• Korean Journal of Animal Reproduction
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• v.12 no.3
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• pp.132-140
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• 1988

These experiments were carried out to examine the development capacity of mouse blastomers separated from 2 to 8-cell stage mouse embryos. The female ICR and C3H mice were subjected to supervolution by intraperitoneal injection of PMSG and HCG and then mated with males of the same strain. Embryos were flushed from oviducts and uteri on a proper time after injection of HCG. After removal of zona pellucida with 0.5% pronase, each embryos were separated into 1/2, 1/4, 2/4, 1/8, 2/8 and 4/8 embryos by pipetting or a fine glass needle in Ca2＋$.$Mg-2＋ free Hoppe& Pitts medium containing 0.02% EDTA. Splitted embryos were cultured in Hoppe & Pitts medium for 48h to 72h. The embryos developed to blastocyst were transferred to recipients on 2 or 3 days of pseudopregnancy. On the other hand, a monozygotic pairs of 1/2 embryos developed to blastocyst after 48h in vitro culture were transferred to recipients on 2 days of pseudopregnancy or pregnancy. The results obtained were summarized as follows. 1. Success rates of separation of blastomeres from 2-, 4- and 8-cell embryos were 91.7%, 68.5-92.4% and 60.8-90.6%, respectively. 2. Development rates of various type of blastomeres to blastocyst after 72h in vitro culture were ranged 64.7-87.1%. 3. Blastocysts obtained after 48h in vitro culture were transferred to recipients on 2 or 3 days of pseudopregnancy. The production rates of live fetuses after transfer on 2 days, only 1/2, 2/4 and 4/8 embryos, were 13.2%, 13.5% and 17.2%, respectively and those of embryos transferred on 3 days were 11.8%, 9.6% and 11.5%, respectively. However, the production rates of live fetuses 1/2 embryos following 72h in vitro culture and transfer to recipients on 2 or 3 days of pseudopregnancy were 7.7% and 12.5%, respectively. 4. From 29 and 31 pairs of 1/2 embryos transferred to recipients on 2 days of pseudopregnancy or pregnancy, 4 sets of monozygotic twins were produced from only pregnant recipients.

• Journal of Embryo Transfer
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• v.20 no.2
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• pp.113-121
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• 2005

This study was conducted to investigate the effects of different hexoses (glucose, mannose, galactose and fructose) on in vitro development of parthenogenetic embryos in the pigs. When the parthnogenetic embryos were cultured in medium with concentrations of 5mM glucose or 1mM galactose, the rates of embyos developed to morula and blastocyst stages were significantly higher than those in another culture conditions (P<0.05). However, high concentration of galactose inhibited development to morula and blastocyst stages. Addition of hexoses at early stage of porcine parthenogenetic embryos were effective for in vitro development. Especially, the embryos cultured in medium with glucose at early stage were effective for development to 2-cell $(72\%)$ and blastocyst $(19\%)$ stages compared with embryo cultured without glucose. From the present results, it is suggested that development of porcine parthenogenetic embryos can improve in medium with 5mM glucose. The concentration of 1mM galactose was also effective for development of porcine parthenogenetic embryos. It also show that parthenogenetic embryos cultured with glucose at early stage can improve in vitro development.

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

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.15-21
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• 1994

