ACROSOME REACTION PROGRESS IN FROZEN-THAWED AND CAPACITATED BOAR SPERMATOZOA INFLUENCES THE EFFICIENCY OF IN VITRO FERTILIZATION

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1 H G BDG, 2, 28 (1) G Z-HWD D D B Z LU H Y V LZ rtečíková. 1, 2, Hulínská. 1, ečková Z. 1, Hnzlová K. 1, Ješet. 1, chtková. 1 1 Veterinry eserch nstitute, Brno, zech epublic 2 sryk University, culty of cience, Brno, zech epublic bstrct n this study functionl sttus in bor spermtozo collected from frozen-thwed semen nd cpcitted in vitro by cffeine ws monitored. he motile spermtozo of bors (, B nd ) were isolted by ercoll grdient, treted with 1m or 2m cffeine nd incubted in V medium or co-incubted with mtured porcine oocytes for 3 hours. otility, vibility, crosome integrity nd fertilizing bility of spermtozo were evluted. otility nd vibility of spermtozo lightly decresed during cpcittion in ll tested bors independently of cffeine tretment (from 5.4±8.5 to 38.8±5.8 nd from 56.±6.9 to 43.9±5.5, respectively). he crosome rection ws fster for 1m cffeine-treted compred with 2m cffeine-treted spermtozo in ll sires but its progress ws different in ech bor. he men penetrtion rte ws higher for 2m cffeine-treted compred with 1m cffeine-treted spermtozo. n the other side, monospermy ws higher nd the men % ±D of norml fertiliztion ws significntly higher (p<.5) for 1m cffeine-treted compred with 2m cffeinetreted spermtozo for ll tested bors (29.8±6.1 vs. 12.7±5.6). t cn be concluded tht the progress of crosome rection ws specific for spermtozo of ech bor nd influenced the efficiency of norml V process. Keywords: Bor, frozen spermtozo, cffeine, crosome rection, in vitro fertility he use of cryopreserved bor spermtozo is very importnt becuse higher number of insemintion doses with the stndrd fertilizing bility of spermtozo prepred from the sme ejculte nd useful for in vitro fertiliztion is possible to obtin. rerequisite for successful oocyte fertiliztion nd embryo production is good functionl sttus of frozen-thwed spermtozo. ny ssys for spermtozo evlution hve been developed for these purposes (Johnson et l., 27). he crosome rection progress in spermtozo is necessry for fertiliztion process becuse only spermtozo with rected crosome cn penetrte the oocytes. or tht reson the fertiliztion medium usully contins cffeine, n gent which is known to induce cpcittion nd crosome rection of bor spermtozo (unhshi et l., 1999) nd increse their penetrtion into oocytes (Wng et l., 1991). lthough more effective techniques for porcine embryo production hve been widely developed during the pst yers, low incidence of normospermy nd high incidence of polyspermy in inseminted oocytes remin mjor problem (unhshi, 23). t ws documented tht one of the resons of polyspermic fertiliztion could be the qulity of spermtozo (irrd et l., 1993). ome differences in in vitro fertilizing bility of fresh or frozen spermtozo mong individul bors hve been described (Long et l., 1999; ellés et l., 23). But only little informtion is vilble for prediction of fertilizing bility of frozen-thwed bor spermtozo (Gde et l., 21). 7 herefore effective methods in the field of bor sperm function evlution re still needed. he im of this study ws to monitor the functionl sttus of bor spermtozo collected from frozen-thwed semen nd cpcitted in vitro by cffeine. ur ttention ws focused on the reltionship between crosome integrity chnges of spermtozo nd the efficiency of in vitro fertiliztion. teril nd methods emen cryopreservtion he sperm-rich frctions were collected from ejcultes of 2-5 yer old bors of ietrin () nd Lrge White (B nd ) breeds with proven fertility. he semen with t lest 7% motility nd 9% crosome integrity of spermtozo ws frozen ccording to n djusted procedure described by Westendorf et l. (1975) nd subsequently modified by vrjl et l. (24). fter the equilibrtion in cooling extender (11 % egg yolk (v/ v),.12 lctose nd.11 trehlose) from 17 to 5, semen were resuspended in freezing extender (,22 lctose, 22.8% egg yolk (v/v), 1.3% (v/v), quex, initübe, iefenbch,germny nd 7.5 % glycerol (v/v) to finl concentrtion of.5 9 of spermtozo/ml nd frozen in.5 ml strws in liquid nitrogen vpour. he semen thwing ws crried out by holding strws t room temperture for 15 s nd plunging them into wter bth t 37 for 3 s.

