperm survivl during cryopreservtion (38). A suitle semen extender must provide n energy source, uffering ction to regulte ph chnge, provision of suitle osmotic pressure nd it should lso contin components tht protect spermtozo from cold shock (44). Egg yolk-sed extenders re lrgely used in frozen nd chilled semen preservtion in most of the mmmlin species such s got, rm, ull, equine, pigs nd even humn eing (20, 26, 32, 42-43, 48, 53). Although, egg yolk sed extenders show excellent cryoprotective effects on spermtozo during cryopreservtion in lmost ll mmmlin species, risk of xenoiotic contmintion hs rised question on its use (2, 7, 19). As direct consequence, most countries fer the risk of introducing exotic diseses through trnsporttion of milk or egg yolk-sed products. Moreover, endotoxins produced y such contminnts re thought to e reson for reduced fertilizing cpcity of spermtozo (7). Also the presence of egg yolk gloules in the extender 57

7 interfere in microscopic exmintion of diluted semen. To circumvent these demerits, we hve developed non-niml origin, economic, esy to prepre nd pthogen-free, SMT extender to sustitute EYT extender for cttle semen cryopreservtion. Non niml origin, soy milk, is prepred from soy ens nd the finish product is utoclved prior to use in semen extender hence it do not posses risk of xenoiotic contmintion. Moreover, SMT extender is clerer thn EYT extender nd hence there is less interference in microscopic exmintion of diluted smples. It is well known tht the protective effect of egg yolk ginst cold shock during freeze-thw process is lrgely due to low density lipoproteins (28). It hs lso een reported tht the egg yolk lipids like lecithin intercts with sperm memrne (30-31, 41) nd it is elieved tht these ssocitions hve stilizing effect during cold shock (34). Soy en is rich source of lecithin nd there is no surprise why severl commercil compnies introduced lecithin sed new genertion diluents (2, 17). Although, De Leeuw et l., (11) nd Celeghini et l., (8) found egg yolk-sed extender more efficient thn soy lecithin sed extender for ull sperm survivl following freezing. In contrry, severl reserchers clim tht soy lecithin sed extenders re superior thn egg yolk sed extenders (2, 5, 48). We lso found tht SMT extender, developed in our l, is comprle to conventionl EYT extender for liquid preservtion t 5 C s well s cryopreservtion. Spermtozo preserved in SMT extender showed non significnt (P > 0.05) difference in viility nd motility following preservtion s compred to spermtozo preserved in EYT extender. This indictes tht survivl of spermtozo in SMT extender is s good s in EYT extender. Spermtozo in SMT extenders with soy milk concentrtions elow optimum showed reduced qulity during liquid preservtion due to less protective effect nd vilility of energy source. But, ove the optimum concentrtion of soy milk (25%) in semen extender, fll in qulity prmeters were oserved. As explined y Forouznfr et l., (14), fll in sperm motility is possily due to the presence of higher concentrtion of soy lecithin in the extender. Stndrd seminl prmeters like motility, morphology, nd sperm concentrtion re not sufficient to predict fertility (15) nd functionl of plsm memrne ply crucil role in fertiliztion. Revell nd Mrode (40) developed n osmotic resistnce test for ovine semen nd found etter correltion with fertility thn tests sed solely on motility. Results showed tht spermtozo preserved in SMT nd EYT extender do not show significnt (P>0.05) difference in test for osmotic resistnce (HOST) or crosome upto 72 hrs during liquid preservtion t 5 C. This shows tht memrne rchitecture is mintined in SMT extender, similr to EYT extender. Glycerol, permeting