• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.71-76
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• 1996

This experiment was carried out to investigate the developmental ability of bovine embryos matured and fertilized in vitro to the gastrulation stage. The bovine oocytes were collected from 2∼5mm follicles, matured for 20∼24hrs in 5% CO2 incubator and then fertilized with frozen-thawed semen. On day 9 after IVF and after freezing and thawing the hatching abilities of expanding blastocysts were examined. Cleavage rate and production rate to expanding blastocysts were 59.7%(955/1604) and 20.7%(333/1604), respectively. Hatching rate of day-9 expanding blastocysts was 54%(40/74), that after freezing and thawing was 56%(79/141). Also, developmental ability of hatched blastocysts to the primitive streak stage was 26%(6/23).

• 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.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.269-276
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• 1997

The objective of this study was to investigate the effects of thiol compounds with bovine oviduct epithlial crlls(BOEC) co culture on development and intracellular glutathione(GSH) concentrations of bovine embryos derived from IVM /IVF oocytes. In experiment 1 and 2, embryos developed to 2~8 cell stage after in vitro fertilization were co-cultured with BOEC in CR$_1$aa with or without $\beta$-mercaptoethanol($\beta$-ME) and cysteamine. The percentage of embryos that developed to morulae and blastocysts in 0,10, 25 and 5O$\pi$M $\beta$-ME with BOEC was 48.1, 64.0, 72.9 and 75.9%, respectively. Twenty-five and 5O$\pi$M $\beta$-ME groups were significantly higher than in 0 and 1O$\pi$M $\beta$- -ME groups(P$\pi$M cysteamine with BOEC was 50.0, 53.2, 72.0 and 66.7%, respectively. Fifty $\pi$M cysteamine group was significantly higher than any other groups (P$_4$aa with 0 and 5O$\pi$M $\beta$-ME or cysteamine were 68.5, 77.8, 78.7 and 80.0pM, respectively. Fifty $\pi$M $\beta$-ME group was significantly higher than that of control(P<0.05), but cysteamine group was not. Cell numbers of blastocysts were not difference in all experimental groups. These experiments indicate that $\beta$-ME and cysteamine with BOEC co-culture can affect the development and intracellular GSH concentrations of bovine embryos produced by IVM /IVF docytes.

• Journal of Animal Reproduction and Biotechnology
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• v.38 no.3
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• pp.143-150
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• 2023

Background: The successful production of superior or transgenic offspring from in vitro produced embryos in cattle relies heavily on the quality of blastocyst stage embryos. In order to enhance the developmental competency of these embryos, a novel culture method was devised. Methods: This study utilized stem cell culture medium (SCM) from hESCs as a supplement within the culture medium for bovine in vitro produced embryos. To gauge the efficacy of this approach, in vitro fertilized embryos were subjected to culture in CR1aa medium enriched with one of three supplements: 0.3% BSA, 10% FBS, or 10% SCM. Results: The blastocyst development and hatching rates of one-cell zygotes cultured in CR1aa medium supplemented with SCM (23.9% and 10.2%) surpassed those cultured in CR1aa medium supplemented with BSA (9.3% and 0.0%) or FBS (3.1% and 0.0%) (p < 0.05). Furthermore, post-zygotic gene activation, cleaved embryos cultured in CR1aa medium supplemented with SCM (57.8% and 34.5%) exhibited notably higher rates (p < 0.05) compared to those cultured with BSA (12.9% and 0.0%) or FBS (45.7% and 22.5%) supplementation. Furthermore, the microinjection of SCM into the cytoplasm or pronucleus of fertilized zygotes resulted in elevated blastocyst development and hatching rates, particularly when the microinjected embryos were subsequently cultured in CR1aa medium supplemented with SCM from the 8-cell embryo stage onwards (p < 0.05), in contrast to those cultured with FBS supplementation. Conclusions: In conclusion, this study conclusively demonstrated that the incorporation of SCM into the culture medium significantly enhances the developmental progress of preimplantation embryos.

• Reproductive and Developmental Biology
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• v.36 no.1
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• pp.49-53
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• 2012

The present study was conducted to examine the generation of reactive oxygen species (ROS) during micromanipulation procedures in bovine somatic cell nuclear transfer (SCNT) embryos. Bovine enucleated oocytes were electrofused with donor cells, activated by a combination of Ca-ionophore and 6-dimethylaminopurine culture. Oocytes and embryos were stained in dichlorodihydrofluorescein diacetate or 3'-(p-hydroxyphenyl) fluorescein dye and the $H_2O_2$ or $^.OH$ radical levels were measured. $In$ $vitro$ fertilization (IVF) was performed for controls. The samples were examined with a fluorescent microscope, and fluorescence intensity was analyzed in each oocyte and embryo. The $H_2O_2$ and $^.OH$ radical levels of reconstituted oocytes were increased during manipulation (37.2~49.7 and 51.0~55.2 pixels, respectively) as compared to those of mature oocytes ($p$<0.05). During early $In$ $vitro$ culture, the ROS levels of SCNT embryos were significantly higher than those of IVF embryos ($p$<0.05). These results suggest that the cellular stress during micromanipulation procedures can generate the ROS in bovine SCNT embryos.

