• Asian-Australasian Journal of Animal Sciences
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• v.14 no.9
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.1-10
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• 2004

These studies were carried out to improve the reproductive efficiency through embryos transfer of Hanwoo IVM/IVF embryos. Following routine IVM/IVF procedure, oocytes and zygotes were cultured far 40 to 44 h in CRlaa medium with BSA. Then 2 to 8-cell embryos were removed the cumulus cell and were cultured in CRlaa medium containing 10% fatal bovine serum and 2.5 mM taurine in 5% $O_2$ and 5% $CO_2$ at 38.5$^{\circ}C$. The fresh embryos of the morulae and blastocysts cultured for 6 to 9 days in vitro or the frozen-thawed embryos were transferred into recipients. The pregnancy rates of the blastocyst produced for 6, 7, 8, and 9 days in vitro culture were 59.4, 68.2, 66.0 and 100%, respectively. In the developmental stage, pregnacy rates of early blastocysts (61.1%), blastocysts(64.7%) and expanded blastocysts(69.5%) were higher than that of morulae stage(20.0%). The pregnancy rates according to the corpus luteum grades of A, B and C in recipients were 73.6, 62.9 and 50.0%, respectively. Effects of donor-recipients synchrony of after day 2, 1 and 0, before day 1 and 2 on the pregnancy rates were 35.7, 65.5, 72.6, 67.9 and 60.0%, respectively. Pregnancy rates of the body condition score of recipients $\leq$2(71.3%) were higher than those of $\geq$3.0 score(40.0%). The pregnancy rates according to the parity of recipients when embryo was transferred to cow(70.6%) was higher than in heifer(59.1%). The pregnancy rates according to hormone treatment before embryo transfer were 69.9% in hCG + GnRH administration group and 63.0% in control group. Fresh and frozen-thawed embryos on the pregnancy rates were 70.6 and 36.4%, respectively. Pregnancy rates in single and AI+single was 90.0% and 64.8%. Pregnancy rates in twin induction was better than in single. These results indicate that pregnancy rates after transfer were affected on the embryo ages, donor-recipient synchrony, body condition score of recipients, corpus luteum status, parity and hormone treatment to recipients.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.15-20
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• 2010

This study was performed to identify the optimal timing for oocyte donor replacement during OPU procedure. OPU was carried out to collect oocytes from every donor at an interval of $3{\sim}4$ days (2 times a week). The collected oocytes were matured in vitro in TCM-199 supplemented with 10% FBS, 10 mg/ml of FSH and 1 mg/ml of estradiol for 24 h. After 24 h of exposure to sperm, the presumptive zygotes were cultured in CR1aa medium supplemented with 4 mg/ml of BSA for 3 days before being changed to CR1aa medium with 10% of FBS for another $3{\sim}4$ days. The mean numbers of retrieved oocytes were remained constantly up to 3 months ($6.0{\pm}0.5$, $6.2{\pm}0.7$, $5.2{\pm}0.6$), but significantly decreased at over 4 to 6 months ($3.7{\pm}0.5$, $2.8{\pm}0.4$, $1.2{\pm}0.2$) (p<0.05). The blastocyst development potential was also very similar rate from 1 to 3 months (37.2%, 40.4% and 44.6%), but significantly decreased from 4 to 6 months (24.8%, 29.3% and 28.6%, respectively) (p<0.05). The production of OPU derived embryos in periods of 1 to 3 months ($2.2{\pm}0.3$, $2.5{\pm}0.3$ and $2.3{\pm}0.4$) were significantly higher than those in 4 to 6 months ($0.9{\pm}0.2$, $0.8{\pm}0.2$ and $0.3{\pm}0.2$, respectively) (p<0.05). In conclusion, the efficient periods for the production of OPU derived embryos was until 4 months, twice per week to produce over 64 transferable embryos and then replace new donor after 3 months use. The best replacement time is 3 months and could be maximized production of OPU derived embryos.