• 한국가축번식학회지
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• 제15권2호
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• pp.125-132
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• 1991

The studies on the carried out to investigate the effects of capacitation method of spermatozoa on the in vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible of diameter 3~5mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and FCS for 24~48hrs in an incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18hrs with motile capacitated sperm by preincubation of mKRB, treatment of HIS(high strength ion), Ca-IA(Inophore A), BFF(bovine follicular fluids) and heparin. The results obtained in these experiments were summarized as follows : 1. The in vitro fertilizatin and cleavage rate offollicular oocytes fertilized with capacitated spermatozoas in BO solution by preincubation of mKRB, treatment of HIS, Ca-IA, BFF and heparin method were 53.1%, 33.9%, 50.8%, 48.1%, 58.8% and 28.1%, 17.7%, 26.2%, 22.8%, 32.8%, respectively. And the fertilization and cleavage rate of heparin method was of highest of all. 2. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solutin by both caffeine, BSA and heparin methods were 65.8%, 70.3% and 40.8%, 47.3%, respectively, and those rates were higher treatment of heparin＋BSA, heparin＋caffeine than treatment of heparin. 3. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoa in BO solution with heparin concentrations of 2, 5, 10, 20, 40$\mu\textrm{g}$/ml were 50.0%, 54.7%, 58.1%, 51.7% and 27.9%, 32.8%, 37.1%, 30.0%, respectively. And the fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution with 10$\mu\textrm{g}$/ml of heparin was the highest of all. 4. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with caffeine concentraton of 10, 20, 30, 40$\mu\textrm{g}$/ml were 71.4%, 74.3% and 70.6%, 70.0% and 45.7%, 47.3%, 44.1%, 41.4%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with caffeine and heparin together(70.3~74.3%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%). 5. The in vitro fertilization and cleavage rate of follicular oocytes fertilized with capacitated spermatozoas in BO solution containing heparin with BSA concentration of 5, 10, 20, 30$\mu\textrm{g}$/ml were 63.6%, 62.9%, 66.7%, 60.3% and 44.1%, 43.5%, 48.5%, 42.7%, respectively. The fertilization and cleavage rate of spermatozoa fertilized in BO solution with BSA and heparin together(60.3~66.73%) was higher than that of spermatozoa fertilized in BO solution with heparin(58.8%).

• 한국가축번식학회지
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• 제21권2호
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• pp.95-102
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• 1997

The studies on the carried out to investigate the effective concentration of cryoprotectant agents and sucrose by one-step straw method of bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1$\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ 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 various concentrations of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as devellpmental rate on in vitro culture or FDA-test. The results are smmarized as followes : 1. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was attained 2.0M glycerol, 2.0M DMSO, 1M or 2.0M propanediol. 2. The high in vitro developmental rates of bovine frozen embryos after rapidly thawed in freezing medium was obtained single cryoprotectant(6.7~17.4%) than mixed cryoprotectants(6.7~16.7%). 3. In vitro developmental rate of bovine embryos after rapid frozen-thawing in the freezing medium added 0.25M and 0.50M sucrose were higher cleavage rate than those of sucrose concentration of 0.75M and 1.00M. 4. The freezing methods on in vitro developemental rates of bovine embryos was attained slow freezing method(9.70~15.6%) higher than rapid freezing method(9.4~13.3%).

• 한국가축번식학회지
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• 제21권2호
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• pp.103-109
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• 1997

The studies on the carried out to investigate to determine the optimum thawing temperature and equilibration time and 1 step straw method of frozen bovine embryos. The follicular oocytes were cultured in TCM-199 medium containing 10 IU/ml PMSG(Sigma, USA), 10 IU/ml hCG(Sigma, USA), 1 $\mu\textrm{g}$/ml $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% CO2 in air at 38.5$^{\circ}C$ 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 various concentration of sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water. Survival and in vitro developmental rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as followes : 1. The equilibration time on in vitro developmental rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20 min.). 2. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher in vitro developmental rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$. 3. The thawing time on in vitro developmental rates of bovine embryos was attained after short period of time(1~5 min.) in the freezing mediuim higher than long period of time(10min.). 4. The in vitro developmental rates of bovine embryos after rapid frozen-thawing by 1 step straw method in the freezing medium added 1.5M, 2.0M glycerol, DMSO, propanediol and 0.25M, 0.50M, 0.75M, 1.00M sucrose were 12.5~19.4%, 10.0~15.6%, 9.1~13.8% and 6.7~12.9%, respectively.

