Factors influencing in vitro fertilization in goats 3

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1 50 (2) : , 2004 A cta Zoologica S inica 3 33, : 1) / ; 2) /, ; 3) h / h ; 4) 3 4 / 0 1 ; 5) / ; 6) / [ 50 (2) : , 2004 ] Factors influencing in vitro fertilization in goats 3 ZHOU Jia2Bo, WU Yan2Guang, L UO Ming2Jiu, HAN Dong, L IU Li2Qing, CHAN G Zhong2Le, TAN Jing2He 33 College of Animal Science and Technology, Shandong Agricultural University, Taian , Shandong, China Abstract We studied the effect of buck individuality, epididymal origin of spermatozoa, presence and absence of cumu2 lus during in vit ro maturation and fertilization and length of maturation culture on in vit ro fertilization and embryo devel2 opment in goats. The results are summarized as follows : 1) semen from different bucks produced significantly different rates of fertilization, cleavage and morulae/ blastocysts ; 2) rates of fertilization, cleavage and morulae/ blastocysts by spermatozoa from cauda epididymidis were not different significantly from those of ejaculated spermatozoa but were markedly higher than those of spermatozoa from corpus epididymidis, and those of spermatozoa from corpus epididymidis were significantly higher than those of spermatozoa from caput epididymidis ; 3) there was no significant difference in fer2 tilization rates among oocytes in vit ro matured for 24, 27 and 30 h, but fertilization rates of oocytes matured in vit ro for 21 h declined significantly ; rates of morulae/ blastocysts in oocytes matured for 21 and 30 h were significantly lower than rates among oocytes matured for 24 and 27 h ; 4) rates of fertilization and morulae/ blastocysts in oocytes with 3 or 4 grade cumulus expansion were significantly higher than those of oocytes with 0 or 1 grade cumulus expansion ; 5) me2 chanical removal of cumulus prior to in vit ro maturation severely affected in vit ro fertilization and embryo development to morulae/ blastocysts of goat oocytes ; 6) complete or partial removal of cumulus prior to in vit ro fertilization did not affect fertilization and cleavage of oocytes, but complete removal of the cumulus significantly decreased the rate of morulae/ blas2 tocysts. In summary, in vit ro maturation and fertilization of goat oocytes were affected by many factors [ Acta Zoologica Sinica 50 (2) : , 2004 ]. Key words Oocyte, Spermatozoa, In vit ro fertilization, Cumulus oophorus, Goat,,, ( In vit ro maturation, fertilization and embryo culture, IVM2IVF2IVC) IVM2IVF2IVC Hanada (1985) , ( G ) ( 0) [ This research was funded by the 973 Project of China Science and Technology Ministry ( Grant No1G ) and the Good Breeds Extension Program of Shandong Province, China ] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail :

2 2 : 217, (1992) COC,, COC IVM2IVF2IVC, 10 % ( v/ v) FCS ( GIB2 CO) 10 IU/ ml PMSG ( ), TCM2199 ( GIBCO) (, 2003) ( Sigma, M28410) 100 l, Eppig and Downs (1989) ( Cumulus2oocytes complex2, 3-4 h COC es, COC) (Denuded oocyte, DO) % CO % CO 2, ( Germinal vesicle breakdown, GVBD) COC Hawk et al. 112 (1992),, 6 5 ml 2 ml DM (Defined medium, ) ( mdm,, (Crosby Younis et al., 1991), 70 l et al., 1981 ; Fukui and Sakuma, 1980) ( Parrish et al., 1986) 1 h,, 017 ml,, 200 g 10 min (Schoreder and Eppig, 1984 ; Sirard et al., 1988 ; Vanderhyden and Armstrong, 1989), 200 g 1,, DPBS (Meizel, 1985 ; Salustri et al., 1989) Hunter and Moor (1987) / ml, min, Gliedt et al. (1996) 24 h 28 h, Marquant2Le Guienne COC 100 l et al. (1990), 3, 18-22,, 5 l, , / ml, 3815, 5 %CO 2 17 h,,, (1 3) 24 h, 1 % ( 24 h, 100 IU/ ml 50 g/ ml ) 3 h, 10 ml (8 ) 2-6 mm 100 IU/ ml 50 g/ ml 20 %, 011 % PVA, 15-20, 3, DPBS ( 50 g/ ml mdm) 1 g/ ml TAL P 1 min, ( TCM mg/ ml mg/ ml 315 mg/ mlbsa ( GIBCO) ) l Dulbeccoπs PBS ( GIBCO) 48 h

