牛腔前卵泡的简易机械分离方法

A simple Mechanical method for the isolation of intact preantral follicles from adult bovine

采用两种机械法分离牛腔前卵泡。方法一 (M -1) ,皮肤移植刀切割、剪碎、过滤镜下直接捡卵法。方法二 (M -2 ) ,皮质片剪碎、离心、悬浮、过滤镜下捡卵法。M -1平均每卵巢采卵数为 46 9个± 18 2个 ,M -2为 6 8 4个± 12 5个 ;处理时间M -1为 2 5 4min± 6 7min ,M -2为 46 3min± 16 8min ,两种方法采集腔前卵泡数量及处理时间均存在显著差异。平均每小时采集卵泡数分别为 99 5和 87 2个 ,M -1多于M -2。两种方法获得腔前卵泡大小分布规律也有明显差异 ,M -1采集腔前卵泡直径为 6 0～ 15 0 μm ,绝大部分是次级卵泡 ,而M -2获取腔前卵泡则偏小。总的看来 ,M -1处理时间短 ,回收效率高 ,方法简便 ,且分离卵泡直径较大 ,适于体外培养 ,是机械分离牛腔前卵泡的一种理想方法。

Two mechanical methods were used to isolate the bovine ovarian preantral follicles.Method one (M-1):ovarian cortical slices were dissected from ovaries with skin-graft razor,then the cortical slices were cut into pieces,filtrated through nylon mesh filters (￠450 m).The filtrated medium were sought out directly for preantral follicles under a stereomicroscope at a low magnification. Method two(M-2):Ovarian cortical slices were dissected and cut into pieces.The solution was centrifuged at 1000g for 5 minutes.The suspensions of ovarian fragments were suspended and filtered.The mean numerbers of preantral follicles isolated from each ovary of cows were 46.9±18.8(M-1) and 68.4±12.5(M-2),respectively.While the process time was 25.4min±6.7min(M-1) and 45.3min±16.8min (M-2) for each ovary.There are different distribution of the size of preantral follicles collected in the two methods.The diameters of the preantral follicles isolated by M-1 are 60～150 μm,most of them are secondary follicles,and they are available to be cultured in vitro.In conclusion,this study showed that a simple,rapid mechanical method can be used effectively to isolate intact preantral follicles from adult cow ovaries.