• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.7-13
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• 1994

To verify in vivo viability of IVF-derived bovine embryos, morula and blastocysts that developed from in vitro matured and fertilized ova were transferred to the uteri of recipient cows and normal calves were produced. To produce IVF-derived bovine morula or blastocysts, ova matured and fertilized in vitro were cultured in culture medium for 7~8 days at 39$^{\circ}C$ under the humicified atmosphere of 5% CO2. Two different culture systems, a co-culture system with TCM-199 and bovine epithelial cells (BOEC) and CR1aa without somatic cell support, were compared. Cleavage rates to 2~8 cell stage and developmental rates of IVF-derived bovine embryos to blastocyst stage were not different between co-culture system (51.3 and 14.0%) and CR1aa medium (60.4 and 22.1%), respectively. Embryos were classified into three grades by embryo quality and then one or two embryos in higher quality(A and B grades) were transferred to the uterus of recipients. In this study Korean Native calf was first born after transfer of IVF-derived embryos. Total four live calves were normally developed to term from IVF-derived bovine blastocysts and one female fetus was still-born approximatedly 8 months of gestation, but there was no pregnancy after transfer of morula. Therefore, normal calves could be produced after transfer of IVF-derived bovine embryos cultured in CR1aa medium without somatic cell support. In addition, our results suggest that in transfer of IVF-derived bovine embryos blastocyst stage is better than morula.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.161-169
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• 1995

To investigate the metabolism of various substrates in preimplantation bovine embryos, uptake of glucose and pyruvate, and lactate production were measured in single IVF-derived bovine embryos by a non-invasive method. When the embryos were incubated for 5 h in culture medium supplemented with 1 mM glucose and 0.4mM pyruvate as substrates at each developmental stage, glucose uptake was increased with more advanced developmental stages while pyruvate uptake was decreased. Total lactate producton of 2-cell embryos was significantly higher than that of blastocysts (p<0.05). Both of glucose uptake and lactate production in normal morulae produced in vitro was significantly high compared to the degenerated embryos(p<0.05). The results obtained in the study suggest that pyruvate as an exogenous substrate may be support in bovine embryos until 8-cell stage, whereas glucose may be effective as an energy source after morula stage. In addition, it was proven thatlactate was not effective as an energy source in preimplantation development of IVF-derived bovine embryos.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.7-14
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• 1994

The present experiments on cryopreservation were designed to examine the effects of solution toxicity, equilibration time and cell stages on the post-thaw survival of bovine IVF embryos. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g /ml FSH, 10 $\mu$g /ml LH, 1 $\mu$g /ml 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. The bovine IVF embryos were exposed to the EFS solution in one step at room temperature, kept in the EFS solution during different period for toxicity test, vitrified in liquid nitrogen, and thawed rapidly. 1. after the bovine blastocysts were exposed to EFS solution for 2 min. at room temperature and then they were washed in 0.5 M sucrose solution and TCM-199, they were cultured to examined cryoprotectant induced injury during exposure, Most of the embryos(95.0%) developed to reexpanded blastocoels. However, when the exposure time was extended to 5 and 10 min, these development rates dropped dramatically in 5 min. (69.5%) and 10 min. (47.4%), respectively, 2. When the bovine IVF embryos were vitrified in EFS solution after the equilibration for 1 and 2 min. exposure, The embryos to have reexpanded blastocoels following thawing, washing and culture processes were found to he 82.6 and 73.9%, respectively. However, when the exposure time was extended to 3 min, this survival rate dropped to 18.2%. The optimal time for equilibration of bovine IVF blastocysts in EFS solution seemed to he 1~2 min. 3. When the bovine IVF embryos were equilibrated for 1 min. the significantly (P<0. 05) higher post-thaw survival rates were obtained from the embryos of blastocyst stage(81.3%) than morulae stage(5. 1%). The optimal cell stage for viterification with EFS solution proven to he blastocyst stage in bovine IVF embryos. 4. The number of blastomeres of blastocyst stage was examined with nuclear staining with Hoechst 33342 during 7 to 9 days post-insemination. The cell counts of frozen bovine IVF embryos were found significantly(P$\geq$7.5 and those of the fresh embryos 76.6$\geq$7. 1, which were cultured in the sarne period and conditions as frozen embryos.

