직류통전의 강도와 기간 및 공핵란의 일령이 소 핵이식배의 융합과 발달에 미치는 영향 = Effects of Strength and Duration of Direct Current ( DC ) Pulse , and Age of Donor Embryo on Fusion and Development of Bovine Nuclear Transfer Embryos

다국어 초록 (Multilingual Abstract)

The nuclear transfer technique is a biotechnique of life science that produces cloned animals with same genetic performance by transplantation of a blastomere of a donor embryo with multiplied cells into an enucleated oocyte. In this experiment, effects of strength and duration of a DC pulse at activation and fusion as well as age of donor embryos on fusion and development of OBCs were investigated for improvement of production efficiency of nuclear transfer embryos. When bovine enucleated oocytes were activated with a DC pulse of 0.6 KV/㎝ for 50 μsec or 1 KV/㎝ for 90 μsec and fused with in vitroderived day 4 donor blastomere by a DC pulse of 1 KV/㎝ for 30 μsec, the fusion rates, the cleavage rates and the developmental rates to morula plus blastocyst of oocyte blastomere complexes were 90 or 83.3%, 59.3 or 56.7%, and 7.4 or 6.7%, respectively. There was no significant (P$gt;0.05) difference between the two DC pulse treatments in the percentages of fusion, cleavage and development. In the subsequent experiment, enucleated oocytes were activated with DC pulse of 0.6 KV/㎝ for 50 μsec and cultured for 6 h in CRlaa supplemented with 10 ㎍/㎖ cycloheximide. Then, a blastomere of an in vitro-derived day 4 donor embryo was introduced into the enucleated oocyte. The OBCs were fused with a DC pulse of 0.6 KV/cm for 50 μsec, or 1 KV/㎝ for 30 μsec, or 2 KV/㎝ for 15 μsec. The fusion rate (82.9%), the cleavage rate (55.2%) and the developmental rate (10.3%) of the OBCs fused with a DC pulse of 1 KV/㎝ for 30 μsec were higher than the OBCs fused with a DC pulse of 0.6 KV/㎝ for 50 μsec or in 2 KV/㎝ for 15 μsec. The degeneration rate was significantly (P$lt; 0.01) lower with 1 KV/㎝ for 30 μsec (2.9%) than with 2 KV/㎝ for 15 μsec pulse (55.6%). When day 3, 4, and 5 embryos after in vitro fertilization were used as donor nuclei and in vitro matured oocytes that were enucleated and activated with a DC pulse of 0.6 KV/㎝ for 50 μsec and cultured for 6 h in CR 1 aa supplemented with 10 ㎍/㎖cycloheximide were used as recipient cytoplasms, the fusion rates by a DC pulse of 1 KV/㎝ for 30 μsec were 81.3, 84.9, and 85.7%, the cleavage rates were 46.2, 64.4, and 50%, and the developmental rates were 7.7, 8.9, and 6.7%, respectively. The fusion rates, cleavage rates, and developmental rate were not significantly (P$gt;0.05) different among the age groups of embryos. In this experiment, the results indicate that the enucleated oocytes activated with a DC pulse of 0.6 KV/㎝ for 50 μsec and cultured for 6 h in CRlaa supplemented with 10㎍/㎖ cycloheximide should be fused with day 5 donor blastomeres with a DC pulse of 1 KV/㎝ for 30 μsec for production of many cloned embryos.