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Effect of Fusion Condition on In Vitro Development of Caprine Cloned Oocytes with Nuclear Transfer  

박희성 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센터)
김태숙 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
이윤희 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
정수영 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
이명열 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
홍승표 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
박준규 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
김충희 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센)
정장용 (진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센터)
Publication Information
Reproductive and Developmental Biology / v.28, no.2, 2004 , pp. 127-132 More about this Journal
Abstract
This study was conducted to examine the effects of electric stimulation conditions on in vitro developmental ability of caprine embryos after somatic cell nuclear transfer. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean native goats. The caprine ear cells were cultured in vitro in serum-starvation condition (TCM-l99 + 0.5% FBS) for 3 to 5 days of cell confluence. The zona pellucida of in vivo and in vitro matured oocytes were partially drilled using laser system. Single somatic cell was individually transferred into the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol. After the electofusion, embryos were activated by electric stimulation or Ionomycin + 6-DMAP. Nuclear transfer embryos were cultured in mSOF medium supplemented with 0.8% BSA 6∼7 days at 39 , 5% $CO_2$, 5% $O_2$, 90% $N_2$. The fusion rate of donor cells was 60.4% and 40.3 % in ear cell and fetal fibroblast, and cleavage rate were 40.6% and 48.2%, respectively. No significant difference was found in the fusion and cleavage rate in different donor cells. Nuclear transferred oocytes were fused by electric pulses of 1.30∼1.40, 2.30∼2.39 and 2.40∼2.46 ㎸/cm. There was no significant difference among different electric pulses in fusion rates (26.7, 34.8 and 43.8%). The cleavage rate was higher (p<0.05) in 1.30∼1.40 ㎸/cm (82.9%) than 2.30∼2.39 ㎸/cm (43.8%) and 2.40∼2.46 ㎸/cm. (51.8%). The fusion rates of recipient oocyte source were 1st (43.5% and 23.6%), 2nd (55.7％ and 39.2%) and 3rd (66.1% and 52.8%) in in vivo and in vitro oocytes. However, fusion ratee were significantly higher (p<0.05) in in vivo than in vitro oocyte. The cleavage rate of fused oocytes from in vivo and in vitro sources were 52.6% and 54.4%, respectively. No significant difference was found in the cleavage rate according to the recipient oocyte source. These results suggest that factors such as field pulse of electric stimulation and oocyte source could affect in vitro developmental ability of nuclear transplanted caprine oocytes.
Keywords
Somatic cell; Nuclear transfer; Fusion; Activation; Cleavage; In vivo; Caprine;
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