3 %CO 2 95 %,, 1, 3, h ( 1), 7 1 / Table 1 Rates of fertilization, cleavage and morulae/ blastu2 114 lae of different buck individuals , 11412,, ( ) ( ) h, h COC 0-4 0,, ; 1, 1-2 ; 2,, 2,, ; 3,, ; 4, /, / Table 2 Fertilizing capacity of goat spermatozoa from differ2 3 ent parts of the epididymis, 27 h / 3 27 h,,, SPSS 810, Kolmogrov2Smirnow, Levene,,, P < / 213 ( ) / Bucks oocytes / Embryo developed/ cultured embryo h / ( P < 0105) ( P > 0105), 21 h ( ) 1 25/ 31 (8016) a3 105/ 146 (7119) a 42/ 146 (2818) a 2 19/ 40 (4715) b 34/ 98 (3416) b 8/ 98 (8116) b 3 17/ 26 (6514) ab 50/ 79 (6313) ab 15/ 79 (1819) ab 3 : 212 Sources of spermatozoa oocytes / Embryo developed/ cultured embryo ( ) 25/ 31 (8016) a 3 85/ 131 (6418) a 27/ 131 (2016) a Ejaculate Cauda 19/ 25 (7610) a 59/ 94 (6217) a 14/ 94 (1419) a Corpus 4/ 11 (3613) b 10/ 36 (2717) b 2/ 36 (515) b Caput 0/ 10 (010) ab 0/ 35 (010) ab 0/ 35 (010) ab 3 :

4 2 : ( P < 0105) h 30 h / 24 h 27 h ( 3) ( P < 0105) / 1 Table 3 Fertilization and embryo development of oocytes in - 2 vitro matured for different time / ( P > 0105) / / Embryo developed/ cultured 5 Maturation embryo time(h) oocytes ( ) 21 4/ 12 (3313) a 3 10/ 38 (2613) a 2/ 38 (513) a 24 8/ 11 (7217) b 14/ 22 (6316) b 4/ 22 (1812) b 27 16/ 21 (7612) b 33/ 49 (6713) b 10/ 49 (2014) b 30 11/ 15 (7313) b 24/ 31 (6010) b 3/ 31 (917) b 3 : 4 Artifi2 29/ 66 57/ / 160 cially denuded (4319) b (3516) b (1016) b Table 4 Effect of the degree of cumulus expansion on fertil2 ization and embryo development / / Embryo developed/ cultured Degree of embryo cumulus expansion ( ) oocytes 5, Table 5 Effect of cumulus during in vitro maturation on fer2 tilization and embryo development Cumulus status Natu2 rally denuded / / Embryo developed/ cultured embryo oocytes 1-2 With 1-2 layers of cumulus cells 3 With 3 or more layers of cumulus cells ( ) 5/ 40 6/ 72 0/ 72 (1215) a (813) a (010) a / 40 88/ / 135 (7715) ab (6512) ab (2017) ab 56/ / / 200 (8213) ab (6710) ab (2315) ab 0 0/ 61 (010) a 3 0/ 85 (010) a 0/ 85 (010) a 1 1/ 30 (313) b 0/ 60 (010) a 0/ 60 (010) a 2 6/ 40 (1510) c 13/ 106 (1213) b 0/ 106 (010) a 3 : 3 25/ 40 (6215) ab 43/ 90 (4717) ab 11/ 90 (1212) b 4 49/ 60 (8116) ac 83/ 125 (6614) ac 27/ 125 (2116) ab 3 : 214, 0, 1,, (3 4 ) Marquant2Le Guienne et al. / ( P < 0105), 3, ( 4) 216,,, / 3 (1990), (36 % - 95 %)

