关键词: 猪, 卵母细胞, 手工克隆, 体细胞核移植

Abstract: The objective of this study was to explore the optimal conditions of electrofusion combined with artificial activation influencing the development of zona-free porcine cloned embryos, and to lay the foundation for Bamamini pig somatic cell nuclear transfer embryo by handmade cloning (HMC). Cloning embryos were fused by one-step or two-step electrofusion and activated in CHX+CB for 4, 5 and 6 h, then cultured in WOWs for 7 d to observe the fusion and embryo development. The results were as follows：(1) The fusion rate of oocytes subjected to one-step electrofusion with parameter 2 (180 V·mm^-1, 30 μs×1) was higher than that with parameter 1 (160 V·mm^-1，30 μs×1) (65.31% vs 58.33%，P<0.05). (2) There was no significant difference (75.88% vs 81.20%，P>0.05) in fusion rate between the groups of oocytes subjected to one-step electrofusion with parameter 3 (the first：180 V·mm^-1，10 μs×1; the second: 85 V·mm^-1，80 μs×1 with an interval of 60 minutes) and parameter 4 (the first：200 V·mm^-1，10 μs×1 ; the second: 85 V·mm^-1，80 μs×1 with an interval of 60 minutes). (3) There was no significant difference in cleavage and blastocyst rates of cloned embryos between the groups of oocytes subjected to electrofusion with the parameter 4 and parameter 2 ((79.98% vs 72.66%), (13.14% vs 9.36%)，P>0.05). (4) There was no significant difference in the rate of cleavage and blastocyst development from oocytes, which were subjected to electrofusion, activated by CB+CHX for 4, 5 and 6 h (P>0.05). The results revealed that the procedure with parameter 4 of electrofusion activated by CB+CHX for 5 h was relatively suitable.

Key words: porcine, oocytes, handmade cloning, somatic cell nuclear transfer