• Journal of Embryo Transfer
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• v.13 no.1
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• pp.37-41
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• 1998

This study was conducted to investigate the survival and hatching rates after refrozen-thawed bovine IVF blastocysts. The survival rates after refrozen-thawed bovine IVF blastocysts produced on day 7, day 8 and day 9, were 66.6%(16/24), 62.5%(15/24) and 65.3%(17/26), respectively. The survival and hatching rates after the first frozen-thawed bovine JVF blastocysts were 90.0%(27 /30) and 70.0%(21 /30), but in refrozen-thawed bovine IVF blastocysts were 66.2%(49 /74) and 45.9%(34 /74), respectively. The results of this study were suggest that refrozen-thawed bovine IVF embryos had survival ability.

• 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.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.299-309
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• 2000

This study was performed 1) to establish the optimal PCR condition of sex determination in Hanwoo IVM/IVF embryos, 2) to examine the sex determination and sex ratio to the developmental stages of Hanwoo IVM/IVF embryos by two-step PCR method. The sexing of bovine IVF embryos were accurately determined by PCR methods using Y chromosome specific DNA primer(BOV 97M, 141bp) and bovine specific DNA primer(216bp). The fregment size were shown at 141 and 216 base pairs(bp) in male, and 216 bp in female. Two-steps PCR method in which the samples were amplified by 15 cycles with Y chromosome specific DNA primer and then amplified by additional 30 cycles with bovine specific DNA primer was effective in the sexing of bovine IVF embryos. The zona-free embryos were more effective than zona-intact embryos in bovine IVF embryo sexing. The appearance of Y chromosome specific band was 45.2% in embryos treated with protease K and 53.3% in embryos treated with freezing and thawing repeatedly. The optimun volume of DNA for sexing of Hanwoo IVF embryos were 2 to 10 $\mu$1 in Zona-free embryos and 12 to 13 $\mu$1 in zona-intact embryos. The sexing rate of bovine IVF embryos by PCR was 96.0% and questionable rate not identified sex was 4.0%, respectively. Among the sexed embryos, the percentage of male and female was 49.7% and 46.3%, respectively, the sex ratio was 1: 1.1. The successful rate of embryo sexing was increased to the developmental stages.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.443-451
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• 1997

These studies were conducted to determine the sex of preimplantation Hanwoo embryos produced in vitro using polymerase chain reaction(PCR). Y chromosome specific and bovine speicific DNA primers were synthesized and tested for embryo sexing. Bovine IVF embryos were produced in TCM 199 and CR1aa medium, and classified by developmental stages on Day 7 to 9. The effects of developmental rates to bovine IVF blastocysts on sex ratio were also investigated using PCR methods. The results obtained in this study were as follows; 1. Developmental rates to blastocyst from IVM/IVF embryos in TCM 199 and CR1aa medium for 9 days were 23.5 and 30.2%, respectively, and there was significant difference between the media(P<0.05). 2. Male to female ratio of early, mid, expanded and hatching balstocyst produced on Day 7 were 0.7:1, 1.4:1, 2.2:1, and 2.5:1, respectively, and male embryos was significantly higher proportion in expanding and hatching blastocysts(P<0.01). 3. On Day 8, male to female ratio of early, mid, expanded and hatching blastocysts were 0.6:1, 1:1, 2.5:1, and 2.7:1, respectively. Both expanded and hatching blastocysts obtained a significantly higher proportion of males(P<0.01). 4. The male : female ratio of early, mid, expanded and hatching blastocyst produced on Day 9 was 0.6:1, 0.8:1, 1:1, and 2.2:1, respectively. Hatching blastocysts had a significantly higher ratio of males(P<0.01). The developmental rate of IVM/IVF embryos to blastocyst for 9 day culture was higher in CR1aa than that in TCM 199 medium. For the sex ratio by developmental stages of IVF embryos, male ratio was higher in expanded blastocyst but female in early blastocysts.