5 (2000), 21 h 30 h /, 1716 % %, 24 h 27 h, 110 % %,,, 6, Table 6 Effect of cumulus during in vitro fertilization on fer2, 1 tilization and embryo development / /, Embryo developed/ cultured, Cumulus embryo, morphology Com2 pletely denuded Partially denuded Intact oocytes ( ) 24/ 30 63/ 96 14/ 96 (8010) a 3 (6516) a (1415) b 26/ 32 62/ 90 21/ 90 (8111) a (6818) a (2313) a 40/ / / 150 (8416) a (6713) a (2210) a 3 :,,,, Harayama et al. (1993),, ; ( Yanagimachi, 1994) 74 % ( Itagaki and Toyoda, 1991 ) ( Bavister et 93 %, al., 1982 ) ( Kikuchi et al., 1993 ) Williams et al. (1991), ( Fatehi et al., 2002), A23187,,,,, (Behalova and Greve,, 1993 ; Cox, 1991), Hawk et al. ( 1992) 8 h,,,,, Gliedt et al. (1996),, / 24 h 28 h,, 24 h 28 h,, (2 ),, (2002), ( ), 1-2 (Vanderhyden and Armstrong, 1989) ( Zhang et al., 1995 ; Leibfried2Rutledge et al., 1989),, ( Cox et al., 30 h, 21 h 1993) ( Chian et al., 1995), ( Fukui, 1990), ( Katska

