卵母细胞去核及卵丘细胞移核方法对猪体细胞核移植的影响

以猪体外成熟卵母细胞周围分离得到的卵丘细胞作为猪体细胞核移植的供核细胞,研究了卵母细胞不同去核方法(盲吸法、活性荧光染色去核法、盲吸法和活性荧光染色法联合去核法)的去核率差异,并比较了卵丘细胞透明带下注核和胞质内注核两种重建胚构建方法的胚胎裂解率、4 8细胞发育率和桑椹胚率的差异。结果表明:(1)以荧光染料染色法的去核率(87.18%)最高,与其他试验组相比差异显著(P<0.05);盲吸法去核所用时间最少(1.2 min/个),与其他试验组相比差异显著(P<0.05)。(2)透明带下注核比卵母细胞质内直接注核的卵裂率(40.11%/29.75%)和4 8细胞率(11.86%/6.81%)高,差异显著(P<0.05);桑椹胚率(1.75%/1.79%)无明显差异(P>0.05)。

Effects of methods of oocyte enucleation and those of cumulus cell injecting into enuclated oocyte on porcine somatic cell nuclear transplantation

Cumulus cell obtained from in vitro mature porcine cumulus oocyte complexs(COCs) was used as donor of porcine somatic nuclear transfer.Different enucleation methods(blind aspiration(BA),bisbenzimide stain method(BS) and blind aspiration combined with bisbenzimide stain method(BABS)) were studied on enucleation rate of oocyte.Two methods of cumulus cell injecting into enuclated oocyte,perivitelline microinjection(PM) and intracytoplasmic microinjection(IM) were compared on embryo cleavage rate,48 cell developmental rate and morula rate.The results showed:(1) BS had the highest enucleation rate(87.18%),significantly different from other two groups(P<0.05);BA cost the least time(1.2 min per oocyte),significantly different from other two groups(P<0.05);(2) PM had higher cleavage rate(40.11%/29.75%) and 48 cell developmental rate(11.86%/6.81%) than those of IM,two groups were significantly different(P<0.05),but morula rate(1.75%/1.79%) of two groups was not significantly different(P>0.05).