• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.76-76
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• 2001

The objective of this study was to optimize the vitrification method of in vitro produced bovine embryos. Thus, in vitro produced embryos at 8 cell, morula and blastocyst stages were vitrified on electron microscope grids (EM grids) or in open pulled straws (OPS) with EG5.5 (5.5 M ethylene glycol, 1.0 M sucrose and 10% FBS in m-DPBS medium) freezing solution and their survival rates after thawing were compared. The embryos on EM grids or in OPS were briefly exposed to EG5.5 freezing solution and plunged directly into liquid nitrogen within 30 to 35 sec. Post-thawed embryos were serially diluted in 0.5, 0.25 and 0.125 M sucrose in m-DPBS, each for 1 min, and then cultured in CRI aa medium supplemented with 10% FBS. Embryonic survival rate was assessed as re-expanded and hatched rates of those embryos after warming. The rates of re-expansion embryos did not significantly different between EM grid (8 cell: 42.10%, morula: 66.66% and blastocyst: 77.08%) and OPS (8 cell: 47.36%, morula: 61.90% and blastocyst: 83.33%) methods. In addition, the hatched rates in EM grid (8 cell: 31.57%, morula: 57.14% and blastocyst: 72.91%) were similar to those in OPS (8 cell. 34.21%, morula: 50.00% and Blastocyst: 77.08%). Interestingly, even at the same blastocyst stage, the in vitro survival of day 7 embryos (EM grid: 79.48 and OPS: 87.18%) was higher than those of day 8 embryos (EM grid: 72.10 and OPS: 82.06%). The total cell number of blastocyst developed in vitro after vitrification was examined with Hoechst 33342 staining to compare the embryo quality among different treatment groups. The total cell number of blastocyst was not significantly different between vitrified groups (EM grid: 162.4$\pm$8.0 and OPS: 158.4$\pm$7.1) and unvitrified control (168.0$\pm$5.6). These results indicate that both vitrification containers can provide the high rate of embryo survival. Moreover, the OPS container may not need a cap to protect the container from floating after immersion in L$N_2$. Therefore, this study suggest that bovine embryos can be cryopreserved easily, effectively and successfully by vitrification method using EM grid or OPS with EG5.5 freezing solution. In the future, the Pregnancy rate would be investigated after transfer of our vitrified embryos into the appropriated recipients.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.229-234
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• 1991

Thc chimeric morulae were produced following aggregation of the half embryos which were microsurgically bisected at 8-cell and early morula stage. Different phenotypic embryos were obtained by mating ICR female mice with ICR or CBA male mice. The early morula stage was thc desirable stage for the aggregation of mouse embryos after bisection. The post-thawed survival rates of bisected-aggregated embryos that developed into normal blastocyst after conventional freezing in DMSO and ethylene glycol were 30.5 and 32.896, respectively. One offspring was produced by transferring the 67 frozen-thawed bisected-aggregated embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.2 no.4
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• pp.595-598
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• 1989

Bovine embryos/ova obtained from in-vitro fertilization were either co-cultured on a monolayer of bovine cumulus cells or cultured in medium alone. Embryos/ova co-cultured with cumulus cells developed to 8-cell (30.9%), morula (29.8%) and blastocyst stages (26.6%) after 3-4, 5-6, and 7-8 days of culture, respectively, while embryos/ova cultured in medium alone failed to develop beyond 8-cell (0-13.3%), morula (0-1.5%) and blastocyst stages (0%). The results of this study demonstrated the beneficial effect of cumulus cells on the development of bovine embryos.

• Korean Journal of Animal Reproduction
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• v.17 no.2
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• pp.121-125
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• 1993

Early development of bovine oocytes fertilized in vitro in the medium with caffeine and heparin was examined in different culture systems. When the oocytes were transferred into culture medium 8 h after insemination, 12%(7/60) of penetrated oocytes cleaved to 4-cell stage 24 h after insemination. The proportions of oocytes cleaved to 80to 16-cell stage 48 h after insemination had also a to be higher in oocytes transferred into culture medium 8 h (29%) than 16 h(10%) or 24 h(4%) after insemination. 52% of the 4-cell embryos developed to morula and blastocyst stages when they were co-cultured with oviductal epithelia, whereas only 5% of embryos cultured without the epithelial cells(P<0.001). In another experiment, embryos were co-cultured with ampulla, isthmus or utero-tubal junction of oviducts. There are no significant differences in the proportions of embryos developed to morula and blastocyst stage.