2 H G BDG, 2, 28 (1) otile spermtozo seprtion fter thwing, motile spermtozo were seprted on 4 vs. 6 ercoll-grdient by centrifugtion t 52g for 4 minutes. he pellet ws removed nd wshed in B medium (initübe, iefenbch, Germny). he spermtozo were resuspended ( 6 spermtozo/ml) in mb fertiliztion medium (modified ris-buffered medium contining 113.1m l, 3m Kl, m, m l 2 H 2, 2m ris, 11 m glucose, 5m sodium pyruvte nd.4% B) supplemented with 1m or 2m cffeine. he cffeine treted spermtozo were either cpcitted or used for oocyte insemintion. permtozo cpcittion Both the 1m cffeine- nd 2m cffeine-treted spermtozo were cpcitted in humidified tmosphere of 5% 2 t 39 for 3 hours. permtozo ssessment hirty minutes fter thwing (th), immeditely fter cffeine tretment (), nd t 1h-intervls during cpcittion (1, 2, 3h) the sperm motility, vibility nd crosome integrity were ssessed. otility ws evluted subjectively using phse contrst microscope. or vibility, the spermtozo were stined with bisbenzimide Hoechst nd evluted immeditely by epifluorescence. or crosome sttus evlution, stining ccording to relly ws used (Věžník et l., 24). he percentges of motile nd vible spermtozo, nd spermtozo with intct nd non-intct crosome or without crosome were determined. ocyte mturtion nd fertiliztion vries were collected from cyclic sows t locl bttoir nd oocyte-cumulus complexes were isolted by slicing of ovrin cortex. ocytes were mtured in medium with.2 m sodium pyruvte,.57 m cystemin, 5 U ml -1 penicillin, 5 mg ml -1 streptomycin, % fetl clf serum nd gondotropins (.G.6 15 U ml -1 ; ntervet, Boxmeer, Hollnd) in n tmosphere of 5% 2 t 39 for h. hey were inseminted in mb medium with 1mcffeine- or 2m cffeine-treted spermtozo of ech bor t stndrd gmete rtion nd co-cultured for 3hours. ssessment of fertiliztion fter co-culture, oocytes were incubted in Z-3 medium (Yoshiok et l., 22) for 15 hours, fixed, stined with bisbenzimide Hoechst nd exmined by epifluorescence. hey were considered s penetrted when the oocytes hd t lest one swollen sperm hed nd/or mle pronucleus in cytoplsm. hose with more thn one swollen sperm hed or mle pronucleus were considered s polyspermic. nly the oocytes with one mle nd one femle pronucleus nd two polr bodies were clssified s monospermic. he dt were expressed s rtes of penetrted oocytes from inseminted, s monospermic or polyspermic oocytes from penetrted nd norml fertilized oocytes from inseminted. ttisticl nlysis Dt from in vitro fertiliztion expressed s mens ± D were nlyzed by the tudent s t-test. esults he men rtes±d of motile nd vible spermtozo fter semen thwing were 45.1±8.8 % nd 5.4±6.4%, respectively. fter seprtion, the men percentges ±D of motile nd vible spermtozo incresed lightly to 5.4±8.5 % nd 56.