Effect of Fructose Addition in Skim Milk Extender on the Quality of Liquid Nili-Ravi Buffalo (Bubalus bubalis) Semen

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1 Pkistn J. Zool., vol. 42(3), pp ,. Effect of Fructose Addition in Skim Milk Extender on the Qulity of Liquid Nili-Rvi Bufflo (Bulus ulis) Semen Shmim Akhter, Muhmmd Sjjd Ansri*, Bushr Allh Rkh, Syed Murtz Hssn Andri, Muhmmd Anwr nd Nemt Ullh Animl Physiology Lortory, Deprtment of Zoology, Pir Mehr Ali Shh Arid Agriculture University, Rwlpindi-4, Pkistn (SA, MSA, BAR), Animl Reproduction Lortory Animl Sciences Institute, Ntionl Agriculturl Reserch Centre, Islmd 45, Pkistn (SMHA, MA), nd Fculty of Veterinry nd Animl Sciences, Pir Mehr Ali Shh Arid Agriculture University, Rwlpindi-4, Pkistn (NU) Astrct.- This study ws designed to identify the effect of fructose ddition in skim milk extender on qulity of ufflo ull spermtozo (motility, in vitro longevity, plsm memrne integrity, norml picl ridge nd normlities) stored t 5 C for seven dys. For this purpose, semen from five Nili-Rvi ufflo ulls ws collected nd diluted t 37 o C with finl concentrtion of x 6 motile spermtozo per ml in skim milk extender contining 5mM or mm fructose or without fructose (control). The extended semen ws cooled from 37 o C to 5 o C in 2 hours nd stored t 5 C for seven dys. Sperm motility, in vitro longevity nd plsm memrne integrity did not differ (P>.5) in ll three experimentl extenders t 1 st dy of storge. Sperm motility, in vitro longevity, plsm memrne integrity on 3 rd, 5 th nd 7 th dy of storge ws significntly (P<.5) higher in extender contining fructose 5mM nd mm s compred to control. Sperm morphology viz; norml crosomes; hed, mid piece nd til normlities remined similr (P>.5) in ll three experimentl extenders upto seven dys of storge t 5 o C. In conclusion, ddition of 5mM nd mm fructose in skim milk extender improved the preservility of ufflo ull semen stored t 5 o C. Keywords: fructose, semen qulity, liquid semen, ufflo ull spermtozo INTRODUCTION The development of rtificil insemintion (AI) technique hs llowed the rpid dissemintion of genetic mteril from smll numer of superior sires to lrge numer of femles (Vishwnth nd Shnnon, 1997). Bufflo ull semen is routinely preserved in liquid or frozen stte ccording to suitility for AI (Anzr et l., 3). Commonly, tris, citrte nd milk sed extenders re used for the storge of ufflo semen t 4-5 C tempertures (Andri, 9; Snsone et l., ). Skim milk extender hs een recognized s n pproprite storge medium in liquid stte for ufflo ull spermtozo ecuse of its higher ility to conserve semen qulity, vilility nd economic suitility (Akhter, 6). Storge of spermtozo in liquid stte requires suitle energy supplement in extender (Snsone et l., ). Fructose is mjor nturlly occurring energy source for ufflo spermtozo in the * Corresponding uthor: m.sjjd.nsri@gmil.com -9923//3-227 $ 8./ Copyright Zoologicl Society of Pkistn. seminl plsm for metolic processes (Snsone et l., ). Dilution of the semen for freezing purpose decreses the ville source of energy for spermtozo (Akhter, 6). Therefore, ddition of energy source in semen extender is required for proper functioning of the spermtozo. We hypothesized tht ddition of fructose s energy source in skim milk extender cn improve its ility to mintin the qulity (motility, in vitro longevity, plsm memrne integrity, norml picl ridge nd normlities) of Nili-Rvi ufflo ull semen stored t 5 C. Therefore, present experiment ws designed to study the effect of 5mM nd mm fructose ddition in skim milk extender on motility, in vitro longevity, plsm memrne integrity, norml picl ridge nd normlities of ufflo ull spermtozo stored t 5ºC. MATERIALS AND METHODS Extender preprtion Powdered skimmed milk (SKIMZ ; CANDIA) % (w/v) without the ddition of fructose ws used for preprtion of extender s control. Second nd third extenders were prepred

