EFFECT OF DIFFERENT EXTENDERS AND STORAGE PERIODS ON MOTILITY AND FERTILITY OF RAM SPERM

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1 Avilble online t Mc Vet Rev 2015; 38 (1): Originl Scientific Article EFFECT OF DIFFERENT EXTENDERS AND STORAGE PERIODS ON MOTILITY AND FERTILITY OF RAM SPERM Rossen Georgiev Stefnov 1, Georgi Anev 2, Desislv Vsilev Abdjiev 1 ASTRACT 1 Institute of iology nd Immunology of Reproduction-AS, bul. Tsrigrdsko Shose 73, p.c. 1113, Sofi, ulgri 2 Agriculturl Institute, G Turgovishte, ulgri Received 5 July 2014; Received in revised form 23 November 2014; Accepted 1 December 2014 The im of this study ws to test the effect of extenders contining different sugr in their composition on rm sperm motility nd pregnncy rte of ewe s following rtificil insemintion. Semen were collected from ten North-est ulgrin fine-fleece breed nd tested for qulity. Semen ws diluted with different extenders, with di- nd triscchrides. A series of experiments were repeted in triplicte. Totl motility ws determined by using Sperm Anlysis (SCA, Microptic, Spin). A totl of 200 North-est ulgrin fine-fleece breed mture ewes were used for cervicl insemintion with sperm dose t the concentrtion of 100 x 10 6 spermtozo. Pregnncies were dignosed 60 dys fter AI by - rel-time ultrsonic scn device (Allok SSD 500). In conclusion, our experiments demonstrted tht higher sperm motility fter storge t 4 C for 24 hours nd 48 hours hs rm spermtozo diluted with extender 1, with combintion of discchrides (sucrose nd lctose) nd triscchrides (rfinos). This semen extender (number 1) cn be used for successful insemintion of ewes nd to enhnce pregnncy rte fter rtificil insemintion. Key words: rm, sperm extenders, motility, fertility INTRODUCTION Artificil insemintion with chilled-stored semen hs become technique in sheep breeding (1). Efforts hve been mde to improve the preservtion of rm semen by the modifiction of extender composition (2), s well s with the ddition of vrious components to mintin motility, fertilizing cpcity nd preserve sperm membrne integrity (3,4,18). The preservtion of functionl properties of sperm in the storge medium is dependent on the quntities of non-electrolytes. The spermtozo require exogenous substrtes to obtin energy through mitochondril oxidtive phosphoryltion nd glycolysis by the consumption of glycolysble sugrs, such s glucose, fructose, mnnose (5). Corresponding uthor: Assoc. Prof. Rossen Stefnov, PhD E-mil ddress: stefnovrossen@gmil.com Present ddress: Institute of iology nd Immunology of Reproduction- AS, bul. Tsrigrdsko shose 73, p.c. 1113, Sofi, ulgri Copyright: 2015 Stefnov R. This is n open-ccess rticle published under the terms of the Cretive Commons Attribution License which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl uthor nd source re credited. Competing Interests: The uthors hve declred tht no competing interests exist. Avilble Online First: 5 December Fructose is thought to be mjor energy source for ejculted spermtozo, nd together with glucose is found in the seminl plsm in mny mmmlin species. In mny species, glucose nd fructose hve been investigted for their different effects on gmetes in terms of metbolizble energy nd fertility potentil, but the beneficil effects vry between species (6). The effects of sugrs on the metbolism of freshly ejculted spermtozo hs been studied in rms, but metbolize glucose nd fructose use different pthwys, resulting in seprte systems of energy mngement s indicted by their different roles in glycogen metbolism nd motility ptterns (7). Sugrs re the most commonly used components for semen extenders; however, the concentrtion of these sugrs in the extenders vries mrkedly. To optimize the extender medium for the chievement of best storge semen chrcteristics which would infer greter fertility, it is importnt to study the influence of different sugrs on rms spermtozo. The im of this study ws to test the effect of three extenders with different rtios of sugr in their composition on rm sperm motility nd pregnncy rte of ewe s following insemintion. 85

