• 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.

• Journal of Animal Reproduction and Biotechnology
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• v.35 no.1
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• pp.86-93
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• 2020

The establishment of porcine embryonic stem cells (ESCs) from porcine somatic cell nuclear transfer (SCNT) blastocysts is influenced by in vitro culture day of porcine reconstructed embryo and feeder cell type. Therefore, the objective of the present study was to determine the optimal in vitro culture period for reconstructed porcine SCNT embryos and mouse embryonic fibroblast (MEF) feeder cell type for enhancing colony formation efficiency from the inner cell mass (ICM) of porcine SCNT blastocysts and their outgrowth. As the results, porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days showed significantly increased efficiency in the formation of colonies, compared to those for 7 days. Moreover, MEF feeder cells derived from outbred ICR mice showed numerically the highest efficiency of colony formation in blastocysts produced through in vitro culture of porcine SCNT embryos for 8 days and porcine ESCs with typical ESC morphology were maintained more successfully over Passage 2 on outbred ICR mice-derived MEF feeder cells than on MEF feeder cells derived from inbred C57BL/6 and hybrid B6CBAF1 mice. Overall, the harmonization of porcine SCNT blastocysts produced through in vitro culture of the reconstructed embryos for 8 days and MEF feeder cells derived from outbred ICR mice will greatly contribute to the successful establishment of ESCs derived from porcine SCNT blastocysts.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.78-78
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• 2002

The role of heat shock proteins in shielding organism from environmental stress is illustrated by the large-scale synthesis of these protein by the organism studied to date. However, recent evidence also suggests an important role for heat shock protein in fertilization and early development of mammalian embryos. Effects of elevated in vitro temperature on in vitro produced bovine embryos were analysed in order to determine its impact on the expression of heat shock protein 70 (HSP70) by control and frozen-thawed after in vitro fertilization (IVF) or nuclear transfer (NT). The objective of this study was to assess the developmental potential in vitro produced embryos with using of the various containers and examined expression and localization of heat shock protein 70 after it's frozen -thawed. For the vitrification, in vitro produced embryos at 2 cell, 8 cell and blastocysts stage after IVF and NT were exposed the ethylene glycol 5.5 M freezing solution (EG 5.5) for 30 sec, loaded on each containers such EM grid, straw and cryo-loop and then immediately plunged into liquid nitrogen. Thawed embryos were serially diluted in sucrose solution, each for 1 min, and cultured in CRI-aa medium. Survival rates of the vitrification production were assessed by re-expanded, hatched blastocysts. There were no differences in the survival rates of IVF using EM grid, cryo-loop. However, survival rates by straw were relatively lower than other containers. Only, nuclear transferred embryos survived by using cryo-loop. After IVF or NT, in vitro matured bovine embryos 2 cell, 8 cell and blastocysts subjected to control and thawed conditions were analysed by semiquantitive reverse transcription polymerase chain reaction methods for hsp 70 mRNA expression. Results revealed the expression of hsp 70 mRNA were higher thawed embryos than control embryos. Immunocytochemistry used to localization the hsp70 protein in embryos. Two, 8-cell embryos derived under control condition was evenly distributed in the cytoplasm but appeared as aggregates in some embryos exposed frozen-thawed. However, under control condition, blastocysts displayed aggregate signal while Hsp70 in frozen-thawed blastocysts appeared to be more uniform in distribution.

• Journal of Embryo Transfer
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• v.11 no.2
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• pp.137-144
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• 1996

The present study was carried out to obtain the pregnancy and delivery rate following transfer of fresh and frozen-thawed Korean native cattle(KNC) blastocysts(1~4 em-bryos / head) produced in vitro to Holstein recipients. The pregnancy rate of fresh and frozen-thawed KNC blastocysts produced in vitro was 50%(7 /14 heads) and 38.5%(5 /13 heads), respectively. The pregnancy rate of frozen-thawed KNC blastocysts produced in vitro frozen using 1.5M ethylene glycol and 1.4M glycerol for cryoprotectant was 33.3%(2 /6 heads) and 42.9 %(3 /7 heads), respectively. Seven calves including 2 sets of twin were born fiom 5 pregnant recipients receiving eleven fresh blastocysts. Three pregnant recipients were aborted among four pregnant recipients receiving twelve frozen-thawed blastocysts and one calf was born from the rest of one pregnant recipient.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.1
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• pp.15-21
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• 1999