2 228 S. AKHTER ET AL. y dding 5mM nd mm fructose to control extender, respectively. Antiiotics (Streptomycin 1 mg/ml nd Benzyl i.u./ml) were dded in ech extender t room temperture. Semen collection nd initil evlution Semen from five Nili-Rvi ufflo ulls of similr ge group nd known fertility mintined t Semen Production Unit, Qdird, Shiwl, Pkistn ws colleted with rtificil vgin mintined t 42 C, for period of three weeks (replictes). Semen collection ws performed once week nd two ejcultes were collected per collection. After collection, semen ws trnsferred to lortory for initil evlution. Visul progressive sperm motility ws ssessed microscopiclly t X. Sperm concentrtion ws ssessed y Neuuer hemocytometer. Qulifying semen ejcultes were pooled to get sufficient semen for replicte hving motility >% nd split into three liquots for further processing. Semen processing Semen liquots were diluted t 37 o C with skim milk extender without fructose (control) or with extenders contining 5mM or mm fructose. The extended semen ws cooled from 37 o C to 5 o C in 2 hours nd stored t 5 C for seven dys. All the semen qulity ssys were performed t 1 st, 3 rd, 5 th nd 7 th dy of storge. Semen qulity ssys Progressive sperm motility ws ssessed using phse contrst microscope s descried erlier. Sperm in vitro longevity of ech extended ufflo ull semen smple ws ssessed for six hours t 37 C, fter two hours intervl (Akhter et l., 8). Sperm plsm memrne integrity ws determined y hypo-osmotic swelling ssy (HOS). The HOS solution (osmotic pressure ~ 1mOsm/ kg) contined sodium citrte.735g nd fructose 1.351g, dissolved in ml distilled wter. To perform ssy, µl semen smple ws mixed with µl pre-wrmed (37 C) HOS solution nd incuted t 37 C for to minutes. After incution, drop of semen smple ws exmined using phse contrst microscope (X). One hundred spermtozo were counted nd the percentge of cells with curled tils (swollen/intct plsm memrne) ws recorded s HOS positive (Akhter et l., 8). Sperm morphology ws ssessed y mixing µl of semen smple with µl of 1% forml citrte. A drop of semen smple ws exmined under phse-contrst microscope (X). One hundred spermtozo were counted to determine the percentge of intct crosomes, nd hed, mid piece nd til normlities (Andri et l., 8). Sttisticl nlysis The dt re presented s Men (± SE). The dt on semen qulity prmeters were nlyzed using Anlysis of Vrince (ANOVA). When F- rtio ws found significnt, the mens were compred using LSD test. A 5% (P<.5) level ws used to determine sttisticl significnce. RESULTS Motility, in vitro longevity nd plsm memrne integrity of semen The dt on the effect of fructose ddition in semen extenders on motility, in vitro longevity nd plsm memrne integrity of ufflo spermtozo re presented in Figure 1A-C. There ws no difference (P >.5) in sperm motility, in vitro longevity nd plsm memrne integrity on 1 st dy of storge in extender with fructose supplementtions or control. However, sperm motility, in vitro longevity nd plsm memrne integrity on 3 rd, 5 th nd 7 th dy of storge ws significntly etter in extenders contining 5mM nd mm fructose s compred to control. Norml picl ridge of semen The dt on the effect of fructose ddition in semen extenders on norml picl ridge of ufflo spermtozo re presented in Figure 1D. Percentge of sperm with norml picl ridge did not differ (P>.5) in extenders contining 5mM nd mm fructose s compred to control for seven dys of storge t 5 C.

3 FRUCTOSE ADDITION FOR LIQUID STORAGE OF BUFFALO SEMEN 229 Sperm motility (%) Anormlities of semen The dt on the effect of fructose ddition in semen extenders on normlities (hed, mid piece nd til) of ufflo spermtozo re presented in Tle I. There is no difference in sperm hed, mid piece nd til normlities in extenders contining 5mM nd mm fructose s compred to control upto 7 dys of storge t 5 C. Dy of Storge A: Motility DISCUSSION Sperm in vitro longevity (%) Sperm plsm memrne intigrity (% Norml crosomes (%) 1 Dy of storge B: In vitro longevity Dy of storge C: Plsm memrne integrity Dy of storge D: Norml crosome Fig 1: Effect of fructose ddition in semen extender on motility (A), in vitro longevity (B), plsm memrne integrity (C) nd norml crosome (D) of ufflo ull spermtozo on different dys of storge t 5 C. Brs with different letters differ significntly (P<.5) t given dy. Present experiment ws designed to study the effect of fructose ddition s energy source in skim milk extender on the qulity (motility, in vitro longevity, plsm memrne integrity, norml picl ridge nd normlities) of ufflo ull spermtozo stored t 5 ºC. Motility, in vitro longevity nd plsm memrne integrity re the prmeters used to determine the effect of experimentl procedures on the storge of ufflo ull semen (Akhter et l., 8; Andri et l., 8). It is well recognized tht sperm motility is ffected y the properties of diluting medi (Andri, 9). In vitro longevity ssessment is n indirect indictor of sperm liveility in femle reproductive trct. Hypo-osmotic swelling test hs een widely used to ssess the functionl intctness of the ufflo ull spermtozo plsm memrne (Akhter et l., 8; Andri et l., 8). In our study, motility, in vitro longevity nd plsm memrne integrity of ufflo ull spermtozo on 1 st dy of storge remined similr in ll three experimentl extenders. However, sperm motility, in vitro longevity nd plsm memrne integrity on 3 rd, 5 th nd 7 th dy of storge ws etter (P <.5)) in extenders contining 5mM nd mm fructose s compred to control. In similr studies, El-Az et l. (1984) reported increse in motility of Egyptin ufflo ull spermtozo fter the ddition of fructose nd lctose thn glucose in Tris sed extender. Similrly, improvement in motility nd percent liveility of Murrh ufflo ull spermtozo ws oserved in milk sed extender in frozen nd liquid stte fter the ddition of fructose, sucrose nd rffinose in Tris nd milk sed extender (Kumr et l., 1994,). Moreover, 2% glucose nd 1% fructose were reported to provide

