This study was carried out to determine the factors on achieving good viability of embryos biopsied fur sexing, to investigate pregnancy rate following embryo transfer(ET) with sexed embryos, and to confirm the accuracy for the calves bort following ET with sexed embryos by polymerase chain reaction(PCR). To investigate viability of Hanwoo embryos after biopsy for sexing, fresh and frozen/thawed embryos were biopsied according to different developmental day of blastocysts, different stage of blastocysts, and different biopsy grade and the embryos themselves were incubated for 2 hours in TCM199 after microsection to be evaluated morphologically for recovery as blastocyst. The results obtained were as follows : 1. The rate of oocytes cleaved in vitro and the rate of blastocyst of the cleaved oocytes were 52.5% and 21.6%, respectively. The rate of blastocyst on day 8 was 11.2%, denoting the highest rate during whole culture period posterior to in vitro fertilization(IVF) 2. After biopsy for sexing, the viability rate of blastocyst on day 7, 8 and 9 was 75.0%, 88.4%, and 100.0%, respectively and the viability of early, mid, and expanded blastocyst after biopsy was 75.0%, 88.9%, and 91.1%, respectively The viability rate of fresh and frozen/thawed embryos was 89.9%, 71.4%, respectively. And the viability of expanded, hatching, and hatched blastocyst of frozen/thawed embryos was : 75.0%, 75.0%, and 50.0%, respectively. The viability of embryos according to biopsy grade of 10∼20%, 21∼30%, and 31∼40% was 85.7%, 91.5%, and 71.4%, respectively. 3. Pregnancy rate after transfer with biopsied embryo between flesh and frozen/thawed embryos was 22.6% and 20.0%, respectively. 4. In comparison between sex by PCR method and sex of calves born after embryo transfer, the accuracy of sex deterimination was 92.3% (12/13).

This study was conducted to enhance the efficiency of embryo transfer and embryos produced in vitro according to select good quality blastocyst among in vivo or in vitro produced embryos. After middle blastocys(MB) and late blastocyst(LB) stages embryos that developed in vitro for 7∼8 days were treated hypertonic solution(380 mOsm or 600 mOsm) and the rate of development to hatched blastocyst(HB) was examined. The results obtained were summarized as follows: The proportion of embryos developed to HB was higher than that of control regardless of hypertonic solution treatment. However, it was related to culture time in hypertonic solution The propotrion of embryos developed to HB of morphologically recovered embryos in 380 nOsm solution ws higher than that of morphologically shrinked embryos. However, treatment time was not significantly different the rates of HB development. Especially, the proportion of embryos developed to HB of morphologically recovered embryos of the 30-min-treated group was significantly higher than that of control (p<0.05). The results suggest that hypertonic solution treatment should enhance the efficiency for criterion of transferable blastocyst quality.

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).

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.

This study was conducted to investigate the effects of vitrification solution and developmental stage on the survival rate of vitrified-thawed human blastocyst embryos. Human blastocyst embryos were cryopreserved by vitrification using EFS and GE solution, and their survival rates were examined after thawing and further culture. EFS solution was consisted of 40% ethylene glycol, 18% Ficoll 70 and 0.3M sucrose. GE solution was consisted of 25% glycerol and 25% ethylene glycol. Embryos were exposed to EFS and GE solution by 2 steps and 3 steps, respectively, and plunged into liquid nitrogen after loading into 0.25ml plastic straws. Blastocysts were classified into 4 groups in accordance with their developmental stage: into 1) EEB, 2) MEB and 3) EdB, of blastocysts developed on day 5, and 4) 6d-Bla(the blastocysts which formed on day 6). The blastocysts at each stage were vitrified by GE solution and cryopreserved in LN2. After thawing them, we examined their survival rates, respectively. The resulted of this study were as follows: 1. The survival rate of blastocysts vitrified by GE solution was 64.4%, significantly higher than that (5.7%) vitrified by EFS solution (P<0.001). 2. When the blastocysts were vitrified by GE solution according to each developmental stage, the survival rates of EEB, MEB, EdB and 6d-Bla were 65.9%, 65.9%, 73.2% and 58.1%, respectively. In conclusion, the cryopreservation of human blastocysts by vitrification is likely to have a marked advantage in terms of cost, work and time as compared to the conventional slow freezing in IVF-ET programs.

체외생산된 소 배반포 및 탈출 배반포강 내의 유리 아미노산 농도를 측정하였다. 체외 배양은 소 혈청알부민이 함유된 합성난관배양액(synthetic oviduct fluid; SOF)에서 실시하였으며 수정 후 180시간의 배반포 및 216시간 후의 탈출 배반포를 실험에 공여하였다 아미노산 측정은 알부민 대신 Polyvinyl alcohol이 함유된 SOF 미소적에 수정란을 분주하고 미세조작기를 이용하여 배반포강 내의 액을 추출하여 20종류의 아미노산을 측