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KMID : 0900920000240040365
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Korean journal of Animal Reproduction
2000 Volume.24 No. 4 p.365 ~ p.373
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Effect of Fusion Procedure on the Development of Embryos Produced by Somatic Cell Nuclear Transfer in Hanwoo (Korean Cattle)
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Im G.-S.
Yang B.-S.
Park S.-J.
Chang W.-K.
Park C.-S.
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Abstract
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The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco′s modified Eagle medium supplemented with 10% fetal bovine serum at 38.5^{circ}C in a humidified atmosphere of 5% CO_2in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 4mutextrm{s}, two pulses of 70 volts for 40mutextrm{s} and one pulse of 180 volts for 15mutextrm{s}. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15mutextrm{s} or 30mutextrm{s}. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 muM calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 {mu}ell well of modified CR1aa supplemented with 3 mg/mell BSA in th varepsilon four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/mell BSA and 5% FBS for 4 days. The incubation environment was 5% CO_2, 5% O_2, 90% N_2 at 38.5^{circ}C. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 mutextrm{s} yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30mutextrm{s} yielded significantly higher fusion rate compared with that for 15 mutextrm{s}(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.
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KEYWORD
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Somatic cell nuclear transfer, Fusion pulse, Enucleated oocytes
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