• Journal of Embryo Transfer
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• v.4 no.1
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• pp.35-40
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• 1989

The effects of cryoprotectants (glycerol, DMSO and ethylene glycol) and the concentrations (0, 0, 25, 0.5and 1.0 M) of sucrose in the diluent on the is vitro survival of mouse morulae froaen rapidly in liquid nitrogenvapour were examined. When the embryos were equilibrated in 1.5 M cryoprotectants +0.25 M sucrose in one-step or in 3.0 M cryoprotectants +0.25 sucrose in two-step and diluted with 0, 0.25, 0.5, or 1.0 M sucrose solution after thawing, high survival rates were obtained in ethylene glycol (48.0% to 88.2 %) or in glycerol (35.0 % to 77.8 %). These results show that 1.5 M ethylene glycol is a highly efficient cryoprotective agent for the rapid freezing of mouse morula embryos and 0.5 M sucrose was optimal concentration in the diluent after thawing.

• Korean Journal of Animal Reproduction
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• v.9 no.1
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• pp.36-39
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• 1985

This experiment was carried out to investigate the morphology of bovine embryos thawed after deep freezing at -196$^{\circ}C$ and the development of frozen-thawed embryos after in vitro culture in Ham's F-10 medium with 10% NBCS. The results obtained were summarized as follows: 1. The propotion of embryos which a, pp.ared mophologically normal was averaged 77.5% (79/102). 2. The morphologically normal rate of frozen-thawed blastocyst (78.6%) was higher than that of morula (76.7%), but there was no significant difference. 3. Normal development was observed in 20 of 68 embryos cultured for 24-72hr in medium and overall survival rate was 29.4%. 4. Survival rate fo blastocyst (33.3%) was higher than that of morula (25.7%).

• The Korean Journal of Zoology
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• v.34 no.3
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• pp.389-393
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• 1991

To obtain monozygotic multiplets from 8-cell mouse embryos, we artificially constructed chimeric embryos by introducing one blastomere (donor) of 8-cell embryos of Fl hybrid (C57BL/6 X CBA) mice into 4-cell ICR mouse embryos (carrier) of which one blastomere had been previously removed with a micromanipulator. After 42 h of culture, the developmental frequency of chimeric embryos to normal morula and blastocyst was 95% (310/328). When chimeric embryos at morula or blastocvst stage were transferred to pseudopregnant mice,39%, (70/180) of them were born. Most of the offspring (56/70) were the carrier type in coat color, whereas only three of them were the donor type, of which ho were assumed to be derived from single 8-cell donor embryo. Because the two donor type mice Ivere the same sex and produced only the donor type offspring from a testcross, they are probably monozvgotic multiplets of 8-cell mouse embryos. However, since their internal chimerism was not able to be examined, it remains to be determined if their genetic constitutions are identical.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.2
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• pp.125-131
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• 1992

The possible effect of human follicular fluid(hFF) on the growth and development of fertilized oocytes and embryos is important because the fallopian tubes are exposed to FF after follicular rupture and the processes of fertilization and embryo cleavage occur inside the fallopian tubes. Previously, it was suggested that human FF might adversely affect on the development of early mouse embryos. In order to investigate the effect of hFF on the development of embryos, early mouse embryos were cultured in media containing various protein sources as bovine serum albumin(BSA), fetal cord serum(FCS) and FF. And we evaluated the development of early mouse embryos in terms of the morphology, cleavage rate, and cell count of blastcysts. There were no significant differences in the morula and blstocyst formation rates of 2-cell mouse embryos cultured in the media containg three different protein sources and three different concentrations of FF. The blastocyst formation rate of 1-cell mouse embryo cultured in FF group was significantly higher than that cultured in BSA group(P<0.05). The morula and blastocyst formation rates of 2-cell mouse embryos of the group cultured in the media containing FF were comparable with those of other two groups, in addition, the cell count of blastocysts of FF group in the 2-cell embryo culture was higher than those of BSA group and HCS group(P<0.01), and this finding was also noted in 1-cell embryo culture. There was no difference in the morula and blastocyst formation rates of the 2-cell mouse embryos cultured in the media containing different concentrations of FF. These results suggest that mature human follicular fluid has no inhibitory activity on the development of early mouse embryos even in high concentration and may be a good protein source which is positively associated with the development of mouse embryos in vitro especially in 1 cell embryo culture.

