The possible use of micromanipulative biopsy and PCR of the biopsied embryonic cells was tested to produce sexed bovine embryos in practical terms. By micromanipulation and PCR techniques, higher survival rate and accurate sexing of demi-embryos were btained. Bovine oocytes matured and fertilized in vitro were co-cultured with bovine oviductal epithelial cell (BOEC) monolayer in USU-6 medium supplemented with 15% FBS, and the embryos of 37% (327/885) were developed to blastocysts. Among 111 blastocysts produced by invitro, only 7 (6.3%) embryos were found unable to determine their sex, probably due to the loss of cells, since no PCR product was found from those cells. All the remaining 104 (93.7%) demi-embryos survived micromanipulation and demonstrated male-specific product or bovine-specific product alone suggesting that correct sexing of the sample. Forty-three point one percent(25/58) of manipulated and cryopreserved demi-embryos after thawing were survived. Final verification of the sexed embryos is necessary to make sure the same sex in fetus and newborn calf upon embryo transfer. The established sexing method on a large number of bovine embryos from previous and this study suggests that this a could be used practically in the field.

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 +-, +-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.

수정란이식의 주변기술인 초기배의 미세조작및 성 판정은 가축의 경우, 경제 형질의 유전적 개량에 크게 기여하였다. 본 연구는 미세조작 biopsy와 PCR에 의한 체외생산 소 초기배의 급속. 정확한 성 판정 기법을 확립하기 위해 실시하였다. 체외성숙 및 체외수정에 의해 생산된 소수정란은 소 난관상피세포와 공배양을 통해 8-세포부터 배반포시시까지 체외발생시킨 후 미세조작 biopsy에 이용되었다. 미세조작 biopsy 과정중에 약간의 형태적인 손상이 관찰되

This study was experimented that developmental effects of bovine in vitro fertilized embryos by coculture system and supplementation of energy materials into simple media. With the ovaries from slaughter house in vitro maturation by 24h, in vitro fertilization was performed with sperms collected by Percoll gradient method. Fertilized embryos were cocultured in 15% FCS+CZB medium with BOEC(bovine oviductal epithelial cell), GCM (granulosa cell monolayer) and MEFC(mouse embryonic fihrohlast cell). And also in this study, there was trying to improve the early developmental rate of embryos by addition of concentration-controlled Na-pyruvate, D-glucose which were used as energy sources into CZB medium. In vitro developmental rate was confirmed by the cleavage rate of 48h post-IVF and the embryo development rate at 240h culture. In the coculture system BOEC had 20.0% of blastocysts rate, which was higher than that of other coculture systems. To determine the optimum concentration for early embryo developmental rate rapidly, through the gradient of concentrations of Na-pyruvate and D-glucose, we focused on the cleavage rate at 48h and blastocysts rate at 240h. In case of Na-pyruvate, cleavage rate and developmental rate over 3-cell were lower at the concentration of 1.OOrnM than the other treatment concentrations, otherwise the blastocysts rate was higher as 23.2% than the others. That result showed that as like reported group which had higher develop-mental rate over 3-cell was also higher to the blastocysts rate. In case of D-glucose, there was no effects through the concentration changes. It was the result of this study for which the use of BOEC coculture system and 1.OOmM Na-pyruvate as an energy source had an effect upon embryo development.

This study was performed to establish the condition and the methods for the techniques of insertion the isolated blastomere cells into cytoplasm, in order to research the develop-mental ability of bovine embryo blastomere cells in vitro produced. After 24h in vitro ovary maturation with the ovaries from a slaughter house, in vitro fertilization was performed to the vital sperms which their mobility were decided by percoll gradient method, with 2~8 cell stage embryos, the blastomeres were isolated in +. +-free PBS, and following that embedded into agar and alginate solution, respectively. The rates of in vitro develop-ment are as follows ; in agar embedded 11 among 120(9.2%) 1 /2~1 /3 blastomers cleaved and 6 among 93(6.5%) 1 /4~1 /8 blastomeres cleaved. In sodium alginate-embedded 14 among 84(16.7%) 1 /2~1 /3 blastomeres cleaved and 6 among 85(7.1%) 1 /4~1 /8 blastomeres cleaved. In case of Na-alginate, the rate of the cells were better than those of agar. The results suggest that the techniques for embeeding the isolated blastomeres into gel may help cloning of bovine early embryo without nuclear transplantation.

In the experiment I for maas production of bovine early embryos, 18~20hpi fertilized eggs (756 eggs) and parthenogenic eggs (618 eggs) which were treated by 10% ethanol were cultured in both TGM and CZB. In the experiment II, suppiment effects each in CZB and CRlaa were tested by matured and fertilized oocytes which were after 18~20hpi. In the case of experiment I after 48hr, the cleavage rates of normally fertilized eggs were 66.6% in TCM treatment and 77.7% in CZB treatment, and after 240h the blastocysts were 7.5% in TCM and 14.1% in CZB. In the parthenogenic eggs, the deavage rates at 48hr were 39.6% in TCM and 57.5% in CZB, and at 240h, the blastocysts were 0.9% in TCM and 4.4% in CZB. These results showed that the effects of CZB on developmental ability to parthenogenic eggs as well as nomally fertilized eggs are relatively high. In experiment W, the effect of exposing the cleaved embryos to CZB for 30h on the blastocyst formation was examined. Similar rates of blastocyst formation were obtained both in TCM and CZB, suggesting that CZB exposure. during ealry development is critical. In experiments III ~ V, the effects of supplements were examined. The cleavage rates of CZB treatments at 48h were 83.8% in control, 78.1% in BSA+A.A+SIT, 75% in 5% FCS+A. A+SIT, 88.6% in BSA+A.A+SIT and not co-cultured BSA+A.A+SIT had 85.7% and in the case of 240h blastocysts showed 22.6, 0.0, fl.1, 6.5 and 0%, respectively. As a result, this study showed that CZB was effective culture system for in vitro development, and that CZB and CRaa had no significant differences and effects between them. It may be concluded that in the simple media containing supplements could replace the co-culture systems of bovine early embryo development.

초기배의 성판정은 대상가축의 성을 선발하는 수단으로써 가축의 육종 및 번식에 있어 가치가 매우 높다. 체세포, 체외수정 또는 처녀발생 초기배의 성을 결정하기 위해 capillary polymerase chain reaction (PCR)을 이용하였으며 성판정에 이용되는 상실배 또는 배반포는 체외수정과 그 후의 난관상피세포와의 공배양에 의해 생산되었다. 초기배의 genomic DNA는 0.2g/L proteinase K를 함유하고 있는 PCR lysis

본 논문은 3개의 강우관측망과 최적 강우관측망을 토대로 호우의 면적 평균강우량 산정용 섬진유역 강우관측망의 개량안을 제안했다. 강우관측망 설계문제는 면적평균강우량추정분산으로 나타내지는 정확도와 자료 수집비로 구성되는 목적함수를 최소화하는 것이다. 익히 알려진 분산경감법으로는 최소화 알고리즘인 SATS기법이 채용되었다. 첫 단계에서, 비용에 부과된 2개의 교환계수값에 따라 최적 관측망과 대안관측망이 얻어졌다. 다음 단계에서, 최적으로 선정된 우량국에 기존