The aim of the present study was to determine the effect of paternal sex chromosome on early development of buffalo embryos fertilized and cultured in vitro. Embryos were produced in vitro from abattoir derived buffalo oocytes. The cleaved embryos were cocultured with buffalo oviductal epithelial cells and evaluated on day 7 under the phase contrast microscope to classify development. The embryos which reached the morula/blastocyst stage were fast developing, the embryos which were at 16-32 cell stage were medium developing and the embryos below 16 cell stage were slow developing. The embryos which showed some fragmentation in the blastomeres or degenerated blastomeres, were degenerating. Sex of emberyos (n=159) was determined using PCR for amplification of a male specific BRY. 1 (301 bp) and a buffalo specific satellite DNA (216 bp) fragments. The results thus obtained show that 1) X and Y chromosome bearing sperms fertilize oocytes to give almost equal numbers of cleaved XX and XY embryos, 2) male embryos develop faster than female embryos to reach advanced stage and 3) degeneration of buffalo embryos is not linked with the paternal sex chromosome. We suggest that faster development of males is due to differential processing of X and Y chromosome within the zygote for its activation and / or differential expression of genes on paternal sex chromosome sex chromosome during development of buffalo embryos fertilized and cultured in vitro which may be attributed to a combination of genetic and environmental factors.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.229-234
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• 1994

This experiment was carried out to develop the model system for mass production of biomedical and nutritional proteins (human proteins) through mamraary gland of the transgenic cattle produced by gene manipulation and embryological technologies. Human growth hormone gene fused with rat $\beta$-casein gene promoter was microinjected into pronuclei of one cell bovine embryos produced by in vitro fertilization. After microinjection, embryos were cultured in vitro for 6 or 7 days. Twenty embryos reaching to blastocysts were transferred to 10 beef recipients, each receiving two embryos. Recipients were diagnosed for pregnancy by rectal palpation at 76 days after embryo transfer. One of them was pregnant to term and produced a female calf weighing 21 kg at 280 days following embryo transfer. DNA was extracted from umbilical cord tissue and blood of calf born for confirming gene insertion. As determined by Southern hybridization, the transgene was not found.

• Journal of Embryo Transfer
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• v.15 no.3
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• pp.255-261
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• 2000

The objective of this study was to improve the efficiency of bovine embryo transfer by transferring of Hanwoo embryos into Hanwoo or Holstein recipients. The cryopreserved or fresh in vitro produced(IVP) embryos were transferred into uterine horn contralaterally or ipsilaterally to the corpus luteum. The recipients were inseminated by artificially on the next day of estrus. The pregnancy was diagnosed by rectal palpation at 60∼90 days after transfer of the embryos. The pregnancy rate by transfer of one or two embryos was 78%(7/9) and 74%(31/42), respectively. The pregnancy rates according to the grade of corpus lutea of recipients was 75% (20/27) and 82.0%(18/22) at the grade of A and B, respectively. Ten(67.0%) of 15 Holstein recipients transferred with IVP Hanwoo embryos and 5(42.0%) of 12 Holstein recipients transferred with frozen IVP Hanwoo embryos were pregnant. The single and twin calving ratio in Hanwoos was 77.0%(10/13) and 23.0%(3.13) in the recipients transferred with IVP embryos and 64.0%(7/10) and 27.0%(3/10) in the recipients transferred with frozen IVP embryos, respectively. Twenty-four pregnant cows following transfer of IVP embryos, 21(88.0%) calved the normal calves, and 2(8.3%) aborted. When the frozen IVP embryos were transferred, 16 pregnant cows calved 14(88.0%) normal calves and 2(13.0%) aborted. In conclusion, when one or two IVP bovine embryos were transferred into recipients, the A and B grade of corpus luteum resulted in high pregnancy rates. For the production of twin calves, transfer of the IVP or frozen IVP embryos could be suitable.

• Korean Journal of Animal Reproduction
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• v.17 no.1
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• pp.69-74
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• 1993

This study was investigated to examine the effect of conditioned medium from bovine oviductal cell(BOEC) in the co-culture system with BOEC on in vitro development of in vitro produced bovine embryos. Oocyte-cumulus complexes were cultured for 24 hrs in TCM-199 supplemented with 10% fetal calf serum, 1$\mu\textrm{g}$/ml FSH and 21U hCG, 1$\mu\textrm{g}$/ml oestradiol-17$\beta$ at 39$^{\circ}C$ under 5% CO2 in air. In vitro fertilization was performed with epididymal sperm and heparin (10$\mu\textrm{g}$/ml, 15min.) or caffeine(2.5mM)-treated spermatozoa. Oocytes were incubated with 1$\times$106 spermatozoa/ml for 18 hrs and then cultured in various culture system for 7 days. The development rates of 16-cell or blastocyst stages were recorded on 4, 7 days, respectively, after incubating. The proportions ofembryonic development into molulae and blastocysts were higher in cumulus cell co-culture(23.4%) and BOEC co-culture(34.3%) than in M199-FCS(6.1%). Similarily, the development rates into molulae and blastocysts were significantly higher in BOEC-conditioned medium than those in M199-FCS. Therefore, it is suggested that BOEC co-culture and BOEC conditioned medium increase significantly the development of in vitro produced bovine embryos in in vitro system.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2006.10a
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• pp.40-41
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• 2006