牛胚胎体外生产技术的简化研究

IECHNICAL MODIFICATIONS OF IN VITRO PRODUCTION OF BOVINE EMBRYOS

利用屠宰牛卵巢分离的卵母细胞，以卵裂率和囊胚发育率为标准，对牛胚胎体外生产过程进行了简化试验。结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００～２００枚／平皿）可代替加卵丘细胞的标准密度（５０枚／平皿）培养，其卵裂率（５７．６％比６２．５％）和囊胚发育率（２３．７％比２９．１％）没有显著差异（Ｐ＞０．０５），体外授精时间可从成熟培养后２２ｈ延长至２７ｈ，卵裂率和囊胚发育率均没有显著变化，卵母细胞体外成熟后可以不经洗涤直接移入受精滴，原成熟培养皿内残留卵丘细胞再培养４８ｈ形成的单层细胞，可代替标准方法制作的单层小滴用于体外受精胚胎的共同培养，供胚胎发育培养的单层细胞使用多次不影响囊胚发育率，经简化的ＩＶＦ技术获得的胚胎非手术移入情期同步受体牛子宫角２～２．５个月后直检有５８．３％（７／１２）妊娠。我们认为，传统的牛胚胎体外生产技术经过简化可以提高生产效率，不会减少囊胚发育率和妊娠率，值得推广应用。

A series of experiments were performed to make the technique of in vitro produ-ction(IVP) of bovine embryos simple and effective. The rates of cleavage and blasto-cyst development after IVM and IVF of abatoir-derived oocytes was used for estimation.The results showed that:(1) The in vitro maturation density could be raised from 50/dish to 100-200/dishwithout significant changes of the rates of cleavage(62.5%vs 57.6%)and blastocystdeveloped (29.1%vs 23.7%)(P>0.05). (2)The time for in vitro maturation could be extended from 22h to 27h.(3)Washing procedure before insemination of IVM oocytes was not nesesary.(4)The cumulus cell monolayer derived from denuding the in vitro matured oocvtescould substitute for the standard granular cell monolayer to suport the development ofIVP embryos.(5)The cumulus cell monolayer could be used for the co-culture of IVP embryosmore than one times.The developmental potential of the embryos got from the simplified IVP techniquehas been demonstrated by the high pragnancy rate(58.3%,7/12) established aftertransfer of these embryos. These results suggest that the traditional IVP technique ofbovine embryos could be simplified in many ways to increase the production efficiency.