A method for isolation of buffalo preantral follicles 3

50 (2) :291-296 2004 A cta Zoologica S inica 3 33 530005 31 61 (152135 44181) (32162 14181) (8195 3144) (39105 4127 min) (46143 4115 min) (44155 7182 min) ( 41125 %) ( 38179 %) ( 19195 %) 72 h 56138 % (48191 %) (59134 %) 10 20 1 195120 685100 152135 44181 [ 50 (2) : 291-296 2004 ] A method for isolation of buffalo preantral follicles 3 PAN Hong2Ping FEN G Gui2Xue SHI De2Shun 3 3 Animal Reproduction Institute Guangxi University Nanning 530005 China Abstract To identify an efficient method for isolation of buffalo preantral follicles we designed a tool named comb2 scrape which consists of needles a needle fixing and a handle. The comb2scrape method harvested significantly more pre2 antral follicles (152135 44181/ ovary) than cutting (32162 14181/ ovary) and micromanipulation methods (8195 3144/ ovary P < 0105). The average handling time for each ovary (39105 4127min) was less than cutting (46143 4115 min) and micromanipulation methods (44155 7182 min). Most preantral follicles isolated by the comb2scrape method were primordial follicles ( 41125 %) and primary follicles ( 38179 %) and very few were secondary follicles ( 19195 %). The survival rate of preantral follicles isolated using the comb2scrape method at 72 hours after in vit ro culture did not differ significantly from that of cutting and micromanipulation methods (56138 % vs 48191 % and 59134 %). Cat2 tle ovaries harvested significantly more preantral follicles than buffalo ovaries using the comb2scrape method (1 195120 685100 vs 152135 44181). These results indicate that comb2scrape method is suitable for isolating buffalo preantral fol2 licles [ Acta Zoologica Sinica 50 (2) : 291-296 2004 ]. Key words Buffalo ovaries Preantral follicles Isolation Morphological characteristics 9919 % (Britt 1991 ; Saumamde 1991) ( Hirao et al. 1994) ( Carambula et al. 1999 ; Figueiredo et al. 1993) ( Lucci et 2003210203 2003202219 3 863 No12002AA206605 ) (No12002AA206605) ] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica [ This research was funded by National 863 High Technology Development Program E2mail : ardsshi @gxu1edu1cn

92 2 50 al. 1999) ( Katarina and Manuela 1996) ( Katarina and Manuela 1996) ( Katarina and Frank 1995) ( 1 mm) 50-60 ( Telfer 1996 ) ( B ubal us bubalis) (Figueiredo et al. 1993) 11412 (Cutting method) 21 60 mm 5 ml ( < 1 mm) ( 1 mm) 50-60 2-3 min ( ) 250 m 11413 ( Micromanipulation met hod) 1 111 (2000) 60 mm 5 ml 31 1 mm 61 2 112 35-39 4 h 115 113 ( Hulshof et al. 1994) 3 ( 1) 114 75 % 2-3 10 s (M199 + 5 mmol/ L HEPES + 5 mmol/ L NaHCO 3 + 3 % ) 2-3 : 11411 ( Comb2scrape method) 10 250 m 11 10 20 Gutierrez et al. 6 (1) : (2) : (3) : 2 2 20 ( ) 116 60 mm 5 ml M199 + 2612 mmol/ L NaHCO 3 + 5 1 Fig1 1 Comb2scrape designed for isolating buffalo preantral follicles

2 : 293 2 A. ( 400) B. ( 400) C. ( 400) Fig12 Morphological characteristics of stages of buffalo preantral follicles A. Primordial follicle ( 400). B. Primary follicle ( 400). C. Secondary follicle ( 400). mmol/ L Hepes + 5 % (OCS) + 2 % + 011 g/ ml FSH 2 1 ml 212 5 %CO 2 3815 10 117 ( P < 0105) 1-5 ( P < 0105) ; ( P > 0105) ( ) 1 118 10 20 Table 1 Effects of isolation methods on the number of buffalo preantral follicles recovered and isolation speed 119 SPSS One2Way ANOVA Crosstabs 2 211 ( P < 0105) ; Isolation method Comb2scrape 213 3 41125 % 38179 % 19195 % (58198 %) Cutting Ovary 20 21 20 Micromanipulation Preantral follicles recovered Isolation duration (min) 3047 (152135 44181) a 39105 4127 a 685 (32162 14181) b 46143 4115 b 179 (8195 3144) c 44155 7182 b (One2Way ANOVA P < 0105) Values with the different superscripts in the same columns are signifi2 cantly different (One2Way ANOVA P < 0105).

94 2 50 3 Fig13 Stages of buffalo preantral follicles isolated by the three methods 4 Fig14 Number of different size buffalo preantral follicles isolated by three methods ( 26142 % 14160 %) 215 (62101 %) ( 29105 %) 24 h (8194 %) 214 4 ( P < 0105) 48 h 72 h < 50 m 50-90 m ( 2) ( 42104 % 41119 %) > 90 m 216 (16177 %) < 50 m (64196 %) 50-90 m > 90 m ( 25111 % 9193 %) ( P < 0105) > 90 m 50-90 m ( 46193 % 42146 %) ( 3) < 50 m (10161 %) 1 195120 40130 min

2 : 2 Table 2 Development of preantral follicles isolated by the three methods cultured in vitro ( %) Viable numbers of preantral Isolation method Follicles cultured follicles cultured in vit ro Comb2scrape Cutting 94 92 91 Micromanipulation 24 h 48 h 72 h 78 (82198 %) ab 62 (65196 %) 69 (75100 %) a 55 (59178 %) 82 (90111 %) b 63 (69123 %) 53 (56138 %) 45 (48191 %) 54 (59134 %) (One2Way ANOVA P < 0105) Values with the different superscripts in the same columns are signifi2 cantly different (One2Way ANOVA P < 0105). 3 ( ) Table 3 Recovery eff iciency of preantral follicles in cattle and buffaloes using comb2scrape method Animal Cattle Buffalo Ovary Preantral follicles recovered Isolation duration (min) 20 23 904 (1 195120 685100) a 40130 4150 20 3 047 (152135 44181) b 39105 4127 (One2Way ANOVA P < 0105) ( Gospodarowic et al. 1978) ; (3) Values with the different superscripts in the same columns are signifi2 ( Woodly and Sar2 cantly different (One2Way ANOVA P < 0105). 3 ( Figueiredo et al. 1993) : (1) ; (2) ret 1993) Figueiredo et al. (1993) 2 142 254 512 92 298 54 45 min 3 Figueiredo et al. 295

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