• 한국가축번식학회지
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• 제21권3호
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• pp.281-285
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• 1997

The study was conducted to investigate on in vitro developmental rates of bisected bovine embryos co-culture in 10% FCS＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 7 days after bisection. In vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. In vitro developmental rates of bisected bovine embryos co-cultured in 10% FCS＋TCM-199 media containing PMSG＋hCG, PMSG＋$\beta$-estradiol, hCG＋$\beta$-estradiol, PMSG, hCG 0 to 3 days and 4 to 7 days were 16.7~30.0% and 11.1~25.0%, respectively. In vitro developmental rates of bisected embryos co-cultured in 10% FCS＋TCM-199 media containing hormones significantly higher than that of non co-culture. 2. In vitro developmental rates of bisected bovine embryos co-cultured 10% FCS＋TCM-199 media containing oviductal epithelial cells 0 to 3 days and 4 to 7 days were 25.0% and 22.2%, respectively.

• 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%).

• 한국수정란이식학회지
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• 제12권3호
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• pp.283-291
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• 1997

In vitro development of bovine embryos is affected by many factors such as energy substrates, amino acids, and some growth factors. It has been reported that mRNA of insulin, PDGF and their receptors are detected in cow embryos, and that some chelating agents such as EDTA and transferrin have beneficial role on mouse and bovine embryos. The author hypothesized that insulin, transferrin arid PDGF added to a culture medium increase in vitro development of bovine embryos by chelating toxic substance(s) or increasing cell growth and metabolism. Immature oocytes from slaughtered ovaries of Holstein cows and heifers were matured for 24 hours in a TCM199 containing 10% fetal calf serum, FSH, LH and estradiol with granulosa cells in vitro. Matured oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Embryos cleaved to 2- to 4-cell at 30 hours after IVF were selected and cultured in a 30-$\mu$l drop of a synthetic oviduct fluid medium (SOFM) containing 0.8% BSA, Minimum Essential Medium essential and non-essential amino acids, and insulin, transferrin or PDGF for 9 days. Supplementation of a SOFM with insulin, and /or transferrin did not increase develop-mental rate to expanding and hatching blastocyst of 2- to 4-cell bovine embryos compared with control. The highest developmental rate to hatching blastocyst was shown when PDGF was added at the concentration of 10 ng /ml among the supplementing doses tested in the present study (p<0.05). Addition of PDGF without insulin to a SOFM could not increase embrye development, but combined addition of PDGF with insulin significantly increased (p<0.05) embryo development to hatching blastocyst (50%) compared with control (38%). In conclusion, insulin and PDGF supplemented to a SOFM may act synergistically and have beneficial effect on in vitro development of 2- to 4-cell bovine embryos matured and fertilized in vitro.

• 한국가축번식학회지
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• 제19권1호
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• pp.9-14
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• 1995

This study was conducted to investigate the effect of energy source on development of in vitro development of in vitro matured and fertilized porcine 2-cell embryos. The relative preferences of glucose, lactate and pyruvate for in vitro development of porcine 2-cell embryos were determined. The results obtained are as follows. 1. 33.3, 20.8 and 29.2% of porcine embryos reached morula stage in addition to lactate, glucose, and both glucose and lactate in the culture medium as energy source, respectively. 2. 38.5, 15.4 and 26.9% of porcine embryos reached morula stage in addition to pyruvate, glucose, and both glucose and pyruvate in culture medium as energy source, respectively. 3. 42.9, 21.4 and 28.6% of porcine embryos reached morula stage in addition to pyruvate and lactate, glucse alone, and glucose, lactate and pyruvate in culture medium as energy source, respectively.

