소 수정란의 공배양 및 동결이 체외발생에 미치는 영향에 관한 연구 = Studies on the effects of co-culture and freezing of in vitro fertilized bovine embryos on in vitro developmental rates

다국어 초록 (Multilingual Abstract)

The studies on the carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular oocytes, and to determine the optimum thawing temperature and equilibration time of frozen bovine embryos and on survival rate and in vitro developmental rate of bovine embryos.
The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/㎖의 PMSG(Sigma, USA), 10 IU/㎖의 hCG, 1 ㎍/㎖의 β-estradiol(Sigma, USA) and 10% FCS(Sigma, USA) for 24-48 hrs in incubator with 5% CO_2 in air at 38.5℃ and then matured oocytes were again cultured for 12-18 hrs with motile capacitated sperm by preincubation of heparin. The bovine embryos following dehydration by cryoprotective agents and a various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 30℃ water.
The results are summarized as followes :
1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM-199 media were 75.0%-76.8% and 17.3%-27.6%, respectively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%) were significantly(p<0.05) higher than cumulus-denuded oocytes(23.1%).
2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with 1 ×10^4 cells/㎖, 1 ×10^6 cells/㎖, 1 ×10^8 cells/㎖ and 1 ×10^15 cells/㎖ oviduct epithelial cells in TCM-199 media were 74.5%-77.8% and 15.7%-21.2%, respectively.
3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured in TCM-199 media containing PMSG, hCG, PMSG + hCG, PMSG + β-estradiol, hCG + β-estradiol 0 to 40 hrs after insemination were 74.0%-77.4% and 18.9% - 23.1, respectively.
4. The survival rates of bovine embryos thawed after rapid freezing in the media containing various kinds of cryoprotective agents added 0.25M and 0.50M sucrose were 14.7% - 35.1% and 17.6% - 31.6%, respectively. The survival rates of bovine embryos thawed after rapid freezing in the freezing media containing a various concentration of sucrose added 1.5M and 2.0M glycerol, 1.5M and 2.0M DMSO, and 1.5M and 2.0M propanediol were 23.5% -31.4% and 20.6% - 34.1%, respectively.
5. The temperature thawed at 30℃ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 20℃ and 35℃.
6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5-5 min.) in the freezing medium higher than ling period of time(10-20min.).