Effect Different Concentrations of DMSO and Dilution Rateson Motility Parameters of Stellate (Acipenser stellatus) Spermatozoa

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1 Worl Journl of Fish n Mrine Sciences 5 (4): , 2013 ISSN IDOSI Publictions, 2013 DOI: /iosi.wjfms Effect Different Concentrtions of DMSO n Dilution Rteson Motility Prmeters of Stellte (Acipenser stelltus) Spermtozo Ali Seghi, Mohm Rez Imnpoor n Mohsen Khlili 1 Deprtment of Fisheries, Fculty of Fisheries n Environmentl Sciences, Gorgn University of Agriculturl Sciences n Nturl Resources, Gorgn, Irn 2 Young Reserchers Chub, Bnrgz Brnch, Islmic Az University, Bnrgz, Irn Abstrct: Milt obtine from four stellte mles (Acipenser stelltus) ws cryopreservtion using extener; Tris-sucrose-KCl (30mM Tris, 23.4mM sucrose, 0.25mM KCl, ph 8.0) supplemente wi DMSO tconcentrtion of 5%, 10% n 20%. Semen ws ilute, respectively, wi rtios of 1:0.5, 1:1, 1:2 n 1:5 wi extener n frozen in liqui nitrogen vpor.frozen sperms fter 2 n 15 yswere exclue from freezing. Experiment showe ehighest motility urtion n e mostmotility percentge of post-we sperms fter 2 ysws relte to e tretments wi econcentrtion ofdmso 10% n e ilution rtio of 1: 1 (260±25.62 s n 32.28±5.36%; P<0.05), s well s e upmost mobility n e motility urtion of post-we sperms fter 15 ys wsrelte to e tretments wi e concentrtion of DMSO 10% n e ilution rtio of 1:1(251.19±23.54s n 23.37±5.51%; P<0.05). Theresults showe t holin time from 2 to 15 ysof frozen sperm hs negtive impct on Motility urtion n Motility percentge of post-we sperms. Results lso showe t highestmotility urtion n motility percentge of post-we sperms ws for tretments t hve been ilute wi 1:1 rtio. Key wors: Stellte Sperm DMSO Dilution Rtios Motility Durtion Motility Percentge INTRODUCTION Stellte sturgeon (Acipenser stelltus) re mong e commercilly vlule sturgeon species in e Cspin The cretion of sperm bnks of e selecte stock Se t eir stocks hve ecline rsticlly in e to prevent outbreks, ctstrophes n genetirift is recent eces [3]. essentil to evelop genetic selection progrms in Cryopreservtion inuces severe stress to fish commercil quculture, but it is lsonecessry for spermtozo t in turn ffect sperm qulity in conservtion of strins or species in nger of extinction, termsof fertiliztion ility. Sperm qulity is influence by until e environmentl conitions re recovere (1275 severl fctors such s temperture, iet [4], time of fish species retene ccoring to e Interntionl smpling [5] n elys cuse fter e injection of Union for Conservtion of Nture, hormones [6]. Reprouctive technologies re being evelope to Importnt prmeters for cryopreservtion inclue recover popultion just from frozen sperm, pplying types of exteners n cryoprotectnts, e ilution rtio, furer hybriiztion progrms of using nrogenic e freezing n wing rtes n type of extener use proceures [1, 2]. Biotechnology, ecotoxicology n for fertiliztion. bsic sciences coul lso benefitfrom cryobiology The most efficient permeting cryoprotectnt for rough e preservtion of gmetes or embryos from fish milt ppers to be imeyl sulfoxie (DMSO). Oer moel species or strins s well s from sperm or embryos cryoprotectnts such s glycerol, eylene glycol, geneticlly moifie wi reserch or commercil propne-iol, imeyl cetmie n menol re prouction purposes (polyploiy, trnsgenic). less populr or hve been use wi limite success. Corresponing Auor: Ali Seghi, Deprtment of Fisheries, Fculty of Fisheries n Environmentl Sciences, Gorgn University of Agriculturl Sciences n Nturl Resources, P.O. Box: Gorgn, Irn. Tel:

