猪手工体细胞核移植电融合/激活参数的优化

Optimization of Electrofusion and Artificial Activation Protocols of Porcine Handmade Somatic Cell Nuclear Transfer

通过对猪体细胞核移植中电融合及联合化学激活对猪重构胚发育能力进行探讨,为广西巴马小型猪的手工克隆搭建平台。利用手工克隆(HMC)技术对猪体细胞核移植胚胎采用不同的电融合方法和融合后化学激活不同时间,研究猪手工克隆胚胎的发育能力。结果表明:(1)对重构胚进行一步法电融合时,参数2(180V·mm-1,30μs×1次)与参数1(160·mm-1,30μs×1次)两组间的融合率差异显著(65.31%vs58.33%,P<0.05);(2)对重构胚进行两步法融合时,参数3(第一次:180V·mm-1,10μs×1次,融合60min后进行第二次融合;第二次:85V·mm-1,80μs×1次)和参数4(第一次:200V·mm-1,10μs×1次,融合60min后进行第二次融合;第二次:85V·mm-1,80μs×1次)两组间的总融合率差异不显著(75.88%vs81.20%,P>0.05);(3)采用参数4与采用参数2融合的重构胚其分裂率和囊胚率均差异不显著((79.98%vs72.66%)和(13.14%vs9.36%),P>0.05);(4)在电激活和CHX+CB联合激活4、5、6h这3组中,其分裂率和囊胚率均差异不显著(P>0.05)。相对而言,重构胚采用参数4的两步融合法和CHX+CB激活5h效果较好。

The objective of this study was to explore the optimal conditions of electrofusion combined with artificial activation influencing the development of zona-free porcine cloned embryos,and to lay the foundation for Bama-mini pig somatic cell nuclear transfer embryo by handmade cloning（HMC）.Cloning embryos were fused by one-step or two-step electrofusion and activated in CHX＋CB for 4,5 and 6 h,then cultured in WOWs for 7 d to observe the fusion and embryo development.The results were as follows：（1） The fusion rate of oocytes subjected to one-step electrofusion with parameter 2（180 V·mm-1,30 μs×1） was higher than that with parameter 1（160 V·mm-1,30 μs×1）（65.31% vs 58.33%,P0.05）.（2） There was no significant difference（75.88% vs 81.20%,P0.05） in fusion rate between the groups of oocytes subjected to one-step electrofusion with parameter 3（the first：180 V·mm-1,10 μs×1;the second：85 V·mm-1,80 μs×1 with an interval of 60 minutes） and parameter 4（the first：200 V·mm-1,10 μs×1;the second：85 V·mm-1,80 μs×1 with an interval of 60 minutes）.（3） There was no significant difference in cleavage and blastocyst rates of cloned embryos between the groups of oocytes subjected to electrofusion with the parameter 4 and parameter 2（（79.98% vs 72.66%）,（13.14% vs 9.36%）,P0.05）.（4） There was no significant difference in the rate of cleavage and blastocyst development from oocytes,which were subjected to electrofusion,activated by CB＋CHX for 4,5 and 6 h（P0.05）.The results revealed that the procedure with parameter 4 of electrofusion activated by CB＋CHX for 5 h was relatively suitable.