2 Stefnov R. et l. MATERIAL AND METHODS Animls The experiment ws crried out on June nd July in the Experimentl sttion of griculture (Trgovishte, ulgri). Ten cliniclly helthy rms from north-est ulgrin fine-fleece breed, round three yers old nd kg of live weight were used s semen source. Semen ws collected using n rtificil vgin two times week. Fresh semen with 70% motility ws eligible for experimenttion. Extenders The extenders were used to dilute the smples of semen to be chilled. Semen ws diluted with two different extenders (Tble 1), which gve the best result fter prior experiment with twelve extenders with mono-, di- or three dischride. The third extender served s control (8). All series of experiments (I III) were repeted in triplicte. Prior to sugr mesurement, ech egg-yolk ws mixed in distilled wter to the sme finl concentrtion s in the extenders, i.e., 20% (v:v). To mke the extenders, fresh egg-yolks were pooled nd mixed to reduce the biologicl vrition found in the eggs. nd concentrtions by using sperm nlyzer SCA (Microptic, rcelon, Spin). For ech smple, t lest 200 sperm cells from four rndomly selected fields were evluted. Pregnncy rte experiment A totl of 200 North-est ulgrin fine-fleece breed mture ewes (2-3 yers old) were used for insemintion. The detection of ewes in cliniclly mnifested oestrus ws performed in the morning by rm tester. The ewes were cerviclly inseminted with sperm dose t the concentrtion of 100 x 10 6 spermtozo. The insemintion ws reiterted second time fter 10 h with the sme sperm, which ws stored 48 h. Pregnncy ws dignosed 60 dys fter AI by ultrsonic scn device (Allok SSD 500). Sttisticl nlyses The sttisticl nlyses were performed by GrphPd softwre, using the pirwise t-tests for comprison of the lest-squres mens. The pregnncy rte (number of pregnnt ewes to number of inseminted) in ll groups ws sttisticlly nlyzed using chi-squre test. A vlue of p<0,05 ws considered sttisticlly significnt. Tble 1. Compositions of semen extenders used in the experiments Ingredients I II III N-citrte (g) Scchros (g) Lctose (g) Rfinos (g) Egg-yolk (ml) Distilled wter (ml) to 100 to 100 to 100 Experimentl design The pooled semen ws divided into three equl liquots nd plced in three screw cp closed sterile plstic tubes. The resulting sperm smples were divided nd re-suspended in rtio 1:4 with different extenders to rech finl sperm concentrtion of 2 x 10 6 cells/ml. To void cooling too rpidly, the extended semen smples were plced in room temperture cooler, which reched temperture of 4 C within 45 min. Subsequently, ll smples were stored t 4 C in refrigertor for 24 nd 48 hours nd fter tht semen smples were evluted dily during the first three dys, s motility ws determined on 15 min; 60 min; 120 min; 180 min; 240 min; 300 min nd 360 min, fter incubtion in the wter bth t 39 C. The semen smples were evluted microscopiclly which included percentge of motile spermtozo 86 RESULTS Three different extenders were used for the motility of the spermtozo t different postdilution intervls. Totl motility (%) decresed with storge time in ll cses (Tble 2). However, the type of extender significntly (P<0.05; P<0.001) ffected the percent sperm motility. Combintion of sucrose, lctose nd rfinos in extender 1 showed highest vlues thn other extenders (P<0.001) regrding this chrcteristic fter 240 min to 360 min. Dt indicte tht the survivl of sperm cells on fresh rm semen in extender 1 nd 3 hd similr vlues fter 6 hours incubted t 39 C (P<0.001). Extender 2 showed significntly lower protective qulities (P<0.001). After 48 hours, only semen diluted with extender 1 showed living nd motile

3 Semen extender Effect of different extenders nd storge periods on rm sperm Tble 2. Effect of extender with different sugr contents on the sperm motility in rms on 24 h nd 48 h t 39 C Totl motility (%) After 24 h 1 M 77.0± ± ± ±0.62 D, E 34.67±0.81 b, F 33.60± ±0.33 A 1, 1 D 1, c 19.83±0.16 F 1, A 2 2 M 77.8±0.66 n.s 72.2± ±0.17 C 50.00±0.32 D 9.00±1.40 b 25.00± ± ±0.60 A 1, C 1 D 1, E 1 F 1, 2 3 M 78.0± ± ±0.28, C 49.50±0.40 E 39.83±0.45 F 30.93± ± ±0.19 1, C 1 c, E 1 A 2, 2 After 48 h 1 M 70.0±0.11 n.s 65.33±1.36 A 62.4± ± ±0.98 d 22.3±1.32 A, e 18.0± ± M 70.0± ±1.28 A, 57.1± ± ± ±0.96 A M 70.0± ± ± ± ±1.65 d 15.6±1.85 e 9.3± vlues with sme superscripts re significnt а, b, c P 0.05; d, e- P 0.01; А n-2, В n-2, С, D, E, F P sperm cells (totl motility =10.1%). Smples, which were diluted with extenders 2 or 3 showed no protective effect on rm semen, since ll spermtozo were ded. The beneficil effect of more sugr concentrtions in mintining higher TM% ws consistent. Vlues for pregnncy rtes re presented in Tble 3. There were significnt differences between the extender 1- sucrose, lctose nd rfinos (80 %) nd extender 2 sucrose nd rfinos (71 %), lso between extender 1 nd control extender 6A- with sucrose nd lctose (71.20 %; Tble 3). different metbolism (7). Sperm motility gives mesure of the integrity of the sperm xoneme nd til structures s well s the metbolic mchinery of the mitochondri, nd sperm morphology is surrogte mesure of the integrity of DNA pckging nd the qulity of spermtogenesis (9). The results from the present study clerly demonstrted the mjor effect of sucrose, lctose nd rfinos in semen extenders on chilled rm sperm motility. Totl motility is n importnt indictor of sugr utiliztion by spermtozo s the sugrs provide the externl energy source essentil Tble 3. Ewe s pregnncy rte fter cervicl insemintion with rm sperm diluted in extenders Semen extender No of inseminted ewes (n) No of pregnnt ewes (n) Pregnncy rte (%) %, b % % b Vlues with sme superscripts re significnt а, b p 0.05 DISCUSSION Different sugrs were tested nd compred to the untreted control becuse they hve been shown to hve protective effects on motility ptterns nd for mintining motility. In this study, TM% (>70%) remined high during the first 48 hours nd subsequently, s could be expected, decresed with storge time. The decrese in the percentge of totl motile spermtozo in 0-15 (71-78%) my 87

