JARB Journal of Animal Reproduction and Biotehnology

OPEN ACCESS pISSN: 2671-4639
eISSN: 2671-4663

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Reproductive & Developmental Biology 2004; 28(2): 127-132

Published online June 1, 2004

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

재래산양에 있어서 핵이식란의 융합조건이 융합 및 체외발달에 미치는 영향

박희성,김태숙,이윤희,정수영,이명열,홍승표,박준규,김충희,정장용

진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센터

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% CO₂, 5% O₂, 90% N₂. 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 ㎸/㎝. 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 ㎸/㎝ (82.9%) than 2.30∼2.39 ㎸/㎝ (43.8%) and 2.40∼2.46 ㎸/㎝. (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

Article

Reproductive & Developmental Biology 2004; 28(2): 127-132

Published online June 1, 2004

Copyright © The Korean Society of Animal Reproduction and Biotechnology.

재래산양에 있어서 핵이식란의 융합조건이 융합 및 체외발달에 미치는 영향

박희성,김태숙,이윤희,정수영,이명열,홍승표,박준규,김충희,정장용

진주산업대학교 동물생명과학과ㆍ동물생명산업지역협력연구센터

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% CO₂, 5% O₂, 90% N₂. 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 ㎸/㎝. 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 ㎸/㎝ (82.9%) than 2.30∼2.39 ㎸/㎝ (43.8%) and 2.40∼2.46 ㎸/㎝. (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