提高山羊卵丘细胞核移植效果的研究

 【目的】提高山羊卵丘细胞核移植效果。【方法】采用秋水仙胺提高山羊卵母细胞去核率,5-氮-2'-脱氧核苷（5-aza-dC）和曲古菌素A（TSA）影响山羊卵丘细胞核移植胚胎。【结果】0.5 μg?ml-1秋水仙胺处理山羊卵母细胞效果最好,胞质突起率达91.7%（P＜0.05）;通过比较盲吸去核法与化学辅助去核法的去核率及构建卵丘细胞核移植胚胎的体外发育能力发现,化学辅助去核法的去核率达100%,所构建的重构胚体外发育能力较高,桑椹胚率和囊胚率分别达25.2%和13.1%,显著高于盲吸去核法（P＜0.05）。采用0.01 μmol?L-1的5-aza-dC处 理山羊卵丘细胞,核移植胚胎桑椹胚率和囊胚率分别达30.4%和17.0%,显著高于其它各组（P＜0.05）;采用 400 nmol?L-1TSA处理山羊卵丘细胞,核移植胚胎桑椹胚率、囊胚率分别达31.5%和15.2%。【结论】化学辅助去核法的去核率显著高于盲吸去核法,采用0.01 μmol?L-1的5-aza-dC或400 nmol?L-1 TSA处理山羊卵丘细胞,效果最佳。

Research on the Efficiency of Goat Cumulus Cell Nuclear Transfer Technique

【Objective】 This study was performed to increase the efficiency of goat cumulus cell nuclear transfer. 【Method】Based on the problem of low efficiency of goat somatic nuclear transplantation (SNT), the application of demecolcine was used to increase goat oocytes enucleation rate, the effects of 5-aza-dC and Trichostatin A (TSA) were also investigated when cumulus cells were treated with them on the development rate of goat cloned embryos. 【Result】 The optimum concentration of demecolcine for chemically assisted enucleation was 0.5 μg?mL-1 and the extrusion cones rate of goat oocytes was 91.67% (P＜0.05). Compared with blind enucleation method, the enucleation rate of demecolcine assisted-enucleation method was 100% and increased significantly (P＜0.05). Furthermore, the morula rate and blastula rate of cloned embryos reconstrusted by demecolcine assisted-enucleation method were 25.23% and 13.08%, which were significantly higher than that of embryos reconstrusted by blind enucleation method (P＜0.05). At the same time, it was found that the blastula rate and morula rate of goat cloned embryos reconstructed by cumulus cells treated with 0.01μmol?L-15-aza-dC was 16.96% and 30.41%, which were extremely higher than that of other groups (P＜0.05). When the goat cloned embryos reconstructed by cumulus cells treated with 400 nmol?L-1 TSA, the rate of morula and blastula was 31.52% and 15.22%. 【Conclusion】The enucleation rate of chemically assisted enucleation method was significantly higher than that of blind enucleation method. The effect of goat cloned embryos reconstructed by cumulus cells treated with 0.01 μmol?L-1 5-aza-dC or 400 nmol?L-1 TSA was the best.