2 Worl J. Fish & Mrine Sci., 5 (4): , 2013 Unlike most teleost fish, informtion concerning relile connecte to Nikonmicroscope (Optiphot-2, Jpn) t technology for cryopreservtion of sturgeon semen is 100 mgnifiction t combine wi CCD color vieo not ville. cmer (moel SPC-2000P, Jpn). Sperm motility n Cryopreservtion success ws usully mesure s urtion of spermmotilitywere evlute fromsperm wi post-w sperm motility [7, 8] or s fertiliztionsuccess forwr movement. Immotile sperm were efine s sperm uring erly embryo evelopment [9]. DMSO or eylene t i not show forwrmovement fter ctivtion. glycol ws use s cryoprotectnt. Sturgeon Vieo recors were set t 30 frmes/s using vieo cmer (Acipenser sp., Chonrostei) spermtozo re mounte on microscope. Percentge of sperm motility significntly ifferent from freshwter teleost fish sperm. wsetermine uring 0-15 s post-ctivtion. Motility These ifferences concern morphology (more complex urtion ws evlute by counting e timefrom sperm structure, presence of crosome), physiology (longer ctivtion wi ctivtion solution until sperm stoppe urtion of motility, crosome rection) n biochemistry moving [14]. Anlyses were repete ree times for ech (presence of crosin, rylsulftse) [10, 11]. tretment. Anoer striking ifference between milt properties of sturgeons n teleost fish is e lowosmollity of Extener n Sperm Cryopreservtion: in ese sturgeon seminl plsm [12]. These peculir experiment using extener Tris- sucrose-kcl(30mm Tris, chrcteristics of sturgeon milt myexplin e lck of 23.4mM sucrose, 0.25mM KCl, PH 8.0) [15] were success in sturgeon of cryopreservtion meos supplement wi 5%, 10% n 20% DMSO [16]. Semen estlishe for teleost fish. The objectives of our work n extener h temperture of 4 C. Milt ws ilute t wereto test e effect of(1) DMSO in ifferent rtios of 0.5:1, 1:1, 2:1 n 5:1 wi extener. concentrtions on e mobilityn motility urtion of Suspensions of extene milt were rwn into stelltesturgeon sperm; (2) severl ilute rtes in 0.25-mlstrws. Sperm freezing ws conucte in combintion wi ifferent DMSO concentrtions on e Styrofom box wi liqui nitrogen. Strws (of 0.25 ml mobility n motility urtion of stelltesturgeon sperm. volume) wi ilute sperm, prior kept toequilibrte for 5 minutes t room temperture, were plce forslow MATERIALS AND METHODS freezing uring 3 minutes on 3 cm high floting frme me from e sme mteril s e box. Strws were Sperm Collection for Cryopreservtion: Milt ws subsequentlyplunge into liqui nitrogen [17]. collecte from eight stellte (Acipenser stelltus) mles using syringe wi n ttche polyeylene rigi Mesurement Sperm Mobility, Durtion of Spermmotility tubing inserte into e urogenitl opening. Semen from n Concentrtion: Thwing of sperm ws conucte in ese mles ws obtine in Mrch. Before stimultion, wter b t 40 C for 5 sec [17]. Sperm mobility n fish were trnsferre from pons to tnks wi urtion of sperm motility of we semen ws observe wtertemperture of 18 C for 3 ys n e temperture fter 2 n 15 y of storge in liquin 2. ws rise to 20 C for n itionl 7ys. Post-w mobility n motility urtion ws Spermition ws inuce by injecting of sturgeon observe n evlute by ree opertors using 1 pituitry extrct in ose of 2-3mgkg boy weight [6]. monitor connecte to microscope.sperm concentrtion Spermtozo were collecte wiin 15-24h (epening ws mesure by e opticl ensity meo [18]. on e wter temperture) post hormonl injection. Semen ws store on ice n use wiin 2 h of storge Sttisticl Softwre: All vlues re shown s men±sd. for cryopreservtion. Dt for