• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.127-132
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• 2004

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.

• Korean Journal of Animal Reproduction
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• v.23 no.2
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• pp.149-157
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• 1999

The functional role of cumulus cells on the penetration and polyspermy during in vitro fertilization in porcine was examined. The penetration rate was significantly higher (P＜0.01) in oocytes with (61%) than without (25%) cumulus cells, but significant differences in polysper-my rates were not observed. When hyaluronidase was added to the fertilization medium with different concentrations, penetration rates in oocytes with cumulus cells were higher than in oocytes without cumulus cells at 0 (61% vs 34% ; P＜0.05), 0.01 (56% vs 35% ; P＜0.05), 0.1 (66% vs 30% ; P＜0.05) and 1.0mg/$m\ell$ (39% vs 27%). The polyspermy rates were lower in oocytes without than with cumulus cells, and had a tendency to decrease with high concentrations of hyaluronidase. In another experiment, the penetration and polyspermy rates had a tendency to increase as time of sperm-oocyte culture was prolonged. At 16 and 20 h after insemination, the penetration rates were significantly higher (P＜0.05) in oocytes with (48 and 62% for 16 and 20 h) than without (25 and 31% for 16 and 20 h) cumulus cells in medium containing hyaluronidase. Polyspermy rates were significantly (P＜0.05) lower in oocytes without (13% and 16%) than with (37% and 48%) cumulus cells at 16 and 20 h after insemination. In cumulus-free oocytes inseminated in medium containing different concentrations of cumulus cells, the penetration rates were significantly (P＜0.05) higher in medium with than without hyaluronidase. The proportion of polyspermy was lower in medium without than with hyaluronidase at 0 (10% vs 0%), 10$^2$(25% vs 0%), 10$^4$(24% vs 14%) and 10$^{6}$ (29% vs 10% ; P＜0.05) cumulus cells/$m\ell$. These results suggest that cumulus cells can have a positive influence on sperm penetration, its action on polyspermy control does appear to function primarily on zona pellucida by co-culture of cumulus cells and oocytes in medium without hyaluronidase.