cryoprotectnt, ws first used y Polge et l., (35) for cryopreservtion of chicken spermtozo. Since then glycerol hs een used in ll type of semen extenders for lmost ll niml species. Sperm survivl from cryodmges lrgely depends on composition nd concentrtion of cryoprotectnt in the extender. EYT extender contins 7% glycerol for ovine semen cryopreservtion. Optimum concentrtion of glycerol for SMT extender ws stndrdized nd found to e 6.4%. Following cryopreservtion of Krn Fries spermtozo in SMT extender with 6.4% glycerol non significnt differences in qulity prmeters were oserved with spermtozo cryopreserved in EYT extender with 7% glycerol. Frozen thwed spermtozo results in memrne modifictions, similr to cpcitted spermtozo, like incresed memrne fluidity, phospholipid scrmling (50) nd efflux of cholesterol (9, 25), collectively termed s cryocpcittion, fctor ssocited with the reduced longevity of cryopreserved sperm in the femle reproductive trct (36). The degree of cryocpcittion ws ssessed y LPC rection followed y doule stining nd counting the crosome rected cells. Results showed non significnt (P > 0.05) difference in degree of cryocpcittion of spermtozo in SMT extender nd EYT extender. Hence, it is nticipted tht spermtozo cryopreserved in oth the extenders hve similr functionl life in femle reproductive trct. 58

8 CONCLUSION Spermtozo preserved in the SMT extender showed comprle semen qulity like motility, viility, memrne nd crosome with spermtozo preserved in conventionl EYT extender. Hence, the SMT extender, with 25% soy milk cn sustitute EYT extender for liquid semen preservtion. Moreover, this SMT extender long with 6.4% glycerol cn sustitute EYT extender for cryopreservtion of Krn Fries semen. Acknowledgements: We sincerely thnk Indin Council of Agriculture Reserch (ICAR), New Delhi nd Director, Ntionl Diry Reserch Institute, Krnl, Indi, for providing necessry fcilities during course of study. This study ws supported y World Bnk funded Ntionl Agriculturl Innovtion Project (C4/C30014). The Scientific stff of Artificil Breeding Reserch Complex, NDRI is cknowledged for providing semen smples. REFERENCES 1. Ahmed K nd Foote RH (1986) J Diry Sci 69, Aires VA, Hinsch KD, Mueller-Schloesser F, Bogner K, Mueller-Schloesser S nd Hinsch E (2003) Theriogenology 60, Akhter S, Ansri MS, Rkh BA, Ullh N, Andri SMH nd Khlid M (2011) Reprod Dom Anim 46, Amirt L, Anton M, Tinturier D, Chtgnon GR, Bttut I nd Courtens JL (2005) Reproduction 129, Amirt L, Tinturier D, Jenneu L, Thorin C, Gerrd O, Courtens JL nd Anton M (2004) Theriogenology 61, Bloom E (1950) Anim Breed Ast 18, Bousseu S, Brillrd JP, Mrqunt-Le Guienne B, Guerin B, Cmus A nd Lecht M (1998) Theriogenology 50, Celeghini ECC, Arrud RP, Andrde AFC, Nscimento J, Rphel CF nd Rodrigues PMH (2008) Anim Reprod Sci 104, Chkrrty J, Bnerjee D, Pl D, De J, Ghosh A nd Mjumder GC (2007) Cryoiology 54, Dvis IS, Brtton RW nd Foote RH (1963) J Diry Sci 46, De Leeuw FE, De Leeuw AM, Den Ds JHG, Colenrnder B nd Verkleij AJ (1993) Cryiology 30, FAO (2007) Rischkowsky, B., Pilling, D., (Eds) Rome, Itly. 13. Foote RH (1998) Ithc, New York. 14. Forouznfr M, Shrfi M, Hosseini SM, Ostdhosseini S, Hjin M, Hosseini L, Aedi P, Nili N, Rhmni HR nd Nsr-Esfhni MH (2010) Theriogenology 73, Gde J, Selles E nd Mrco MA (2004) Reprod Dom Anim 39, Glntino-Homer HL, Visconti PE nd Kopf GS (1997) Biol Reprod 56, Gil J, Jnuskusks A, Hrd MC, Hrd MGM, Johnisson A, Soderquist L nd Rodriguez-Mrtinez H (2000) Reprod Dom Anim 35, Gil J, Lundeheim N, Soderquist L nd Rodriguez-Mrtinez H, (2003) Theriogenology 59, Gil J, Rodriguez-Irzoqui M, Lundeheim N, Soderquist L nd Rodriguez-Mrtinez H (2003) Theriogenology 59, Hmmdeh ME, Greine RS, Rosenum P nd Schmidt W (2001) J Androl 22, Hncock JL (1952) J Exp Biol 29,