• 한국가축번식학회지
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• 제18권1호
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• 한국가축번식학회지
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• 제20권3호
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• pp.299-305
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• 1996

This study was conducted to investigate the effects of co-culture for the development rate to morula/blastocyst stages of early porcine embryos, derived from oocytes matured and fertilized in vitro, with porcine oviductal epithelial cell monolayers(POEC) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8∼16-cell and morula/blastocyst stage were 57.2, 48.2, 37.2 and 19.3% in Ham's F-10 with POEC, and 51.4, 41.2, 31.1, and 15.5% in TCM-HEPES with POEC, respectively. The above development rates to morula/blastocyst stages were significantly higher than those of the embryos cultured in the Ham's F-10 and TCM-HEPES with out POEC(P<0.05). The in vitro development rates to the morula/blastocyst stage of 1-cell embryos cultured in Ham's F-10 and TCM-HEPES without POEC were 1.1∼1.2%. Especially, most of embryos were observed to arrest the development beyond 4-cell stages. As shown in the above results, the co-culture of in vitro produced porcine embryos with POEC in the two different media enhanced the development of fertilized eggs to morula/blastocyst stages in vitro. However, we didn't find out any difference for the in vitro development to morula/blastocyst stages between Ham's F-10 and TCM-HEPES media.

• 한국수정란이식학회지
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• 제4권1호
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• pp.28-34
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• 1989

These experiments were carried out to determine the effect of cell stage in embryo bisection on the sub-Sequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgicaily bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using a microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stage. The successful rate of mouse embryo bisection at 4-cell stage (59.0%) was significantly (p <0.05) lower than those at 8-cell (75.6%), 2ce11 (80.7%) or morula stage (84.8%), and highest at blastocyst stage (95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst,the in vitro de'velopment of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-em-bryos bisected at 2- to 8-cell stage was signiflcantiy (p <0.05) lower.The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage,respectively and not significantly (P <0.05)different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2- or 8-cell stage were significantly (P <0.05) lower than the result from the intact embryos of the corresponding stage.

• 한국가축번식학회지
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• 제22권1호
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• pp.35-42
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• 1998

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19\ulcorner20hr after superovulation induction and incubated at 39\ulcorner in 5% CO2 for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA a, pp.ared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of medium. BSA and taurine together in RDH medium showed the additive effects on embryos development and cell number.

• 한국수정란이식학회지
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• 제12권1호
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• pp.27-36
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• 1997

The studies were 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 cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 $\beta$-estradiol and 10% FCS for 24~48 hrs in incubator with 5% $CO_2$ in air at 38.5$^{\circ}C$. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3$0^{\circ}C$ water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. 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 l$\times$ l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+$\beta$-estradiol, hCG+$\beta$-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3$0^{\circ}C$ after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2$0^{\circ}C$ and 35$^{\circ}C$.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 long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

• 한국가축번식학회지
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• 제15권2호
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• pp.141-147
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• 1991

This stduy was carried out in order to investigate the effects of cryoprotective concentration and equilibration time on survival rate of ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucorese were directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blaqstocyst stage after in vitro culture of by FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucroese added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M glycerol were 75.0%, 72.0%, 67.6%, 44.8% and 18.3% respectively. 2. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M DMSO were 64.0%, 66.7%, 70.8%, 52.7% and 18.6, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 0.25M sucrose added 2.0M, 2.5M, 3.0M, 3.5M, 4.0M propanediol were 68.4%, 64.9%, 63.2%, 62.2% and 34.7%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing medium of 2.50M glycerol added 0.1M, 0.25M, 0.5M, 0.75M, sucrose were 60.5%, 72.2%, 70.1% and 54.9%, respectively. The survival rate of in vitro fertilized embryos after ultrarapid frozen-thawing in the freezing medium of 2.5M glycerol added 0.25M sucrose were higher than concentration of 0.10M, 0.50M and 0.75M sucrose. 5. The equilibration time on the survival rate of in vitro fertilized bovine embryos was attained after short period of time(2.5~5min.) in the freezing medium added 0.25M sucrose and 3.0M DMSO higher than long period time(1~20min.).