水牛体细胞核移植方法比较及激活前的时间间隔对全细胞胞质内注射法核移植效果的影响

本研究旨在比较水牛2种体细胞核移植(Somatic Cell Nuclear Transfer,SCNT)方法的效果以及激活前的时间间隔对全细胞胞质内注射法(Whole-Cell Intracytoplasmic Microinjection,WCICSI)核移植效果的影响.采用水牛胎儿成纤维细胞作为供核,比较了透明带下注核法(Perivitelline Microinjection,PM)和WCICSI核移植效果.另外,试验了不同类型的供体细胞进行全细胞胞质内注射后与激活前的受体胞质的最适宜作用时间.结果,WCICSI构建核移植重构胚的成功率显著高于PM(87.1%vs 81.1%,P＜0.05),虽然其重构胚的分裂率极显著低于PM(49.5%vs 71.8%,P＜0.01),但囊胚率、核移植的效率无显著差异(P＞0.05).卵丘细胞和胎儿成纤维细胞在注射后3 h激活,重构胚的囊胚发育率最高;颗粒细胞注射后与激活前的最佳时间间隔可在1.5～3 h,但3 h是最佳的作用时间.结果表明,(1)WCICSI可用于水牛体细胞核移植的研究;(2)水牛胞质内注射供体细胞后3 h进行激活,核移植重构胚的发育效果最好.

Comparison of Somatic Cell Nuclear Transfer of Buffalo and the Effects of Time Interval before Activation on Nuclear Transfer by Whole-Cell Intracytoplasmic Microinjection

The aim of the study was to compare two methods of somatic cell nuclear transfer(SC-NT), and explore the effects of time interval before activation on nuclear transfer(NT) by Whole-Cell Intracytoplasmic Microinjection (WCICSI). NT effect of Perivitelline Microinjection (PM) and WCICSI are compared, when the fetus fibroblasts were used for donor cells. And effect of activation time interval after donor cell by WCICSI on buffalo SCNT was also examined.The results showed that the embryo reconstructing efficiency by WCICSI was higher than that by PM (87.1% vs 81.1%, P＜0.05), but the cleavage rate of reconstructed embryos was lower in former than that in latter (49.5% vs 71.8%, P＜0.01). The blastocyst development rate of reconstructed embryos and the total efficiency of nuclear transfer were not significantly different each other (21.6%vs 19.3%, P＞0.05;18.8% vs 15.7%, P＞0.05). The blastocyst development rate of reconstructed embryos derived from cumulus cells and fibroblasts were the highest when the reconstructed embryos were activated at 3 h after injection. There was no significant difference in the development of blastocyst derived from granular cells between the time interval from 1.5 to 3 h, but the best time interval before activation was 3 h. The results indicated the WCICSI method can be employed in buffalo SCNT. Development rate of reconstructed embryos is best when activation at 3 h after buffalo cytoplasmic injection of donor cells.