• 한국가축번식학회지
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• 제17권2호
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• pp.133-139
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• 1993

The studies were carried out to investigate the effects of co-culture with cumulus cell, oviduct epithelial cells and uterine endometrial cells on the in-vitro fertilization and cleavage rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows : 1. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with cumulus cells in TCM-199 meidum were 64.6%~74.5% and 37.5%~55.3%, respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(51.5%) were significantly(p<0.05) higher than cumulus-denuded oocytes(21.7%). 2. The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 53.5% and 37.2%, 61.7% and 46.8%, 54.5% and 31.8%, 42.2% and 26.7%, respectively. 3. The in-vintro maturation and fertilization rate of porcine oocytes co-cultured with 1$\times$106/ml, 1$\times$108/ml, 1$\times$1015/ml uterine endometrial cells in TCM-199 medium were 54.3% and 39.1%, 58.3% and 43.8%, 55.5% and 33.3%, and 45.7% and 30.4%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with porcine cumulus cells, ovdiduct epithelial cells and uterine endometrial cells, the development rate to the blastocyst stage was 9.5%, 10.7% and 11.8%, respectively and the rates were higher than that of control, 2.1%(p<0.05).

• 한국수정란이식학회지
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• 제12권1호
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• pp.21-27
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• 1997

The studies were carried out to investigate the effects of bisection method and with and without-zona pellucida of embryos on in vitro developmental rate bisected embryos by micromanipulator, micropipette and pipetting. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TCM-199 mediurn containing 10 IU /ml의 PMSG(Sigma, USA), 10 IU /ml의 hCG, 1$\mu$g /ml의 $\beta$-estradiol(Sigma, USA) and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$ and then, matured oocytes were again cultured for 12~ 18 hrs with motile capacitated sperm by preincubation of heparin. Bisected embryos cultured for 1~5 days in 20% FCS + TCM-199 medium. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are summarized as follows :1. The survival rates of bisected bovine embryos by micromanipulator and micropipett were 29.2% and 19.1%, respectively. The rates of non-bisection embryos(46.7%) were significantly higher than those of bisection embryos. 2. The in vitro developmental rates of bisected bovine embryos by micromanipulator, micropipett and pipetting method were 32.4%, 19.4% and 25.6%, respectively.3. The in vitro developmental rates of with and without-zona pellucida of bisected bovine embryos by raicromanipulator were 30.8% and 25.0%, respectively. The rates of nonbisection embryos(53.1%) were significantly higher than those of bisection embryos.

• 한국가축번식학회지
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• 제17권2호
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• pp.141-148
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• 1993

This studies were carried out to investigate the effects of co-culture with cumulus cells, oviduct epithelial cells and uterine endometrium cells on the in-vitro fertilization and cleavage rate of bovine follicular oocytes. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% FCS for 24~48 hrs in a incubator with 5% CO2 in air at 38.5$^{\circ}C$ and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation. The results obtained in these experiments were summarized as follows: 1. The in vitro maturatin and fertilization rate of bovine oocytes co-cultured with cumulus cells in TCM-199 medium were 64.0~74.1% and 40.0~58.6% respectively. And in-vitro fertilization rate of cumulus-enclosed oocytes(55.4%) were significantly(p<0.05) higher than cumulus-denuded oocytes(23.1%). 2. The in-vitro maturatin and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml oviduct epithelial cells in TCM-199 medium were 59.3% and 40.7%, 64.0% and 48.0%, 58.3% and 37.5%, 52.0% and 32.0%, respectively. 3. The in-vitro maturation and fertilization rate of bovine oocytes co-cultured with 1$\times$104 cells/ml, 1$\times$106 cells/ml, 1$\times$108 cells/ml and 1$\times$1015 cells/ml uterine endometrium cells in TCM-199 medium were 56.0% and 36.0%, 60.7% and 42.9%, 59.3% and 37.0%, 52.0% and 36.0%, respectively. 4. When the in-vitro fertilized oocytes were co-cultured with cumulus cells, oviduct epithelial cells and uterine endometrium cells, the development rate to be blastocyst was 12.2%, 15.6% and 11.7%, respectively and rates were higher than that of control, 2.1%(P<0.05).