6 2 : et al., 1989) (Cox et al., 1993 ; Fukui, 1990),,, ( References) Bavister BD, Kinsey DL, Lane M, Gardner D K, Evidence for a role of post2ovulatory cumulus components in supporting fertiliz2 ing ability of hamster spermatozoa. J. Androl. 3 : Behalova E, Greve T, Penetration rate of cumulus2enclosed versus denuded bovine egg fertilized in vit ro. Theriogenology 35 : 191. Chian RC, Okuda K, Niwa K, Influence of cumulus cells on in vit ro fertilization of ovine oocytes derived from in vit ro matura2 tion. Anim. Reprod. Sci. 38 : Cox J F, Effect of the cumulus cells on in vit ro fertilization of in vit ro matured cow and sheep oocytes. Theriogenology 35 : 193. Cox J F, Hormazabal J, Maria AS, Effect of cumulus on i n vit ro fertilization of bovine matured oocytes. Theriogenology 40 : Crosby IM, Osborn J C, Moor RM, Follicle cell regulation of protein synthesis and development competence in sheep oocytes. J. Reprod. Fertil. 62 : Eppig JJ, Downs SM, Chemical signals that regulate mam2 malian oocyte maturation. Biol. Reprod. 30 : Fatehi AN, Zeinstra EC, Kooij RV, Colenbrander B, Bevers MM, Effect of cumulus cell removal of in vit ro matured bovine oocytes prior to in vit ro fertilization on subsequent cleavage rate. Theriogenology 57 : Fukui Y, Effect of follicle cells on acrosome reaction, fertiliza2 tion and developmental competence of bovine oocytes matured i n vit ro. Mol. Reprod. Dev. 26 : Fukui Y, Sakuma Y, Maturation of bovine oocytes cultured i n vit ro : relation to ovarian activity, follicular size and the presence or absence of cumulus cells. Biol. Reprod. 22 : Gliedt DW, Rosenkrans CF, J r, Rorie RW, Rakes J M, Ef2 fects of oocyte maturation length, sperm capacitation time, and heparin on bovine embryo development. J. Dairy Sci. 79 : Hanada A, In vit ro fertilization in goat. Jpn. J. Anim. Re2 prod. 31 : Harayama H, Kusunoki H, Kato S, Capacity of goat epididy2 mal spermatozoa to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane. Reprod. Fertil. Dev. 5 : Hawk HK, Nel ND, Waterman RA, Wall RJ, Investigation of means to improve rates of fertilization in vit ro of bovine oocytes. Theriogenology 38 : Hunter AG, Moor RM, Stage2dependent effects of inhibiting ribonucleic acids and protein synthesis on meiotic maturation of bovine oocytes i n vit ro. J. Dairy. Sci. 70 : Itagaki Y, Toyoda Y, Factors affecting fertilization in vit ro of mouse eggs after removal of cumulus oophorus. J. Mammal Ova. Res. 8 : Katska L, Kauffold P, Smorag Z, Duschinski V, Torner H, Kanitz W, Influence of hardening of zone pellucida on in vit ro fertilization of bovine oocytes. Theriogenology 32 : Kikuchi K, Nagai T, Motlik J, Shioya Y, Izakie Y, Effect of follicle cells on in vit ro fertilization of pig follicular oocytes. Theriogenology 39 : Leibfried2Rutledge ML, Critser ES, Parrish JJ, First NL, In vit ro maturation and fertilization of bovine oocytes. Theri2 ogenology 31 : Liu DJ, Chen HW, Brandon M, Xu RG, Effects of semen from different bulls on in vit ro fertilization of bovine oocytes. J. Inner Mongolian University 31 : ( In Chinese). Marquant2Le Guienne B, Humblot P, Thibier M, Thibault C, Evaluation of bull semen fertility by homologous in vit ro fertiliza2 tion tests. Reprod. Nutr. Dev. 30 : Meizel S, Molecules that initiate or help stimulate the acrosome reaction by their interaction with the sperm surface. Am. J. Anat. 174 : Parrish JJ, Susko2Parrish JL, Critser ES, Eyestone WH, First NL, Bovine in vit ro fertilization with frozen2thawed semen. Theriogenology 25 : Salustri A, Yanagishita M, Hascall VC, Synthesis and accu2 mulation of hyaluronic acid proteoglycans in the mouse cumulus cell2 oocyte complex during FSH induction mucification. J. Biol. Chem. 23 : Schoreder AC, Eppig JJ, The development capacity of mouse oocytes that matured spontaneously in vit ro is normal. Dev. Biol. 102 : Sirard MA, Parrish JJ, Ware CB, Leibfried2Rutledge ML, First NL The culture of bovine oocytes to obtain developmentally competent embryos. Biol. Reprod. 39 (3) : Sun XS, Yue KZ, Ma SF, Liu ZH, Liu Y, Tan J H, Rela2 tionship between cumulus expansion and nuclear maturation in pig oocytes. Scientia Agricultura Sinica 35 : ( In Chinese). Sun XS, Yue KZ, Ma SF, Li JL, Zou Y, Tan J H, The Ef2 fect of pre2incubation of porcine COCSs in medium without go2 nadotropins on the chromatin configuration and quality of oocytes matured in vit ro. Acta Zool. Sin. 49 : ( In Chinese). Vanderhyden BC, Armstrong OT, Role of cumulus cells and serum on the in vit ro maturation, fertilization and subsequent de2 velopment of rat oocyte. Biol. Reprod. 40 : Williams RM, Graham J K, Hammerstedt RH, Determination of the capacity of ram epididymal and ejaculated sperm to undergo the acrosome reaction and penetrate ova. Biol. Reprod. 44 : Yanagimachi R, Mammalian fertilization. In : Knobil E, Neill JD ed. The Physiology of Reproduction. 2ed edn. New York : Raven Press, Younis AI, Zuelke KA, Harper KM, Oliveira MA, Brackett B G, In vit ro fertilization of goat oocytes. Biol. Reprod. 44 : Zhang L, Jiang S, Wozniak PJ, Yang XZ, Godke RA, Cu2 mulus cell function during bovine oocyte maturation fertilization and embryo development in vit ro. Mol. Reprod. Dev. 40 : ,, Mal Brandon,, : ,,,,,, : ,,,,,, :

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