关键词: 猪, 体细胞核移植, 重构胚, 发育

Abstract:

【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.

Key words: swine, somatic cell nuclear transfer, reconstructed embryos, development