• Journal of Veterinary Clinics
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• v.9 no.1
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• pp.323-332
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• 1992

The present study was carried out to examine the effect of oviduct epithelium and its conditioned medium on e development of early bovine embryos in vitro. Oocytes obtained from ovarian follicles of slaughtered cows were cultured in TCM199 with 10% fetal calf serum for 22-24hrs and then fertillzed in vitro using frozen-thawed semen treated with BO-caffein, BO-BSA(20mM heparin added). Oviduct epithelium was collected in each stage of the estrus cycle and conditioned medium was the medium in which oviduct epithelium in early luteal stage was cultured. In vitro fertilized bovine embryos of 1～2 cell were co-cultured with oviduct epithelium from different estrus cycles, cultured in conditioned medium, and cultured in rabbit oviduct. The cleavage rates of in vitro fertilized early bovine embryos co-cultured with oviduct epithelial cell from early luteal, luteal and follicular phase of estrus cycle(67.2～70.8%) and cultured in conditioned medium(56.7%) were significantly(p<0.05) higher than that of the control(44.2%) The rate of development to morula or blastocyst stage in oviduct epithelial cell co-culture(15.3～32.5%) from three phase of estrus cycles and conditioned medium(14.5%) were significantly(p<0.05) higher than that of the control(5.2%). The oviduct epithelial cell from early luteal phase gave a significantly( p<0.05) higher rate of development to morula or blastocyst stage than both luteal and follicular phase. The results of in vivo culture in rabbit oviduct of early bovine embryos were 52.1% for the cleavage rate and 26.7% for the rate of development to morula or blastocyst stage.

• Journal of Embryo Transfer
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• v.13 no.2
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• pp.191-197
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• 1998

Immature nocytes and in VitrO matured Oocytes collected from the slaughtered Korean cattle were frozen slowly with 10% ethylene glycol+5% polyvinyl pyrolidine+0.05M trehalose (l0EPT), 10% ethylene glycol+5% ficoll+0.05M sucrose (1OEFS), or 10% ethylene glycol+5% ficoll+0.05M trehalose (l0EFT) by cell freezer (experiment 1). And also,They were ultra-rapidly frozen with 30% ethylene glycol+10% polyvinyl pyrolidine+0.5M trehalose (3OEPT) or 30% ethylene glycol+18% ficoll+0.5M sucrose (3OEFS) using electron microscope grid (experiment 2). In experiment 1, the cleavage rate was 23.0% when immature oocytes were frozen slowly using various cryoprotectants descrihed above, and 5.1% of cleaved oocytes developed to over morula stage after in Vitro fertilization (IVF). There were no significant differences among these groups. When matured oocytes were frozen slowly, the total cleavage rate was 19.7%, and over morula stage was 3.2%. lOEPT (4.8%) and EFS (4.4%) were slightly more effective than l0EFT (0.0%) for development in vitro. Only in l0EFT treated group, immature oocytes have higher developmental capacity than matured ones, when they were frozen slowly and IVF after thawing. In experiment 2, oocytes were ultra-rapidly frozen using the electron microscope grid with two kind of cryoprotectants described above. In immature oocyte group, the cleavage rate was 13.9% and 5.8% of cleaved oocytes developed to over morula stage after IVF, and in matured group, 25.7 and 7.6%, respectively. There were no significant differences between two kind of cryoprotectants, but in ultra-rapid freezing using electron microscope grid, the efficiency is slightly higher in matured oocyte group.