±6.9 % nd decresed to 38.8±5.8 nd 43.9±5.5 respectively during cpcittion. he crosome rection progress ws different in ech bor nd this process ws fster for 1m cffeine-treted spermtozo compred with 2m cffeine-treted spermtozo in ll tested bors. n bor, the rte of crosome intct spermtozo ws similr before nd fter seprtion nd spermtozo response to cffeine ws continul during cpcittion. he increse in the rte of crosome non-intct spermtozo nd decrese in the rte of crosome intct spermtozo were observed either between h nd 2h or between 1h nd 3h of cpcittion in the 1m cffeinetreted spermtozo or 2 m cffeine-treted spermtozo (igure 1). n bor B, the rte of crosome intct spermtozo mrkedly incresed fter seprtion nd spermtozo response strted quickly, immeditely fter cffeine supplementtion. he rte of crosome non-intct spermtozo ws higher in 1m cffeine-treted spermtozo compred with 2m cffeine-treted spermtozo from h to 2 h nd ws the sme for both cffeine tretments in the of end cptittion (igure 2). n bor, the rte of crosome intct spermtozo lightly incresed fter seprtion nd spermtozo response to cffeine hd one- or two-step chrcter ccording to cffeine tretment. he first decrese in the rte of crosome intct spermtozo occurred between h nd 1h in both cffeine tretment nd the second decrese ws observed between 2h nd 3h of cpcittion but only in 1 m-cffeine tretment when higher rte of spermtozo without crosome ws determined compred with 2 m cffeine-tretment (igure 3). fter oocyte insemintion, the penetrtion rte ws higher for 2 m cffeine-treted spermtozo compred with 1 m cffeine-treted spermtozo in bors nd B nd it ws similr for 1 m nd 2 m cffeine-treted spermtozo in bor. he men ±D vlues of penetrtion nd fertiliztion rtes for oocytes inseminted with spermtozo of ll tested bors re showed in the igure 4. n contrst to penetrtion, the rte of normlly fertilized oocytes ws significntly (p<.5) higher fter insemintion with 1m cffeine-treted spermtozo thn the rte of those inseminted with 2 m cffeine tretedspermtozo. 8

3 H G BDG, 2, 28 (1) igure 1. crosome rection progress in cffeine treted spermtozo of bor permtozo (%) th ime of incubtion (h) crosome intct crosome intct th= fter thwing crosome non-intct crosome non-intct igure 2. crosome rection progress in cffeine treted spermtozo of bor B permtozo (%) th crosome intct crosome non-intct crosome intct crosome non-intct 9 ime of incubtion (h) th= fter thwing

4 H G BDG, 2, 28 (1) igure 3. crosome rection progress in cffeine treted spermtozo of bor permtozo (%) th crosome intct crosome non-intct crosome intct crosome non-intct ime of incubtion (h) th= fter thwing igure 4. enetrtion nd norml fertiliztion efficiency fter insemintion of oocytes with cffeine treted spermtozo for ll bors (men ± D) 9 7 ocytes (%) b enetrted from insem inted orm l fertilizted from insem inted Vlues for the sme prmeter with different superscripts re significntly different (p<.5).

