• Journal of Embryo Transfer
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• v.12 no.2
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• pp.151-159
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• 1997

In vitro fertilization(IVF) derived morula and blastocyst embryos were bisected by a simple method and cultured in vitro without zona pellucida And also bisected embryos were frozen-thawed and cultured in vitro) to evaluate the survival rate. The results obtained were as follows : The average number of grade I or II immature follicular oocytes recovered by slicing method per ovary was 11.9 from 142 ovaries. Following in vitro fertilization, the rates of cleavage and in vitro development to morula and blatocyst were 61.7 and 32.2% respectively. The successful bisection rate of IVE embryos was 67.51%, and the embryos of blastocyst stage were bisected successfully at significantly(P

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.3
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• pp.205-211
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• 2019

In this study, we investigated the possibility of using mouse embryonic stem cell conditioned medium (ESCM) and embryonic stem cell medium (ESM) for in vitro maturation in the efficient in vitro production of blastocysts from porcine follicular oocyte. Depending on the concentration of supplement of ESCM added to the NCSU-23 solution did not affect 2-cell development rates and blastocysts development. However, in particular, the survival rate (10 days of culture) of blastocyst was significantly higher than that of the control group as the additive concentration (30%) increased (p < 0.05). The survival rate of blastocysts showed a similar tendency even with addition of ESM (30%) alone. On the other hand, the duration of the addition of these additives during IVM (0-44 h) was that the IVM I period (0-22 h) were more effective than the IVM II period (22-44 h). Thus, the effect of these additives is probably due to the combination of the various physiologically active substances of ESCM or the appropriate amino acids and vitamins of ESM. In particular, these additives were more effective during the first half (IVM I) of in vitro maturation. In summary, optimization of ESCM or ESM supplementation may improve in vitro maturation of porcine oocyte and affect developmental competency. Therefore, if more efficient methods of adding ESCM or ESM to basal culture medium can be developed during in vitro maturation of porcine follicle oocytes, high quality blastocysts will be developed from low porcine follicular oocyte compared to other domestic animals.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2004.10a
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• pp.90-90
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• 2004

• Korean Journal of Animal Reproduction
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• v.15 no.2
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• pp.133-139
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• 1991

This stduy was carried out in order to investigate the effects of concentration and equilibration time of cryoprotective agents on survival rate of slow and ultrarapidly frozen in vitro fertilized bovine embryos. In vitro fertilized bovine embryos, following dehydration by cryoprotective agents and sucrose, were slowly freezed(from 2$0^{\circ}C$ to -7$^{\circ}C$/-1$^{\circ}C$/min., from -7$^{\circ}C$ -35$^{\circ}C$/-0.2$^{\circ}C$/min. from -35$^{\circ}C$ to -38$^{\circ}C$/-0.3$^{\circ}C$/min.) by cell freezer and directly plunged into liquid nitrogen and thawed in 38$^{\circ}C$ water. Survival rate was defined by development rate to the morula and blastocyst stage after in vitro cultured and FDA test. The results are summarized as follows : 1. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing medium of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 84.3%, 85.9%, 77.8%, 74.3%, respectively. 2. The survival rates of in vitro fertilized bovine embryos after slow frozen-thawing in the freezing of 0.50M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 83.8%, 85.1%, 71.4%, 74.6%, respectively. 3. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋3.0M propanediol were 69.3%, 70.8%, 63.2%, 67.1%, respectively. 4. The survival rates of in vitro fertilized bovine embryos after ultrarapid frozen-thawing in the freezing of 0.25M sucrose added 2.5M glycerol, 3.0M DMSO, 2.0M propanediol and 2.5M glycerol＋2.0M propanediol were 69.4%, 70.1%, 62.3%, 63.5%, respectively. 5. The survival rates of in vitro fertilized bovine embryos after slow and ultrarapid fromthawing in the freezing medium of sucrose added cryoprotective agents were not significant difference between 5min. and 10min. of equilibration time.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.1
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• pp.31-36
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• 1993

The effects of freezing and 1,2-propanediol on early and late pronucleate stage mouse ova were investigated in terms of survival after thawing and development in vitro. The samples were divided into two groups according to different age in pronucleate ova: ova in(1) early pronuclear stage with two distant pronuclei at 18h after hCG injection, and (2) late pronuclear stage with adjacent pronuclei at 30h. Zygotes in the late pronuclear stage have been proven to be more resistant to 1,2-propanediol, showing a significantly higher developmental rate than zygotes in early stage (80.3 versus 66.3%, <0.05), but survival rate was similar in the two groups (91.0 versus 93.5%). After freezing and thawing, survival and developmental rates were decreased in both groups when compared to the control group (54.3 versus 92.3%, 47.7 versus 73.3%. respectively). And developmental rate in the late pronuclear stage zygotes showed significantly higher than in early (55.4 versus 40.0%) after thawing. In conclusion, early pronucleate mouse ova have a lower developmental capacity in vitro and a lower survival rate after freezing and thawing than late ova. These findings suggest that the timing of freezing could be important for survival and further development in vitro in cryopreservation of human pronucleate ova.