9 22. Iritni A, (1980) In: Proc 9th Int Congr Anim Reprod Artif Insemin, Mdrid, Spin 1, Kmpschmidt RF, Myer DT nd Hermn HA (1953) J Diry Sci 36, Lipr JL, Ketterson ED, Noln VJr nd Csto JM (1999) Gen Comp Endocrinol 115(2), Mldjin A, Pizzi F, Gliozzi T, Cerolini S, Penny P nd Nole R (2005) Theriogenology 63, Mlo C, Gil L, Gonzlez N, Cno R, de Bls I nd Espinos E (2010) Cryoiology 61(1), Mson IL (1996) A World Dictionry of Livestock Breeds, Types nd Vrieties. Fourth Edition. C.A.B Interntionl 273 pp. 28. Mouss M, Mtinet V, Trimeche A, Tinturier D nd Anton M (2002) Theriogenology 57, Nelson AI, Steinerg MP nd Wei LS (1976) J Food Sci 41, Ollero M, Bescos O, Cerin-Perez JA nd Muino-Blnco T (1998) Theriogenology 49, Pce MM nd Grhm EF (1974) J Anim Sci 39, Pgl R, Aurich JE, Muller-Schlosser F, Knkofer M nd Aurich C (2006) Theriogenology 66, Prrish JJ, Susko-Prrish JL, Winer MA nd First NL (1988) Biol Reprod 38, Pettitt MJ nd Buhr MM (1998) J Androl 19, Polge C, Smith AU, Prkes AS (1949) Nture 164, Pommer AC, Rutllnt J nd Meyers SA (2003) Biol Reprod 68, Prthlingm NS, Holt WW, Revell SG, Jones S nd Wtson PF (2006) J Androl 27, Rsul Z, Anzr M, Jlli S nd Ahmd N (2000) Anim Reprod Sci 59, Reddy NSS, Gli JM nd Atrej SK (2010) Anim Reprod Sci 119 (3-4), Revell SG nd Mrode RA (1994) Anim Reprod Sci 36, Ricker JV, Linfor JJ, Delfino WJ, Kysr P, Scholtz EL, Tlin F, Crowe JH, Bll BA nd Meyers SA (2006) Biol Reprod 74, Roc J, Crrizos JA, Cmpos I, Lfuente A, Vzquez JM nd Mrtinez E (1997) Smll Rum Res 25, Roc J, Hernández M, Crvjl G, Vázquez JM nd Mrtínez EA (2006) J Anim Sci 84, Slmon S nd Mxwell WM (2000) Anim Reprod Sci 62, Slisury GW, Fuller HK nd Willett EL (1941) J Diry Sci 24, Snchez R, Risoptron J, Sepulved G, Pen P nd Misk W (1995) Theriogenology 43, Sidhu KS, Dhinds J.S nd Gury SS (1992) Biotech Histochem 67, Strdioli G, Noro T, Syll L nd Monci M (2007) Theriogenology 67, Therien I nd Mnjunth P (2003) Biol Reprod 69, Thoms AD, Meyers SA nd Bll BA (2006) Theriogenology 65, Tomr NS (1997) Artificil insemintion nd reproduction of cttle nd uffloes. 4 th ed. Sroj Prkshn, Allhd, Indi. 52. Vishwnth, R nd Shnnon P (1997) Reprod Fertil Dev 9, Wll RJ nd Foote RH (1999) J Diry Sci 82, Wtson PF nd Mrtin CA (1975) Aust J Biol Sci 28, Accepted for Puliction 27/8/12 60

10 Post thw Sperm Motility (%) c d 6.0% Glycerol 6.2% Glycerol 6.4% Glycerol 6.6% Glycerol 6.8% Glycerol 7.0% Glycerol 0 6.0% Glycerol 6.2% Glycerol 6.4% Glycerol 6.6% Glycerol 6.8% Glycerol 7.0% Glycerol Figure 1. Post thw sperm motility of spermtozo cryopreserved in soy milk extender with different glycerol concentrtion for optimiztion of glycerol percentge. Vlues re the Men ± S.E.M. of five experiments performed with semen smples from five rndomly chosen Krn Fries ulls. Mens with different letters re significntly different (P < 0.05). Percentge (%) 100 Fresh 90 Post thw-eyt 80 Post thw-smt Motility Viility Memrne Acrosome Figure 2. Sperm percent Motility, Viility nd Memrne in fresh nd cryopreserved semen extended in egg yolk nd soy milk extender. Vlues re the Men ± S.E.M. of five experiments performed with semen smples from five rndomly chosen Krn Fries ulls. Mens with different letters re significntly different (P<0.05). Live Cpcitted (%) Fresh Post thw-eytpost thw-smt 61 Fresh Post thw-eyt Post thw-smt Figure 3. Sperm percent live cpcittion in fresh nd cryopreserved semen extended in egg yolk nd soy milk extender. Vlues re the Men ± S.E.M. of five experiments performed with semen smples from five rndomly chosen Krn Fries ulls. Mens with different letters re significntly different (P < 0.05). Accepted for puliction 27/8/2012