• Journal of Embryo Transfer
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• v.1 no.1
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• pp.16-27
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• 1986

Based on the current importing and exporing regulations for disease control of embryo transfer, some important microorganisms and their control possibilities are reviewed. The results reviewed were sumrnarized as follows: 1. Regulations regarding to the import of embryos vary between importing and exporting countries, but exporting countries examine the donor and embryos for the heaith certification by the requirements of importing countries. 2. Organisms that infect the gametes are 5 kinds of viruses and the diseases caused by them could not be controlled or eradicated using embryo transfer. 3. Organisms that do not infect the gametes are 4 kinds of viruses and the causal organisms are potential candidates for control or eradication by embryo transfer. 4. Organisms that penetrate the zona pellucida and infect the embryo are 6 kinds of viruses including bovine viral diarrhea virus. 5. Organisms that cannot penetrate the zona pellucida or do not infect the embryo are 15 kinds of viruses and the removal from their contaminations are recommended by proper washing procedure and antisera treatment. Bovine and porcine parvovirus, porcine pseudorabies virus and vesicular stomatitis virus are included in these organisms. 6. Bovine embryos that artificially exposed to various pathogenic organisms such as bovine herpes virus, IBR virus, bluetongue virus, bovine viral diarrhea virus and Brucella abortus in vitro are discussed about their infection by several treatments.

• Korean Journal of Animal Reproduction
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• v.21 no.3
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• pp.287-292
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• 1997

This study was carried out to investigate the effects of concentration and kinds of cryoprotectants, equilibraction time, thawing temperature and time, sucrose concentration on the survival rates of frozen bovine demi-embryos. The bovine demi-embryos following dehydration by cryoprotectants a various concentration of sucrose were freezed by cell freezer and thawed in 3$0^{\circ}C$ water bath. Survival and in vitro developmental rates was defined as development rates on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rates of demi-embryos after frozen-thawing in freezing medium was attained 2.0M glycerol. The high survival rates of demi-embryos after frozen-thawing in freezing medium was obtained using single cryoprotectant(25.0~30.0%) than mixed cryoprotectants(16.7~19.0%). 2. The survival rates of demi-embryos after frozen-thawing in freezing medium added 1.5M, 2.0M glycerol＋0.25M sucrose(37.5~33.3%) were higher survival rates than those of sucrose concentration of 0.50, 0.75M(12.5~26.7%). 3. The equilibration time on the survival rates of demi-embryos was attained after short period of time(30.0~35.0%) in the freezing medium higher than long period of time(21.1%). 4. The thawing temperature on the survival rates of demi-embryos was attained at 3$0^{\circ}C$ of thawing temperature(26.7~40.0%) higher than $25^{\circ}C$ or 37$^{\circ}C$ of thawing temperature(13.3~20.0%). 5. The thawing time on the survival rates of demi-embryos was attained at 1~5 minutes of thawing time(26.7~33.3%) in the freezing medium higher than 10 minutes of thawing time(13.3~18.8%).

• 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.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.101-104
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• 1998

This study examined the effects of growth factors in TCM199 on bovine 1-cell embryos development in vitro. After 6 day to 11 day in culture, 15.8%(19/120), 15.3%(20/130), 21.8%(35/160), 27.0%(56/207), 26.3%(53/201) and 30.7%(40/130) of the 1-cell embryos developed into expanding blastocysts in su, pp.ementing TCM199 with control, insulin, IGF-I, IGF-II, FGF and EGF, respectively. Hatching rate of 1-cell embryos in su, pp.ementing TCM199 with FGF, EGF and IGF-II were 21.4%(53/247), 20.3%(42/206) and 16.8%(41/243), respectively. The beneficial effect of growth factors on embryo development in vitro could be duplicated. These data indicate that the presence of FGF, EGF or IGF-II in the culture medium is beneficial for embryo development in vitro and accelerate cell differentiation.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.287-287
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• 2004

Sexing from bovine embryos which were fertilized in vitro implicate a possibility of production of the sex controlled cattle. This study was carried out to investigate the possibility of determining of embryo sex by fluorescence in situ hybridization (FISH) technique. FISH was achieved in in vitro fertilized bovine embryos using a bovine Y-specific DNA probe which constructed from the btDYZ-1 sequences. (omitted)