影响奶牛体细胞手工克隆技术因素的研究

Study on Handmade Somatic Cell Cloning in Dairy Cattle

旨在优化奶牛的手工体细胞克隆技术方案,本试验以牛卵母细胞为材料研究了2个半卵不同粘合交流电压、重构胚不同激活方法和不同的COCS采集方法对奶牛手工体细胞克隆胚发育的影响。结果表明,(1)降低2个半卵粘合交流电压能显著提高其随后的卵母细胞融合率(P<0.05);(2)A23187+6-DMAP组激活的无透明带重构胚分裂率和囊胚率显著高于Ionomycin+6-DMAP组(P<0.05);(3)真空蠕动泵抽吸法和刀片切割法获得的卵母细胞总数显著高于注射器抽吸法(P<0.05),而切割法获得的1和2级卵母细胞百分数显著高于其它2种方法(P<0.05)。因此,采用真空蠕动泵抽吸法获得COCS,用较低的粘合交流电压进行半卵粘合,采用A23187+6-DMAP激活法进行融合后的激活能显著提高奶牛手工体细胞克隆效率。

To optimize the technique of handmade somatic cell cloning in dairy cattle,based on bovine oocytes,the different alternating current（AC） voltage,different activation methods of reconstructed embryos and different collection methods of cumulus-oocyte complexes（COCs） were used.The results showed that：（1）The fusion rate of oocytes increased significantly with the decreasing of AC voltage（P〈0.05）.（2）The cleavage rates and blastocyst rates of reconstructed embryos treated with A23187 ＋ 6-DMAP was significantly higher than that treated with ionomycin ＋ 6-DMAP（P〈0.05）.（3）The total number of oocytes collected by negative-pressure aspiration and by cutting ovaries was significantly higher than that collected by injector aspiration（P〈0.05）,and the proportion of COCs of grade 1-2 got by cutting ovaries was higher（P〈0.05）.In conclusion,using lower AC voltage,choosing A23187 ＋ 6-DMAP for reconstructed embryos activating,aspirating COCs with negative-pressure aspiration can significantly improve the handmade somatic cell cloning efficiency of dairy cattle.