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Study on the Improvement of Bovine Somatic Cell Nuclear Transfer Technique  

양윤희 (강원대학교 동물자원과학대학)
최종엽 (강원대학교 동물자원과학대학)
이상영 (경상대학교 첨단양돈연구소 생명공학과)
박춘근 (강원대학교 동물자원과학대학)
양부근 (강원대학교 동물자원과학대학)
김정익 (강원대학교 동물자원과학대학)
정희태 (강원대학교 동물자원과학대학)
Publication Information
Korean Journal of Animal Reproduction / v.27, no.3, 2003 , pp. 233-240 More about this Journal
Abstract
This study was conducted to examine the effects of oocyte maturation period, phytohemagglutinin-P (PHA-P) treatment and activation agent on the enucleation, fusion, activation or in vitro development of bovine nuclear transfer embryos. Bovine oocytes were enucleated at 16∼24 h of in vitro maturation (IVM). Adult ear skin cells treated or non-treated with PHA-P were transferred into enucleated oocytes. Reconstituted oocytes treated or non-treated with PHA-P were fused by a pulse of 1.5 kV/cm for 30 $\mu$sec. Fused oocytes were activated with a combination of calcium ionophore (A23187) and cycloheximide (CHXM) or dimethylaminopurine (DMAP), and cultured in vitro for 7∼9 days. Enucleation rate was significantly increased when oocytes were matured for 16∼18 h (70.2∼92.3%, P<0.05) compared to that of oocytes were matured for 20∼24 h (44.3∼53.4%). The location of metaphase-II plate was far off from the 1st polar body as maturation time was increased. PHA-P treatment of donor cells or reconstituted oocytes significantly improved fusion rate (P<0.05). Cleavage and blastocyst formation rates were significantly increased after activation with a combination of A23187 and DMAP (78.6% and 32.9%, respectively) compared to those of embryos activated with a combination of A23l87 and CHXM (48.5 and 15.2%, respectively). From the present result, it is suggested that high enucleation efficiency can obtained by using oocytes matured for 18 h. It also shows that PHA-P treatment can improve the fusion rate, and activation with a combination of A23187 and DMAP can enhance the embryo development.
Keywords
Nuclear transfer; Efficiency; In vitro development; Bovine somatic cells;
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