• 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.

• Journal of Embryo Transfer
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• v.14 no.3
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• pp.171-176
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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 conception of embryos transferred, and pregnancy and twin birth rates after transfer of fresh or frozen-thawed bovine blastocysts produced in vitro were also evaluated. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol. The results obtained were as the following. The pregnancy rate after transfer was higher in co-culture group than in mTALP group, but was not signficantly different, and there is no difference between fresh embryo group and frozen-thawed embryo group in conception rate. The conception rate was not different whether 3∼4 blastocysts or 2 blatocysts transferred into a recipient, but the production rate of twin calves was significantly higher (p<0.05) when 3∼4 embryos transferred. The average birth weight of twin calves(24.38kg) was numerically, but not significantly lighter than that of single calves(26.68kg).

• 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.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.335-341
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• 1997

The aims of this study are to establish a stable isolation method of blastomeres from bovine early embryos and examine their developmental potential in vitro Early embryos were produced by maturation and fertilizaion in vitro of bovine follicular oocytes. Blastomeres were isolated from 2~8-cell embryos in $Ca^2$+-, $Mg^2$+-free PBS+EDTA after removing the zonae pellucidae Isolated blastomeres were cultured in CZB containing BOEC for upto 240 hpi. Cleavage rates of them were 18.5%(10 /54) in 1 /2 blastomeres, 33.3%(16/48) in 1/4 blastomeres and 34.2%(14 /41) in 1/8 blastomeres, respectively. The rates of blastocystic vesicle formed were 8.7%(4 /46) in 1/2 blastomeres, 26.6% (17/64) in 1/4 blastomeres and 10.3%(8 /78) in 1/8 blastomeres, respectively. Blastomeres developed into various types of blastocystic vesicles and trophoblastic vesicles as evidenced by the Hoechst 33258 staining and morphology. This results suggest that the isolation method used and subsequent culture of isolated blastomeres from bovine early embryos should be useful to obtain extra embryonic cells for various analyses such as PCR and putative ES cell culture.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.141-149
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• 1997

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.171-179
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplant embryos. The oocytes collected from slaughterhouse ovaries were matured for 24 h and then enucleated and cultured to allow cytoplasmic maturation and gain activation competence. And then the donor embryos were treated for 12 h with 10 $\pi$g /ml nocodazole and 7.5 $\pi$g /ml cytochalasin B to synchronize the cell cycle stage at 26 h after the onset of culture. The blastomeres were transferred into the perivitelline space of the enucleated nocytes and blastomeres and oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. The age of the recipient(30 vs 40 h) had no significant effect on the fusion rates(82.4 vs 82.1%) and the developmental rates to morula /blastocyst(9.8 vs 11.0%). Effect of Nocodazole treatment on the donor cell cyle synchronization to improve the developmental rates of bovine nuclear transplant embryos was significantly higher than control group(21.4 vs 10.1%, p<0.05). Significant differences were in the percentage of fusion rates(72.9,77.1vs 61.9%) in three types of fusion medium(PBS(+), mannitol and sucrose, p<0.01). The developmental rates of bovine nuclear transplant embryos appeared to be highest in mSOF medium under 5% 0$_2$ condition, but no significant differences were found when compared with TCM199-BOEC and mSOF under two different oxygen ratio(5 and 20%).

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.191-196
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• 1996