5 H G BDG, 2, 28 (1) Discussion n order to predict the fertilizing bility of cryopreserved bor semen, it is necessry to study functionl sttus of spermtozo. herefore we hve focused our ttention on the chnges of bsic functionl prmeters, i.e. motility, vibility nd crosome integrity in cpcitted spermtozo to compre them with spermtozo in vitro fertilizing bility. ur study proved tht the motility, vibility nd crosome integrity of frozen-thwed spermtozo were not negtively influenced by seprtion or cpcittion. We hve found tht the development of crosome chnges in spermtozo during cpcittion were specific for cffeine tretment becuse no differences in motility nd vibility were observed between both cffeine levels. lthough the spermtozo response to cffeine ws different in ech of the tested bors, in ll sires the crosome rection development ws fster in some of periods of cpcittion for spermtozo treted with 1m cffeine thn for those treted with 2m cffeine. Just s cpcittion so fertiliztion ws more effective fter insemintion of oocytes with 1m cffeine-treted spermtozo compred with 2m cffeine tretedspermtozo. he higher rtes of monospermy nd the lower rtes of polyspermy were detected in oocytes fter their insemintion with 1m cffeine-treted spermtozo in ll tested bors. unhshi nd gi (21) reported tht one of the sources of high polyspermy, tht is usully observed in V system in pig, comes from high rte of spermtozo t erly stge of spontneous crosome rection. We suppose tht in our experiments, the fster development of crosome rection induced by optiml cffeine tretment resulted in the decrese of the rte of spermtozo in erly stge of crosome rection nd consequently in the decrese of polyspermy. n our study, the fster crosome rection progress yielded lesser oocyte penetrtion but the decrese of penetrtion ws much lower in comprison with tht of polyspermy. herefore the ccelerted crosome rection positively influenced norml fertiliztion of oocytes. onclusion t cn be concluded tht reltionship between the kinetics of crosome rection development during cpcittion nd the efficiency of in vitro fertiliztion hs been found in bor spermtozo. orml fertiliztion ws more present t oocytes when fster progress of crosome rection ws induced in spermtozo. esponse of spermtozo to cffeine ws specific for ech bor; it would be importnt to modulte cffeine tretment nd consequently crosome rection progress for individul sires. onitoring of crosome rection chnges in frozenthwed bor spermtozo might be used for improvement of in vitro fertiliztion nd embryo production in pig. eferences vrjl G, uello, uiz, Vázquez J, rtínez, oc J. ffects of centrifugtion before freezing on bor sperm cryosurvivl. J ndrol 24;25: unhshi H, sno, ujiwr, gi, iw K, rser L. Both fertiliztion promoting peptide nd denosine stimulte cpcittion but inhibit spontneous crosome loss in ejculted bor spermtozo in vitro. ol eprod Dev 1999;55: unhshi H, gi. egultion of in vitro penetrtion of frozen-thwed bor spermtozo by cffeine nd denosine. ol eprod Dev 21;58: unhshi H. olyspermic penetrtion in porcine V- V systems. eprod ertil Dev 23;15: Gde J, uiz, elles, omr, tás, oy, oto, eindo B. Use of in vitro fertiliztion for evlution of bor semen freezing procedure. 21;22: Johnson L, xwell W, Dobrinsky J, Welch G. tining sperm for vibility ssessment. eprod Dom nim 27;31: Long, Dobrinsky J, Johnson L. n vitro production of pig embryos: comprison of culture medi nd bors. heriogenology 1999;51: ellés, Gde J, omr, tás, uiz. nlysis of in vitro fertilizing cpcity to evlute the freezing procedures of bor semen to predict the subsequent fertility. eprod Domest nim 23;38: irrd, Dubuc, Bolmb D, Zheng Y, oenen K. ollicle-oocyte-sperm interctions in vivo nd in vitro in pigs. J eprod ertil uppl 1993; 48:3-16. Věžník Z, Švecová D, Zjícová, řinosilová. e p e t i t o r i u m s p e r m t o l o g i e ndrologie, metodiky spermtonlýzy. VÚVeL Brno 24. Wng WH, iw K, kud K. n-vitro penetrtion of pig oocytes mtured in culture by frozen-thwed ejculted spermtozo. J eprod ertil 1991; 93: Westendorf, ichter L, reu H. Zur iefgefrierung von bersperm. Lbor- und besmmingsergebnisse mit dem Hulsenberger illetten-verfhren. Dtsch iererztl Wschr 1975;82: Yoshiok K, uzuki, nk, ns nd wmur. Birth of piglets derived from porcine zygotes cultured in chemiclly defined medium. Biol eprod 22;66: his study ws supported by Grnts QG 552 of the inistry of griculture of the zech epublic, nd 523/H64 of Grnt gency of the zech epublic. 11