percentge n urtion of sperm motility were trnsforme by ngulr trnsformtion prior to sttisticl Assessment of Sperm Qulity: Motility of sperm smples nlysis by SPSS 10.0 softwre. The effects of ws estimte uner light microscope t 100 concentrtions DMSO n ilute rtes on post-w mgnifiction immeitely fter mixing of 5µL of sperm sperm motility n urtion of sperm motility were wi 50µL of ctivtion solution )NCl 3.5 mm, Tris-HCl 12 nlyze using two-wy nlysis of vrince (ANOVA). mm, ph=8.5) [13] on microscope slie. Sperm mobility Mens were seprteby Duncn s New Multiple n urtion of sperm motility ws recore using Rnge test n were consiere significntly ifferent softwre gmei tv home mei v. 330 from note book t P<

3 Worl J. Fish & Mrine Sci., 5 (4): , 2013 RESULTS Tle 2 showe e mximum urtion n e most mobility results were observe intretments where e The urtion of sperm motility use for ilute rte ws 1:1, s well s e lowest motility cryopreservtion exceee 320 s (Tle 1). Likewise, percentge n motilityurtion of post-we sperm only sperm smples showing 80% motility or higher were observe in ilute rte ws 1:5. use for e experiments (Tle 1). Effect of Dilution Rtes wi Concentrtions of DMSO on Effect of Dilution Rtes wi Concentrtions of DMSO Qulity Post-Thwe Sperms After 15 Dys: Mximum on Qulity Post-Thwe Sperms After 2 Dys: motility urtion n e upmost mobility of post-we Highestmotility urtion n e most motility percentge sperms fter 15 ys wsrelte to e tretments wi e of post-we sperms fter 2 ys ws relte to e concentrtion of DMSO 10% n e ilution of 1: 1 tretments wi e concentrtion of DMSO 10% n e (251.19±23.54 s n 23.37±5.51%; Tle 3). ilution of 1: 1 (260±25.62 s n 32.28±5.36%; Tle 2). Tle 3 shows e minimum Durtion n e lowest The lest Durtion n e lowest mobility of post-we motility percentge of post-we spermsws observe sperms ws observe in e tretments wi e in e tretments wi e concentrtion of DMSO 20% concentrtion of DMSO 20% n e ilutionof n e ilution of (137.54±19.84 s n 13.38±4.94%; (183.24±18.86 s n 18.31±4.95%; P<0.05) Tle 2. P<0.05). Tle 1: Mles use for sperm cryopreservtion process Mle Boy weight (g) Totl leng (cm) 9 1 Sperm concentrtion ( 10 ml ) Motility urtion (s) Motility percentge (%) ± ± ± ± ± ± ± ±2.06 Tle 2: Effect of ifferent concentrtions of DMSOn ilute rtes on post-w sperm motility n urtion of sperm motility fter 2 ys of freezing Cryoprotectnt Cryoprotectnt concentrtion (%) Dilute rtes (sperm: extener) Motility urtion (s) Motility percentge (%) ± ± DMSO 5 1: ± ± ± ± ± ± ± ± DMSO 10 1: ± ± ± ± ± ± ± ± DMSO 20 1: ± ± ± ± c ± ± Vlues wiin column followe by ifferent superscript letters were significntly ifferent (P<0.05) Tle 3: Effect of ifferent concentrtions of DMSO n ilute rtes on post-w spermmotility n urtion of sperm motility fter 15 ys of freezing Cryoprotectnt Cryoprotectnt concentrtion (%) Dilute rtes (sperm: extener) Motility urtion (s) Motility percentge (%) ± c 5.27 ± DMSO 5 1: ± ±20.35 c ± c 5.10 ± ± ± ± ± DMSO 10 1: ± ± ± c 4.94 ± ± ± ± c 5.10 ±17.57 DMSO 20 1: ± ± ± c 4.90 ± ± c 4.94 ±13.38 Vlues wiin column followe by ifferent superscript letters were significntly ifferent (P<0.05) 423

