水牛及小鼠腔前卵泡分离的研究

Study on the isolation of buffalo and mouse preantral follicles

对水牛和小鼠腔前卵泡的分离方法进行了系统探索,旨在找出一种水牛和小鼠腔前卵泡的有效分离方法。实验一分别用梳刮法、剪碎法和显微分离法分离回收水牛卵巢腔前卵泡;实验二采用梳刮法分离水牛胎牛、青年牛和成年牛腔前卵泡;实验三分别用浓度为0(CK)、0.02%、0.04%、0.08%的胶原酶消化水牛卵巢20 m in,然后采集腔前卵泡;实验四采用梳刮法、剪碎法和显微分离法分离小鼠卵巢的腔前卵泡。结果表明,梳刮法能有效地分离回收水牛和小鼠卵巢的腔前卵泡。该法分离水牛卵巢所得腔前卵泡的原始卵泡最多,次级卵泡最少,而分离小鼠卵巢所得腔前卵泡的次级卵泡最多,原始卵泡最少;水牛胎牛卵巢可分离得到较多的腔前卵泡;酶消化有助于提高剪碎法的腔前卵泡分离效率。

A tool named comb-scrape which consists of needles, a needle fixing and a handle was designed to investigale the methods for isolation of buffalo and mouse preantral follicles were investigated in this study. Comb-scrape method harvested significantly more buffalo preantral follicles [ （152.35±44.81） / ovary] than the methods of cutting [ （32.62± 14.81） /ovary] and micromanipulation [ （8.95 ± 3.44） /ovary, P〈0.05]. The average manipulative time for each ovary （39.05±4.27） min was also less than that of the methods of cutting （46.43±4.15） min and micromanipulation [（44.55±7.82） min, P〈0.05]. Majority of preantral follicles isolated by comb-scrape method were primordial follicles （41.25% ） and primary follicles （38.79 % ）, and very few of secondary follicles （19.95 %） . These results indicated that comb-scrape method was suitable for isolating buffalo preantral follicles. Fetal buffalo ovaries harvested significantly more preantral follicles [ （943.43±143.13） /ovary] than heifer [ （249.50±40.11） /ovary] and adult ovaries [ （128.50±19.99） /ovary] （P〈0.01）, indicating that more preantral follicles can be isolated from buffalo fetal ovaries. Digestion of small buffalo ovary tissue pieces with 0.02%, 0.04% and 0.08% collagenase harvested significantly more preantral follicles in comparison with without digestion （40.38 ±6.12, 47.75±6.84and 52.69±6.15 vs 33.56±6.15 per ovary, P〈0.05）, indicating that combination of cutting and enzymatic method can harvest more buffalo preantral follicles than cutting method alone. Comb-scrape method harvested significantly more mouse preantral follicles [（61.00 ± 9.12）/ ovary] than the methods of cutting [（26.30±5.06）/ovary] and micromanipulation [（14.10±4.16）/ ovary,P〈0.05].The average manipulative time for each ovary （30.80±3.91）rain was also less than that of the methods of cutting （39.10±4.01）min and micromanipulation [（38.40±3.50）min,P〈 0.05]. Majority of preantral follicles isolated by comb-scrape method were secondary follicles （83.61%） and primary follicles （12.13%）,and very few of primordial follicles （4.26%）. These results indicate that comb-scrape method is also suitable for isolating mouse preantral follicles.