A method for isolation of buffalo preantral follicles 3

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1 50 (2) : A cta Zoologica S inica ( ) ( ) ( ) ( min) ( min) ( min) ( %) ( %) ( %) 72 h % (48191 %) (59134 %) [ 50 (2) : ] A method for isolation of buffalo preantral follicles 3 PAN Hong2Ping FEN G Gui2Xue SHI De2Shun 3 3 Animal Reproduction Institute Guangxi University Nanning 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 ( / ovary) than cutting ( / ovary) and micromanipulation methods ( / ovary P < 0105). The average handling time for each ovary ( min) was less than cutting ( min) and micromanipulation methods ( min). Most preantral follicles isolated by the comb2scrape method were primordial follicles ( %) and primary follicles ( %) and very few were secondary follicles ( %). 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 % and %). Cat2 tle ovaries harvested significantly more preantral follicles than buffalo ovaries using the comb2scrape method ( vs ). These results indicate that comb2scrape method is suitable for isolating buffalo preantral fol2 licles [ Acta Zoologica Sinica 50 (2) : ]. Key words Buffalo ovaries Preantral follicles Isolation Morphological characteristics 9919 % (Britt 1991 ; Saumamde 1991) ( Hirao et al. 1994) ( Carambula et al ; Figueiredo et al. 1993) ( Lucci et No12002AA ) (No12002AA206605) ] 33 (Corresponding author). ν 2004 Acta Zoologica Sinica [ This research was funded by National 863 High Technology Development Program E2mail :

2 al. 1999) ( Katarina and Manuela 1996) ( Katarina and Manuela 1996) ( Katarina and Frank 1995) ( 1 mm) ( Telfer 1996 ) ( B ubal us bubalis) (Figueiredo et al. 1993) (Cutting method) mm 5 ml ( < 1 mm) ( 1 mm) min ( ) 250 m ( Micromanipulation met hod) (2000) 60 mm 5 ml 31 1 mm h ( Hulshof et al. 1994) 3 ( 1) % s (M mmol/ L HEPES + 5 mmol/ L NaHCO % ) 2-3 : ( Comb2scrape method) m Gutierrez et al. 6 (1) : (2) : (3) : ( ) mm 5 ml M mmol/ L NaHCO Fig1 1 Comb2scrape designed for isolating buffalo preantral follicles

3 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 % g/ ml FSH 2 1 ml %CO ( P < 0105) 1-5 ( P < 0105) ; ( P > 0105) ( ) Table 1 Effects of isolation methods on the number of buffalo preantral follicles recovered and isolation speed 119 SPSS One2Way ANOVA Crosstabs ( P < 0105) ; Isolation method Comb2scrape % % % (58198 %) Cutting Ovary Micromanipulation Preantral follicles recovered Isolation duration (min) 3047 ( ) a a 685 ( ) b b 179 ( ) c b (One2Way ANOVA P < 0105) Values with the different superscripts in the same columns are signifi2 cantly different (One2Way ANOVA P < 0105).

4 Fig13 Stages of buffalo preantral follicles isolated by the three methods 4 Fig14 Number of different size buffalo preantral follicles isolated by three methods ( % %) 215 (62101 %) ( %) 24 h (8194 %) ( P < 0105) 48 h 72 h < 50 m m ( 2) ( % %) > 90 m 216 (16177 %) < 50 m (64196 %) m > 90 m ( % 9193 %) ( P < 0105) > 90 m m ( % %) ( 3) < 50 m (10161 %) min

5 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 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) ( ) a ( ) b (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) min 3 Figueiredo et al. 295

6 ; ( References) Britt J H Impacts of early postpartum metabolism on follicular development and fertility. Bovine Prac. 24 : Carambula SF Goncalves PBD Costa L FS Figueiredo J R Wheeler J R Neves J P Mondadori RG Effect of fetal age and method of recovery on isolation of preantral follicles from bovine o2 varies. Theriogenology 52 : Figueiredo J R Hulshof SCJ Van den Hurk R Ectors FJ Fonts RS Nusgens B Bevers MM Beckers J F Develoment of a com2 bined new mechanical and enzymatic method for the isolation of in2 tact preantral follicles from fetal calf and adult bovine ovaries. Theriogenology 40 : Gospodarowic D Greenburg G Birdwell CR Determination of cellular shape by the extracellular matrix and its correlation with the control of cellular growth. Cancer Res. 38 : Gutierrez CG John HR Telfer EE Wilmut I Webb R Growth and antrum formation of bovine preantal Follicles in long2 term culture in vit ro. Biology of Reproduction 62 : Hirao Y Nagai T Kubo M Miyano T Miyake M Kato S In vit ro growth and maturation of pig oocytes. Journal of Reproduc2 tion and Fertility 100 : Hulshof SCJ Figueiredo J R Beckers J F Isolation and charac2 terization of preantral follicles from fetal bovine ovaries. The Vet2 erinary Quarterly 16 : Katarina J Frank G The recovery of preantral follicles from o2 varies of domestic cats and their characterization before and after culture. Animal Reproduction Science 39 : Katarina J Manuela S Isolation of preantral follicles from non2 domestic cats viability and ultrastructural investigations. Animal Reproduction Science 44 : Lucci CM Amorim CA Rodrigues APR Figueiredo J R Bao SN Sil2 va J RV Goncalves PBD Study of preantral follicle popula2 tion in sit u and after mechanical isolation from caprine ovaries at different reproduction stages. Animal Reproduction Science 56 : Saumamde J La folliculogenese chez les ruminants. Rec. Vet. 167 : Telfer EE The development of method for isolation and culture of preantral follicles from bovine and porcine ovaries. Theri2 ogenology 45 : Woodly DT Sarret Y Cutaneous basement membranes. In : Lapiere CM Kriegf T ed. Connective Tissue Diseases of the Skin. New York : Marcel Dekker Inc

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