4 Worl J. Fish & Mrine Sci., 5 (4): , 2013 Tle 3 showe e highest motility urtion n e most motility percentge results wereobserve in tretments where e ilute rte ws 1:1, s well s minimum motility urtion n e lest mobility of post-we sperm observe in ilute rte ws 1:5. DISCUSSION Decline in stocks n limite number of potentil breeers hs le to e estlishment of fish sperm cryobnks which ply crucil role in e genetic mngement n conservtion of qutic resources [19, 20]. The estlishment of sperm bnks from vlule fish species incluing sturgeon is wiely prctice in mnycountries [21, 22]. Accoring to e ove results, by compring Tle 2 n 3 were etermine e ilutionrtios hs significnt ifferences on e urtion of sperm motility (P<0.05), s most of e motility urtion relte to ilution rtio of 1: 1of e tretments n e urtion of sperm motility wi incresing ilution significntly reuce. Becuse of e high ilution of e sperm plsm loses its protective effect, sperm viility reuce n by incresingresulting in reuce sperm viility [23]. The results showe t equlity spermsignificntly reuction fter wing ws similr to e results [24]. These reserchers hve reporte t e qulity of Ponto-Cspin sturgeon spermshrply ecrese fter wing. In is experiment, post-we sperms wi DMSOconcentrtion of 10%n e ilution rte 1:1 hs e highest mobility n motility urtion ws similr to eresults [25]. These reserchers hve reporte t e most suitlecryoprotectnt for sperm cryopreservtion Itlin Cobice sturgeon (Acipensernccrii), isdmso concentrtion of 10%. Also, Lhnsteiner et l. [23], nnounce e most motility of post-we sperm Strlet (Acipenser ruenus) ws wi DMSO 10% (80±7.4%). Horv et l. [16], reporte emximum motility n motility urtion ofpost-we sperm of plli sturgeon (Scphyrinchuslbus) ws wi DMSO concentrtion of 5% (26±13%). Liu et l. [25], reporte t emost suitlecryoprotectnt for sperm Cryopreservtion Chinesesturgeon (Acipenser persicus), ws wi DMSO 12%. Thereson ofifference in suitle ensity of cryoprotectnt in written result wi experiment result my be for selecte species, ifference in extener solution or semen isspecific chrcteristics of is species. CONCLUSION From e ove stuy it cn be conclue tstorge of frozen sperm hs negtive impct on motilityurtion n motility percentge of post-we sperms. Results lso showe t e highest motility urtion nmotility percentge of post-we sperms ws fortretments wi e concentrtion of DMSO 10% n eilution of 1:1. REFERENCES 1. Bik, I., O. Ottesen, G. Ruolfsen n S. Johnsen, Chille storge of semen from Atlntic hlibut, Hippoglossushippoglossus L. I: Optimizing e protocol. Theriogenology, 66: Grunin, A.S., A.V. Recoubrtsky, L.I. Tsvetkov n V. Abrmintsev, Investigtion on ispermicnrogenesis in sturgeon fish. The first successful prouction of nrogenetic sturgeons wicryopreserve sperm. Int. J. Refrig., 29: Pourkzemi, M., Cspin Se sturgeon conservtion n fisheries: Pst, Present n Future. Journl of Applie Ichyology, 22: Billr, R., J. Cosson, F. Fierville, R. Brun, T. Rouult n P. Williot, Motility nlysis n energetics of e Siberin sturgeon, Acipenser berii, spermtozo. Journl of Applie Ichyology, 15: Kopeik, E.F., P. Williot n B.F. Gonchrov, Fctors ffecting e cryoresistnce of sturgeon sperm.in: Abstrcts of e Worl Congress of Cryobioligy. 36 Annul Meeting of Society for Low Temperture Biology, July, , Mrseille, Frnce, pp: Williot, P., E.F. Kopeik n B.F. Gonchrov, Influence of testis stte, temperture n ely in semen collection on spermtozo motility in e culture Siberin sturgeon (Acipenser beri Brnt). Aquculture, 189: Ciereszko, A., G.P. To, S.A. Christ n K. Drowski, Effect of cryopreservtion n eophylline on motility chrcteristics of lke sturgeon (Acipenser fulvescens) spermtozo. Theriogenology, 45: Billr, R., J. Cosson n O. Linhrt, Chnges in e flgellum morphology of intct n frozen/we Siberin sturgeon Acipenser berii(brnt) sperm uring motility. Aquculture Reserch,

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