猪体细胞克隆胚胎的体外生产试验

Experiment on Production in Vitro of Porcine Somatic Cell Cloned Embryos

【目的】通过优化猪体细胞核移植程序,建立获得克隆囊胚的有效方法。【方法】比较不同电激活参数、不同体外培养条件对猪体细胞克隆胚发育能力的影响。【结果】选用1.5kV·cm-1、80μs和1DC的参数组合,对猪核移植重构胚进行电融合和电激活,可获得较高的卵裂率和最高的囊胚发育率(分别为71.4%和14.3%),且囊胚发育率显著高于其它组(P<0.05);0.4%BSANCSU23培养液培养克隆重构胚72h,半量换液并未改善克隆胚的发育,但添加10%FBS可显著提高囊胚发育率(15.1%对10.3%,P<0.05)和DNA完整率(56.8%对46.6%,P<0.05);采用猪卵泡颗粒细胞(pGC)共培养体系未能显著提高克隆胚发育率(P>0.05),但卵丘细胞(pCC)单层共培养时,囊胚发育率显著高于对照组(16.7%对9.8%,P<0.05),培养5d的克隆胚凋亡率也显著低于对照组(39.5%对54.2%,P<0.05)。【结论】采用1.5kV·cm-1、80μs和1DC的参数组合对猪克隆重构胚进行电融合和电激活,以0.4%BSANCSU23培养液作pCC单层共培养72h,然后换用10%FBSNCSU23培养液,可明显提高囊胚发育率。

【Objective】 The purpose of this experiment is to establish an efficient method for harvesting porcine cloned blastocysts via optimization of somatic cell nuclear transfer （SCNT） procedures. 【Method】 The effects of different electrical activation parameters and in vitro culture conditions on the developmental competence of porcine SCNT embryos were examined and compared. 【Result】The comparison study of different electrical activation parameters indicated that the combination of a single direct current （DC） pulse of 1.5 kV·cm^-1 electrical field strength for 80 μs could yield better cleavage rate and the highest blastocyst rate （71.4% and 14.3%, respectively） together with significantly higher developmental rate of blstocysts than that from other groups （P〈0.05）. After culture in vitro for 72 h with 0.4% BSA NCSU23 medium, half medium exchange didn’t improve the development of cloned embryos. However, addition of 10% FBS into culture medium could significantly increase the developmental rate of blstocysts （15.1% versus 10.3%, P〈0.05） and the DNA integrity rate （56.8% versus 46.6%, P〈0.05）. Co-cultured with porcine granulose cells （pGC） didn’t increase the cloned embryo developmental rate （P〉0.05）, but co-culture with cumulus cells （pCC） yielded higher blastocyst rate than that from the control （16.7% versus 9.8%, P〈0.05） and the apoptotic rate of cloned embryos cultured for 5 days was also significantly lower than that of the control （39.5% versus 54.2%,P〈0.05）. 【Conclusion 】Under the present experimental condition, the combination of a single DC pulse of 1.5 kV·cm^-1 electrical field strength for 80 μs was the optimal electrical activation parameters for porcine reconstructed embryos. After co-culture with pCC in 0.4% BSA NCSU23 medium for 72 h, addition of 10% FBS into culture medium could significantly increase the developmental rate of blastocysts.