혈통이 등록된 우수한 한우 공란우로부터 회수한 수정란을 간편한 방법으로 성 판별하여 우수한 암송아지를 생산하고자 실시한 결과는 다음과 같다. 회수한 수정란을 punching 또는 bisection 방법으로 biopsy하여 Loop-mediated isothermal amplification법으로 성 판별하였으며, 암컷으로 예측되는 성 판별 수정란을 6두의 수란우에 이식하여 2두가 임신되었고, 수정란 이식 후 278일과 285일에 정상적인 암컷 송아지를

면양과 염소가 최근 수십년동안 세계여러 나라에서 번식생리의 연구를 위한 모델로 사용되어 왔는데, 체내수정란의 생산에 관한 영역도 유럽을 중심으로 활발하게 연구되어왔다. 수정란생산을 위한 발정동기화방법, 과배란처리 및 수정란회수방법 기술은 현재 상당히 많은 기술진척이 이루어진 상태이나, 우리나라 고유의 재래유전자원인 흑염소에는 이를 위한 기술이 미진한 실정이므로 본 실험에서는 흑염소의 체내수정란생산기술을 확립하여 재래가축 유전자원보존을 위한 기초기술을 마련하고자 한다. 축산기술연구소 남원지소에서 사육하고 있는 체중 20kg 이상의 건강한 흑염소를 이용하여 발정동기화를 위해 controlled intravaginal drug release(CIDR)를 질내에 14일 동안 삽입하고, 과배란처리는 FSH를 CIDR 삽입 12, 13, 14일째에 12시간 간격으로 점감법으로 총20mg을 투여하였으며, PGFa를 13일째 FSH와 함께 투여하였다. CIDR는 14일째의 아침에 제거하였다. 수컷과의 교미는 CIDR제거 24시간후에 GnRH를 투여와 동시에 실시하였으며, 채란은 교미후 3일째에 외과적인 방법으로 실시하였다. CIDR처리경과에 따른 progesterone농도는 CIDR 주입시 바로 수치가 상승하여 제거전까지 6~12ng/m1의 농도를 유지하였으며, 제거즉시 2ng/ml 이하로 떨어졌다. 채란시 평균 배란점은 16.5개, 미배란난포 9.8개였으며, 회수수정란은 6.0개를 나타내어 채란율은 36.4%를 나타내었다. 회수된 수정란의 발달단계는 4-cell 78.9%, 2-cell 5.3%, fragmentation 15.8%를 나타내었다. 이와 같은 체내수정란생산방법을 기반으로 하여 이후 수정란의 동결 및 수정란이식기법에 관한 연구를 수행한다면 우리나라의 재래가축인 흑염소의 유전자원 장기보존과 생산성향상에 기여할 것으로 사료된다.배양액에 30 embryos/50ul 소적으로하여 38.8, 5% 의 탄산가스 배양기에서 각각 7일간 배양을 실시하였다. 조사된 결과는 SAS/STAT를 이용하여 통계분석을 실시하였다. 체외수정 12시간 후에 난자 급속 염색법으로 염색을 실시한 결과, 모든 처리구에서 핵성숙률(76.4~95.2%), 정자침투율(51.1~66.9%), 웅성전핵형성률(95.2~100%), 다정자침입률(18.2~25.6%) 및 평균침입정자수(1.2~l.4개)에서 유의적인 차이가 인정되지 않았다. 체외배양 48시간 난할률을 조사한 결과, 처리구별 차이(53.9~67.9%)는 인정되지 않았으나, 배양 7일째 배반포형성률은 각각 14.5, 25.4, 17.3 및 12.4%로서 25uM의 -ME처리구가 유의적(P<0.05)으로 높은 배발달률을 나타내었고, 총세포수에 있어서는 대조구와 처리구간 유의적인 차이가 인정되지 않았다. 따라서 돼지 난포란을 성숙배양할 때, 25uM -ME를 첨가배양하는 것이 양질의 돼지체외수정란을 생산하는 하나의 방법으로 조사되었다.다.

This experiment was carried out to investigate the factors affecting superovulation in rabbits and to determine the effect of pFSH and PMSG on ovarian superovulatory responses and embryo production, and the effect of superovulation treatment with a single injection of pFSH dissolved in polyvinylpyrrolidone on the ovarian responses and the embryo quality. The results obtained were suonmerized as follows: Superovulatory response resulted in significantly (P<0.05) higher ovulation rates and more embryos in spring or autumn, compared with summer or winter. Repeated superovulatory treatments with PMSG leaded to a significantly(P<0.05) decreased number of total follicles and recovered ova. Superovulation with pFSH resulted in the higher number of ovulated follicles and recovered ova than with PMSG. A single subcutaneous injection of pFSH dissolved in 25% PVP resulted in the more ovulation points(33.2) and recovered embryos(30.2), which were comparable to the multiple injections of pFSH(44.8 vs 37.7).These results indicated that the treatment with a single injection of FSH dissolved in PVP was an efficient and simple alternative method to the conventional multiple FSH injections for superovulation in rabbits.

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 sec) in 0.3 M mannitol solution supplemented with 100 M CaCl and MgCl. The nuclear donor embryos of 8-cell stage were synchronized to G phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

In order to improve the cryopreservation by vitrification or slow freezing of nuclear transplant rabbit embryos, the effects of factors affecting embryo cryopreservation such as cryoprotectants, equilibration, cooling rate and post-thaw dilution on post-thaw survial and development were determined using intact embryos of morular stage. And the post-thaw development of nuclear transplanted embryos cryopreserved under the optimal conditions examined was compared between vitrification and slow freezing. The cryoprotectant solution used was ethyleneglycol-ficoll-sucrose (EFS) or ethyleneglycol-poly-vinylpyrrolidone-galactose- I (EPG- I ) for vitrification, and EPG- II for slow freezing. To examine the viability of frozen-thawed embryos, the nuclear transplanted embryos were co-cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) for 24 hrs and the intact morulae were co-cultured with BOEC for 5 days and 3 days to hatching blastocyst stage in 39 ˚C 5% incubator. The results obtained were as follows: Following vitrification with EFS, the post-thaw development of rabbit morulae to hatching blastocyst was significantly(P<0.05) higher in compacted stage(82.4%) than in early morular stage(60.0%). The post-thaw development of compacted morulae to hatching blastocyst was similarly high in vitrification with EFS(82.4%), EPG- I (85.0%) and in slow freezing with EPG- II (83.3%). Following vitrification with EPG- I, the post-thaw development of intact rabbit morulae to hatching blastocyst was similar as 78.0% and 85.0% in 1-step and 2-step post-thaw dilution, respectively. The post-thaw development of nuclear transplanted rabbit embryos of compacted morulae stage to hatching blastocyst was similarly 43.6% and 40.0% in vitrification with EPG- Iand slow freezing with EPG- II, respectively. These results indicated that the rabbit nuclear transplant and intact embryos of morulae stage could be well cryopreserved with either vitrification or slow freezing procedure.