4 Stefnov R. et l. be due to the fct tht there re subpopultions of rm spermtozo with different sensitivities to chnges in the environment, prticulrly reduced temperture. Decresed sperm motility fter 60 hs been postulted to be due to temperture sensitivity of the ATPse-linked sodium potssium pump nd subsequently lekge of ions (10). Preservtion with incresing vriety of sugrs resulted in better mintennce of sperm motility with extender 1. The combintion of sucrose, lctose nd rfinos s prt of semen extender for rm semen provided beneficil effect on the survivl of sperm more thn s sole component. ohlooli et l. estblish tht only sucrose nd lctose in extenders ws less effective t protecting spermtozo during the storge process, which ws indicted clerly by lower osmotic resistnce of spermtozo (11). In ddition, the incresed concentrtion of buffer solutions in the extender reduces the deleterious effects of the gret mount of hydrogenic ions produced from the metbolic ctivity of spermtozo. In ddition, egg yolk is known to contin mny specific components like lecithin, phospholipids nd lipoprotein frctions (12). The significntly higher sperm motility fter storge for 24 hours nd 48 hours could explin the evolution of fertilizing bility nd ws probbly sufficient to chieve gesttion. A dose of 25 50x10 6 sperm for intruterine insemintion, x10 6 sperm for trnscervicl insemintion nd x10 6 for cervicl insemintion is recommended in ewes (13). In the present study, concentrtion of 100x10 6 rm spermtozo ws selected for in vivo use. The chnge in the motility prmeter ws less drmtic in smples diluted with extender 1 nd significnt difference ws observed between the groups regrding the fertiliztion rte or the estblishment of pregnncy. It ws reported tht low motile sperm injection my hve negtive effect on fertiliztion nd pregnncy rtes (14). On the other hnd Vildn Krpuz et l. found no correltion in fertiliztion nd pregnncy rtes with either morphology or motility (15). The possible physiologicl resons for the decline in motility might be due to extrcellulr oxidtive stress, effects of seminl plsm volume-constituents nd endogenous free rdicl production. Substnces from seminl plsm protect spermtozo from premture ging during storge (16). The most sugrs in content of extender 1. my be one of the resons for better preservtion of functionl nd motility prmeters in our study. In conclusion, our experiments demonstrted tht higher sperm motility fter storge t 4 C for 24 hours nd 48 hours hs the rm spermtozo diluted with extender 1. with combintion of discchrides 88 (sucrose nd lctose) nd triscchrides (rfinos). This semen extender (number 1) cn be used successfully for the insemintion of ewes nd it might improve the pregnncy rte fter rtificil insemintion. REFERENCES 1. Ax, R. L., Dlly, M. R., Didon,. A, Lenz, R. W., Love, C. C., Vrner, D.D., Hfez,., ellin, M. E. (2000). Artificil insemintion. In: Hfez,.Hfez, E.S.E. (Eds.), Reproduction in frm nimls. (7 th Eds.) (pp ). Phildelphi, Le nd Febinger ISN Mrti, J. I., Mrti, E., Cebrin-Perez, J. E., Muino-lnco, T. (2003). Survivl rte of ntioxidnt enzyme ctivity of rm spermtozo fter dilution with different extenders or selection by dextrn swim-up procedure. Theriogenology 60, Rih, L., Apolen, D., Pivko, J., Grfenu, P., Kubovic, E. (2006). Influence of implememtors on sheep fertility out of seson. Slovk J. Anim. Sci., 4, number-4 4. Srlos, P., Molnr, A., Koki, A., Gbor, G., Rtky, J. (2002). Evlution of the effect of ntioxidnts in the conservtion of rm semen. Act Vet. Hung., 50, 2, PMid: Adeoy-Osiguw S., Frser, L. R. (1993). A biphsic pttern of 45 C 2+ uptke by mouse spermtozo in vitro correltes chnging functionl potentil. J. Reprod. Fertil., 99, PMid: Willims, AC, Ford, WCL., (2001). The role of glucose in supporting motility nd cpcittion in humn spermtozo. J. Androl., 22, PMid: Rigu, T., Frrem, M., llester, J., Mogs, T., Peň, A., Rodriguez-Gil, J.E. (2001). Effects of glucose nd fructose on motility ptterns of dog spermtozo from fresh ejcultes. Theriogenology 56, Zhriev Z., Georgiev, G., Chelebiyski, S. (1988). Diluent for storge of rm semen for 24 hours t 3-4 С. Journl of Livestock reeding (G), 6, Pcey, A.A. (2006). Is qulity ssurnce in semen nlysis still relly necessry? A view from the ndrology lbortory. Hum Reprod., 21, PMid:

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