化学辅助去核法对延边黄牛卵母细胞去核率及其重构胚发育率的影响

Effects of Chemically Assisted Enucleation on the Enucleation Rate of Yanbian Cattle Oocytes and the Development of their Reconstructed Embryos

为提高延边黄牛体细胞克隆的效率,采用化学辅助去核法——化学诱导剂脱羰秋水仙碱(Demecolcine,简称Deme)处理体外成熟的MⅡ期延边黄牛卵母细胞,经显微操作构建重构胚后观察发育情况。试验研究了Deme的处理浓度、作用时间对延边黄牛卵母细胞去核效果及其重构胚发育的影响。结果显示:体外成熟的卵母细胞在0.2μg/mL、0.3μg/mL、0.4μg/mL的Deme溶液中处理45 min,显核率与去核率无显著差异,去核率达100%,但是0.2μg/mL处理组的囊胚发育率显著高于其他处理组;45 min处理组的显核率显著高于30 min、60 min处理组,45 min处理组的囊胚率高于其他处理组;化学辅助去核法的囊胚率显著高于盲吸法。化学辅助去核能够准确定位去除细胞核,并提高了延边黄牛体细胞克隆的去核率及重构胚的体外发育率。

In order to enhance the efficiency of somatic cell nuclear transfer,MⅡ oocytes of Yanbian cattle were treated by demecolcine.Nucleus of oocytes were deleted by micromanipulation.These oocytes were prepared for the nuclear transfer receptor.The effects of the concentration of demecolcine,the incubation time of oocytes in demecolcine on the enucleation rates and the development of reconstructed embryos were studied.The results showed that：There were no significant differences in the rates of cytoplast protrusion and enucleation in vitro mature oocytes incubated in demecolcine of 0.2 μg/mL,0.3 μg/mL,0.4 μg/mL for 45 min,the rate of cytoplast enucleation was 100%,the 0.2 μg/mL group blastocyst rate was significantly higher than those of other concentrations.The group treated for 45 min had significantly higher cytoplast protrusion rate than the groups treated for 30 min and 60 min.The group treated for 45 min had higher blastocyst rate than other groups.The blastocyst rate of chemically assisted enucleation was significantly higher than that of blind enucleation.These results demonstrated that the chemically assisted enucleation can precisely locate and enucleate the nuclear,and enhance the enucleation rate of Yanbian cattle oocytes and the early development of reconstructed embryos in vitro.