Factors influencing in vitro fertilization in goats 3

50 (2) :216-221, 2004 A cta Zoologica S inica 3 33, 271018 : 1) / ; 2) /, ; 3) 24 27 h / 21 30 h ; 4) 3 4 / 0 1 ; 5) / ; 6) / [ 50 (2) : 216-221, 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 217018, 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) : 216-221, 2004 ]. Key words Oocyte, Spermatozoa, In vit ro fertilization, Cumulus oophorus, Goat,,, ( In vit ro maturation, fertilization and embryo culture, IVM2IVF2IVC) IVM2IVF2IVC Hanada (1985) 2003207207, 2003209214 3 973 ( G200016107) ( 0) [ This research was funded by the 973 Project of China Science and Technology Ministry ( Grant No1G200016107) and the Good Breeds Extension Program of Shandong Province, China ] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica E2mail : tanjh @sdau1edu1cn

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) 3815 5 % CO 2 100 % 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) 80 10 6 / ml, 3815 45 min, Gliedt et al. (1996) 24 h 28 h, Marquant2Le Guienne COC 100 l et al. (1990), 3, 18-22,, 5 l, 315-4 10 6, / ml, 3815, 5 %CO 2 17 h,,, (1 3) 24 h, 1 % 1 111 113 30-35 ( 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, ( TCM2199 0155 mg/ ml 01146 mg/ ml 315 mg/ mlbsa ( GIBCO) ) 20 100 l Dulbeccoπs PBS ( GIBCO) 48 h

18 2 50 3815 5 %CO 2 95 %,, 1, 3, 2 7 48 h ( 1), 7 1 / Table 1 Rates of fertilization, cleavage and morulae/ blastu2 114 lae of different buck individuals 11411 1 2 3, 11412,, 11413 ( ) ( ) 21 24 27 30 h, 11414 27 h COC 0-4 0,, ; 1, 1-2 ; 2,, 2,, ; 3,, ; 4, /, / 11415 2 1-2 Table 2 Fertilizing capacity of goat spermatozoa from differ2 3 ent parts of the epididymis, 27 h 11416 / 3 27 h,,, 115 3 SPSS 810, Kolmogrov2Smirnow, Levene,,, P < 0105 2 211 / 213 ( ) / Bucks oocytes / Embryo developed/ cultured embryo 1 2 3 24 27 30 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 :

2 : ( P < 0105) 215 21 h 30 h / 24 h 27 h ( 3) ( P < 0105) 1-2 3 3 / 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 17/ 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 219 31/ 40 88/ 135 28/ 135 (7715) ab (6512) ab (2017) ab 56/ 68 134/ 200 47/ 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 %)

20 2 50 (2000), 21 h 30 h /, 1716 % - 7417 %, 24 h 27 h, 110 % - 3216 %,,, 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/ 49 101/ 150 33/ 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,, 24 27 (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

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