The objective of this study were to investigate the effects of culture media and additives on the development of bovine in vitro matured(IVM) and in vitro fertilized(IVF) oocytes. In experiment 1, bovine oocytes were cultured in droplets of TC 199 supplemented with 10% fetal bovine serum(FBS) with or without hormones (5$\mu\textrm{g}$/ml FSH, 5$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml E2). Cleavage rates of embryos cultured for 40~44hrs after IVF were higher when embryos were cultured in TC 199 supplemented hormones (68.1%, 921/35) than without hormones (52.7%, 77/146), but the percentages of development beyond morulae stage were not difference (20.7%, 19.4%). In experiment 2, the effects of various media such as TC 199, synthetic oviduct fluid(SOF), CR1aa with different energy source (fatal bovine serum, FBS; bovine serum albumin, BSA) on developmental capacity of IVM/IVF bovine embryos were investigated. The developmental rates into morulae and blastocysts were 27.1, 10.7, 6.3 and 0%, respecitvely, in CR1aa plus 3mg/ml BSA, SOF plus 10% FBS, TC 199 plus 10% FBS, SOF plus 3mg/ml BSA. In experiment 3, the comparisons of bovine embryos developed to morulae and blastocysts in different culture media (TC 199, SOF, CR1aa, Menezo's B2) were investigated. The developmental capacity beyond morulae stage were 32.9, 26.6, 11.1 and 7.1%, respectively, in Menezo's B2 plus BSA, CR1aa plus BSA, SOF plus BSA, TC 199 plus FBS medium. The cell numbers of the blastocyst were not different in different cultrue media. In experiment 4, bovine embryos were co-cultured with vobine oviduct epithelial cells(BOEC) in TC 199 plus FBS, SOF plus BSA, CR1aa plus BSA, Menezo's B2 plus BSA. The morula and blastocyst rates were 44.7, 32.9, 26.0 and 23.3%, respectively, in CR1aa TC 199, SOF, and Menezo's B2 medium. The cell numbers of the blastocyst were similar to those of blastocyst developed in different culture media.

• Nuclear Engineering and Technology
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• v.47 no.1
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• pp.47-58
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• 2015

Four recycling scenarios involving pyroprocessing of spent fuel (SF) have been investigated for a 600-MWe transmutation sodium-cooled fast reactor (SFR), KALIMER. Performance evaluation was done with code system REBUS connected with TRANSX and TWODANT. Scenario Number 1 is the pyroprocessing of Canada deuterium uranium (CANDU) SF. Because the recycling of CANDU SF does not have any safety problems, the CANDU-Pyro-SFR system will be possible if the pyroprocessing capacity is large enough. Scenario Number 2 is a feasibility test of feed SF from a pressurized water reactor PWR. Thefsensitivity of cooling time before prior to pyro-processing was studied. As the cooling time sensitivity of cooling time before prior to pyro-processing was studied. As the cooling time increases, excess reactivity at the beginning of the equilibrium cycle (BOEC) decreases, thereby creating advantageous reactivity control and improving the transmutation performance of minor actinides. Scenario Number 3 is a case study for various levels of recovery factors of transuranic isotopes (TRUs). If long-lived fission products can be separated during pyroprocessing, the waste that is not recovered is classified as low- and intermediate-level waste, and it is sufficient to be disposed of in an underground site due to very low-heat-generation rate when the waste cooling time becomes >300 years at a TRU recovery factor of 99.9%. Scenario Number 4 is a case study for the recovery factor of rare earth (RE) isotopes. The RE isotope recovery factor should be lowered to ${\leq}20%$ in order to make sodium void reactivity less than <7$, which is the design limit of a metal fuel.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.647-659
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• 1997

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.19-28
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• 2001

The present study was undertaken to investigate the effects of cooling rate and equilibration time on the survival, in vitro maturation and development to embryos of frozen-thawed bovine immature oocytes(Germinal Vesicle Stage). The cryoprotectants are used 10% ethylene glycol(EG) as permeating cryoprotectant and 0.05M soc.ose(S) or trehalose(T) as low molecular weight nonpermeating cryoprotectants and 5% ficoll(F) or polyvinylpyrrolidone(PVP) as high molecular weight nonpermeating cryoprotectants. Four freezing solution were uysed in this experiment(EFT: 10% EG + 5% F + 0.05M T, EFS: 10% EG + 5% F + 0.05M S, EPT: 10% EG + 5% P + 0.05M T, EPS: 10% EG + 5% P + 0.05M S). The best equilibration time and freezing solution was 15 min in EPT(83% survival rate of frozen-thawed bovine immature oocytes). When frozen-thawed bovine oocytes were cultured following IVM and IVF, there was no significant difference in cleavage and development rates among the EFT, EFS, EPT and EPS solutions. When 9 blastocysts derived from frozen bovine oocytes were transferred to 6 recipients, two recipients were pregnant. And one was aborted at 45 days of pregnancy and the other had a stillbirth.