This experiment was investigated the effect of cell stage of embryos at 48 hours post-insemination On in vitro development of IVF embryos. The ovaries of Korean native cows or heifers were obtained from an abattoir and kept on 25 to 28$^{\circ}C$ and transported to laboratorty within 2 hrs. The oocytes were matured in vitro(IVM) for 24 hrs. in TGM-199 supplemented with 35 $\mu$g/$m\ell$ FSH, 10 $\mu$g/$m\ell$ LH, 1 $\mu$g/$m\ell$ estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by epididymal spermatozoa treated with heparin for 24 hrs. , and then the zygotes were co-cultured in vitro(IVC) with bovine oviductal epithelial cells for 7 to 9 days. At 48 hrs. post-insemination, the embryos were classfied into 5 to 8-cell, 3 to 4-cell or 2-cell stage and then were co-cultured in vitro(IVC) with bovine oviductal epithelial cells until the embyos reached blastocyst stage. Embryos developed to blastocyst stage were stained with Hoechst 33342 for cell counting. The embryos of 5 to 8-cell stage at 48 hrs. post-insemination with grade I oocytes were significantly (P<0.05) better developed to blastocysts(63.0%) than 3 to 4-cell(42.0%) and 2-cell stage(2.7%) embryos which delayed in the early cleavage, and those embryos cleaved faster in the very early stage seemed to develop to blastocysts earlier. These results indicate that the embryos cleaved faster at 48 hrs. post-insemination seemed to develop to blastocysts earlier.

• Journal of Embryo Transfer
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• v.11 no.3
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• pp.217-223
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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 endometrial cell monolayers(PEM) in the two different media, respectively. The rates of embryos developed to 2-, 4-, 8~16-cell and morula /blastocyst stage were 49.6, 40.5, 28.2 and 15.3% in Ham's F-10 with PEM, and 55.3, 45.9, 32.7, and 17.6% in TCM-HEPES with PEM, 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 TGM-HEPES without PEM(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 PEM were 0~1.2%. 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 PEM in the two different media enhanced the development of fertilized eggs to morula /blastocyst stages in vitro. However, we didn't find out any differences for the in vitro development to morula /blastocyst stages between Ham's F-10 and TcM-HEPES media.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.221-227
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• 1994

This study was conducted to examine the condition of in vitro culture system and the viability after embryo transfer of in vitro matured-in vitro fertilized (IVM-IVF) bovine embryos. The in vitro development to the blastocyst stage was enhanced by supplying bovine serum albumin(BSA) to co-culture medium with bovine oviduct epithelial tissue(BOET) compared with that in medium supplemented with fetal bovine serum(FBS) (41.2% vs. 26. 3%, P<0.05). After transfer of IVM-IVF blastocysts into the uterine horn of recipient females (Aberdeen Angus), one was pregnant to term and produced a head of male Korean native calf. These results confirm that the in vitro development of IVM-IVF bovine embryos is affected with different protein source in co-culture with BOET, and IVM-IVF embryos can develop to term after in vitro culture and embryo transfer.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.1
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• pp.33-38
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• 2004

This study was aimed at testing the gene expression of antioxidant enzymes and apoptosis genes for in vitro culture in porcine embryos produced by in vitro maturation/in vitro fertilization (IVM/IVF). Pocine preimplantation embryos obtainted from IVM/IVF can be successfully culture in vitro, but they are delayed or stop to develop at specific developmental stage. Many factors such as reactive oxygen species and apoptosis in an IVM/IVF system followed by in vitro culture influence the rate of production of viable blastocysts. Porcine embryos derived from IVM/IVF were cultured in the atmosphere of 5% $CO_2$ and 20% $O_2$ at $38.5^{\circ}C$ in NCSU23 medium. The patterns of gene expression for antioxidant enzymes and apoptosis genes during in vitro culture in pocine IVM/IVF embryos were examined by the modified semi-quantitative single cell reverse transcriptase-polymerase chain reaction (RT-PCR). Porcine embryos produced by in vitro procedures were expressed mRNAs for CuZn-SOD, GAPDH and GPX, whereas transcripts for Mn-SOD and catalase were not detected at any developmental stages. Expression of caspase-3 mRNA was detected at 2 cell, 8 cell 16 cell and blastocyst, but p53 mRNA was not detected at any stages. The fas transcripts was only detected in blastocyst stage. These results suggest that various antioxidant enzymes and apoptosis genes play crucial roles in vitro culture of porcine IVM/IVF embryos.

• 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

• Korean Journal of Animal Reproduction
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• v.21 no.2
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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.

• 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%).