• Reproductive and Developmental Biology
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• v.32 no.3
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• pp.167-173
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• 2008

Somatic cells such as oviduct epithelial cell, uterine epithelial cell, cumulus-granulosa cell and buffalo rat river cell has been used to establish an effective culture system for bovine embryos produced in in vitro. But nitric oxide (NO) metabolites secreted from somatic cells were largely arrested the development of bovine in vitro matured/ in vitro fertilized (IVM/IVF) embryos, suggesting that NO was induced the embryonic toxic substance into culture medium. The objective of this study was to investigate whether BOEC co-culture system can ameliorate the NO-mediated oxidative stress in the culture of bovine IVM/IVF embryos. Therefore, we evaluated the developmental rate of bovine IVM/IVF embryos under BOEC co-culture system in the presence or absence of sodium nitroprusside (SNP), as a NO donor, and also detected the expression of growth factor (TGF-$\beta$, EGF and IGFBP) and apoptosis (Caspase-3, Bax and Bcl-2) genes. The supplement of SNP over 5 uM was strongly inhibited blastocyst development of bovine IVM/IVF embryos than in control and 1 uM SNP group (Table 2). The developmental rates beyond morulae stages of bovine IVM/IVF embryos co-cultured with BOEC regardless of SNP supplement (40.4% in 5 uM SNP+ BOEC group and 65.1% in BOEC group) were significantly increased than those of control (35.0%) and SNP single treatment group (23.3%, p<0.05: Table 3). The transcripts of Bax and Caspase-3 genes were detected in all experiment groups (1:Isolated fresh cell (IFC), 2:Primary culture cell (PCC), 3:PCC after using the embryo culture, 4: PCC containing 5 uM SNP and 5: PCC containing 5 uM SNP after using the embryo culture), but Bcl-2 gene was not detected in IFC and PCC (Fig. 1). In the expression of growth factor genes, TGF-$\beta$ gene was found in all experimental groups, and EGF and IGFBP genes were not found in IFC and PCC (Fig. 2). These results indicate that BOEC co-culture system can increase the development beyond morula stages of bovine IVM/IVF embryos, possibly suggesting the alleviation of embryonic toxic substance like nitric oxide.

• Korean Journal of Animal Reproduction
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• v.17 no.3
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• pp.243-248
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• 1993

Bovine embryos at 2-to 8-cell produced by in vitro matured/in vitro fertilized(IVM/IVF) were cultured in TCM 199 or Synthetic oviduct fluid(SOF) with 10% fetal bovine serum(FBS) or cocultured with cumulus or bovine oviduct epithelial cell(BOEC) in TCM-199 or SOF medium. In experiment 1, the proportions of embryos developed to morula and blastocysts stages in TCM 199 medium were higher when they were co-cultured with cumulus cell(29%) or BOEC(33%) than that of TCM 199 with 10% FBS(12%, P<0.01). In experiment 2, embryos deived from IVM/IVF were cultured in SOF with 10% FBS or cocultured with cumulus cell or BOEC in same medium. The higher development rates of IVM/IVF embryos developed beyond morula stages were obtained in cumulus cell co-culture group(39%) than those of BOEC group(26%) and SOF with 10% FBS group(17%, P<0.01). The present results indicated that the early development of IVM/IVF embryos can be maintained efficienty in SOF with cumulus cell co-culture.

• Korean Journal of Animal Reproduction
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• v.19 no.1
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• pp.49-54
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• 1995

This experiments were carried out to investigate the viabilities and the pregnancy rate of frozen-thawed IVF bovine embryos in various media, cryoprotectants and age of embryos produced. Hanwoo oocyte were collected in size of 2~7mm follicles, matured for 20~22hrs at 38.5$^{\circ}C$ in 5% CO2 incubator and then in vitro fertilized with Hanwoo semen. Blastocysts or more developed embryos at Day 7, 8 and 9 were frozen in 1.5 or 1.8M ethylene glycol. Viability of frozen thawed IVF embryos were identified the reformation of blastocoele after thawing and culture for 24~48 hours at 38.5$^{\circ}C$ in 5% CO2 incubator. Production rate of Hanwoo IVF embryos in TCM 199 and CR1aa ws 21.3%(39/183) and 28.1%(41/146), respectively. The viability of frozen thawed IVF embryos was higher rate in 1.8M ethylene glycol and Day 7 embryos than that in 1.5M and Day 8.53 cows out of 100 Hanwoo receipients transfered IVF embryos were pregnant and twin production rate was 26.3%.