• Journal of Embryo Transfer
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• v.10 no.2
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• pp.115-120
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• 1995

This study was carried out to investigate On in vitro fertilization, survival rate and developmental rate of rapidly frozen bovine immature oocytes. Immature oocytes cultured for 1, 12, 24, 48 hours in 20% FCS + TCM-199 medium and thereafter rapidly freezing-thawed oocytes inseminated with capacitated sperm. The immature oocytes following dehydration by 1.5M DMSO + 2.0M glycerol + 0.25M sucrose + TCM 199 media + 20% FGS were directly plunged into liquid nitrogen and thawes in 3$0^{\circ}C$ water. Rapid freezing embryos co-cultured in 20% FCS + TCM-199 media containing hormones(21U/mL PMSG, 21U /mL hGG and 1 $\mu$g /mL 17$\beta$-estradiol) and cumulus cells(1 x 105-6 cells). Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The in vitro maturation and fertilization rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing4hawed were 57.1%, 45.7%, 37.1%, 25.7% and 40.0%, 31.4%, 20.0%, 11.4%, respectively. 2. The survival rate of immature bovine oocytes on in vitro maturation period(1, 12, 24, 48 hrs) before rapid freezing-thawed were 33.3%, 26.7%, 20.0%, and 10.0%, respectively. The survival rate of rapid freezing4hawed immature oocytes was significantly lower than that of non-freezing oocytes. 3. The survival rate of rapid freezing4hawed excellent and good bovine embryos co-cultured in 20% FCS + TCM-199 media containing hormones(PMSG, hCG, 17$\beta$-estradiol) and cumulus cells 4 to 5 hrs and 20 to 24 hrs were 35.0%, 15.0% and 25.0%, 15.0% and 40.0%, 20.0% and 30.0%, 15.0%, respectively. The survival rate of embryos co-cultured in TCM-199 media containing hormones and cumulus cells was significantly higher than that of non co-culture.

• Korean Journal of Animal Reproduction
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• v.19 no.4
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• pp.259-264
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• 1996

This study was carried out to investigate on the survival rates and in vitro developmental rates of bisected bovine embryos were by manipulator and micropipette. Bisected embryos were co-cultured in 20% FCS(v/v)＋TCM-199 media containing hormones, oviductal epithelial cells and cumulus cells 0 to 72 hrs after bisection. Survival rate and in vitro fertilization rate were defined as development rate on in vitro culture or FDA-test. The results are summarized as follows ; 1. The in vitro developmental rate of biseciton embryos co-cultured in 20% FCS＋TCM-199 medium containing PMSG, hCG, PMSG＋hCG, hCG＋$\beta$-estradiol 0 to 20 hrs and 20 to 40 hrs were 36.7, 26.7, 33.3, 40.0, and 30.0, and 30.0, 33.3, 30.0, 26.7, and 26.7%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing hormones was significantly higher than that of non co-culture(25.0%). 2. The in vitro developmental rates of bisection embryos co-cultured in 20% FCS＋TCM-199 medium containing oviductal epithelial cells 4 to 5 hrs and 20 to 24 hrs were 40.0 and 33.3%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing oviductal epithelial cells was significantly higher than that of non co-culture(25.0%). 3. The in vitro developmental rates of bisection embryos co-cultured in 20% FCS＋TCM-199 medium containing cumulus cells 4 to 5 hrs and 20 to 24 hrs were 43.3 and 36.7%, respectively. The survival rate of bisection embryos co-cultured in TCM-199 medium containing cumulus cells was significantly higher than that of non co-culture (25.0%).

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.163-169
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• 1999

This study was carried out to investigate the effect of co-culture system(bovine oviduct epithelial cells; BOEC) and defined culture system(modified TALP ; mTALP) on the development of IVM-IVF embryos, and survival of in vitro produced blastocysts after freezing and thawing. Occytes from the slaugheterhous ovaries were matured and fertilized using general protocol. The results obtained were as the following: 1. Survival rates of frozen-thawed blastocysts using 10% glycerol as cryoprotectant was higher in day 7 blastocysts than in Day 8 and 9 blastocysts from co-cultrue system, but survival rate of frozen-thawed blastocysts was higher in Day 10 blastocysts than in day 8 and 9 blastocysts from defined culture system. Regardless of their age, survival rate of frozen-thawed blastocysts was significantly higher (p<0.05) in co-culture system than in defined culture system. 2. The cell number of blastocysts was significanlty higher (p<0.05) in Day 7 blasotcysts than in Day 8 and 9 blastocysts from co-cultures, but the cell number of blsstocysts was significantly higher (p<0.05) in Day 10 blastocysts than in Day 8 and 9 blastocysts from defined culture system. Regardless of the culture system, blastocysts with higher cell number showed higher survival rates after freezing and thawing.

• Korean Journal of Animal Reproduction
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• v.20 no.3
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• pp.233-238
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• 1996

This study was conducted to investigate the effects of cryoprotective agents and thawing temperature on the survival rate of the bisected embryos of mouse. The results of this study were summairzed as follows: 1. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed, the rates of normally developed bisected morula which was denuded were 31.7, 39.1, 28.0 and 23.1%, respectively. 2. The survival rates of bisected morula encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 72.4, 68.7, 64.0 and 59.5%, respectively. 3. The survival rates of bisected denuded blastura in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 48.3, 44.8, 32.1 and 28.6%, respectively. 4. The survival rates of bisected blasturae encased into the zona pellucida in in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed were 73.6, 67.4, 53.0 and 49.1%, respectively. 5. In in vitro culture after frozen with DMSO, glycerol, ethylene glycol or propanediol and thawed at room temperature, the rates of the normally developed bisected morula which was encased into the zona pellucida were 67.1, 62.3, 57.7 and 53.0%, respectively, and those of the bisected blasturae encased into the zona pellucida were 70.8, 65.4, 56.6 and 52.1%, respectively. 6. The survival rates of bisected morula which was encased into the zona pellucida in in vitro culture after forzen with DMSO, glycerol, ethylene glycerol or propanediol and thawed at 37$^{\circ}C$ were 74.3, 71.3, 63.9 and 57.4%, respectively, and those of bisected blastura encased into zona pellucida were 76.0, 69.1, 61.1 and 56.1%, respectively.

• Journal of Embryo Transfer
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• v.12 no.1
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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)