4 2 S. AKHTER ET AL. Tle I.- Effect of fructose ddition in semen extender on sperm normlities (hed, mid piece nd til) of ufflo ull spermtozo on different dys of storge t 5 C. Dy of storge Tretment Sperm normlities (%) Hed Mid piece Til 1 Skim milk 1. ±.. ±.6 2. ± 1.2 Skim milk + Fructose 5mM 1. ±. 1. ±.6 3. ±.6 Skim milk + Fructose mm 1. ± ± ±.6 3 Skim milk 2. ± ± ± 1.5 Skim milk + Fructose 5mM. ± ± ±.6 Skim milk + Fructose mm 1. ± ± ±.6 5 Skim milk 1. ±. 1. ±. 4. ± 1.2 Skim milk + Fructose 5mM 1. ± 1.. ± ± 1.5 Skim milk + Fructose mm 1. ± 1.5. ±. 5. ±.6 7 Skim milk 1. ±.6. ± ±.6 Skim milk + Fructose 5mM 1. ± ± ± 1. Skim milk + Fructose mm 1. ± 1.2. ±. 6. ± 1. etter motility of Murrh ufflo ull semen in Tris sed extender t 5 C (Kumr et l., 1992). An ddition of 5mM nd mm of fructose in skim milk extenders improved the survivility of ufflo ull spermtozo in this study. Sperm morphology (crosome, hed, mid piece nd til) is criticl for the successful fertiliztion. The presence of norml crosome on spermtozoon is essentil for the crosoml rection tht is required t the proper time to fcilitte fertiliztion (Thoms et l., 1997). It is elieved tht crosoml integrity ssessment cn e n effective tool to predict the fertilizing ility of ufflo ull spermtozo. A definite reltionship hs een estlished etween fertility of preserved semen nd percentge of sperm normlities (Soderquist et l., 1991). In our study, percentge of sperm with norml picl ridge nd normlities (hed, mid piece nd til normlities) did not differ in ll three experimentl extenders for seven dys of storge t 5 C. It is pertinent to mention tht semen preservtion does not necessrily increse the numer of norml sperm in ovine (Revell et l., 3) nd ufflo spermtozo (Akhter et l., 8). It is concluded tht ddition of 5mM nd mm fructose in skim milk extender used for preservtion of ufflo ull spermtozo t 5 C improved sperm motility, in vitro longevity nd plsm memrne integrity. Therefore, fructose ddition my e used for improving the preservility of liquid Nili-Rvi ufflo semen for rtificil insemintion progrmme. REFERENCES AKHTER, S., 6. Effect of milk sed extenders contining different ntiiotics on sperm motility, longevity, morphology, plsm memrne integrity, in vivo fertility nd cteril control of liquid semen from ufflo ulls. Ph.D. thesis, Deprtment of Zoology, University of Arid Agriculture, Rwlpindi, Pkistn. AKHTER, S., ANSARI, M. S., ANDRABI, S. M. H., ULLAH, N. AND QAYYUM, M., 8. Effect of ntiiotics in extender on cteril control nd spermtozol qulity of cooled ufflo (Bulus ulis) ull semen. Reprod. Domest. Anim., 43: ANDRABI, S. M. H., 9. Fctors ffecting the qulity of cryopreserved ufflo (Bulus ulis) ull spermtozo. Reprod. Domest. Anim., 44: ANDRABI, S. M. H., ANSARI, M. S., ULLAH, N., ANWAR, M., MEHMOOD, A. AND AKHTER, A., 8. Duck

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