본 연구는 재래산양의 핵이식을 실시하여 공여세포의 조건, 전기적 세기 및 융합횟수 등이 융합율과 체외발달율에 미치는 영향을 조사하여 최적의 융합조건을 확립하고자 실시하였다. 공여세포는 귀 유래 섬유아세포와 태아 유래 섬유아세포 2종류를 분리 배양하여 사용하였으며, 수핵란의 채취는 성숙한 미경산 재래산양에 과배란을 유기하여 hCG 투여 후 제 35시간째에 외과적인 방법으로 in vivo (체내성숙)난자는 난관을 관류하는 방법으로 회수하고 in vitro (체외성숙)난자는 난포로부터 흡입하여 난포란을 채취하여 약 22시간 체외성숙을 실시하였다. 수핵난자는 난구세포를 제거한 다음 0.05 M sucrose를 처리하여 세포질이 양호하고 극체가 뚜렷하게 보이는 난자만을 선별하여 핵이식을 실시하였다. 핵이식란의 융합은 전기자극방법으로 융합을 실시하였으며, 핵이식 조작 후 약 3시간 동안 전배양을 실시한 다음 활성화를 유도하였다. 복제수정란은 0.8% BSA가 첨가된 mSOF 배양액으로 6∼7일 동안 체외 배양을 실시하였다. 귀 유래 섬유아세포를 공여세포로 사용하였을 때 융합율은 60.4%로서 태아 유래 섬유아세포의 40.3%보다는 높게 나타났다. 분할율에 있어서는 귀 유래 섬유아세포와 태아 유래 섬유아세포가 각각 47.6 및 48.2%로서 차이가 없었다. 2.40∼2.46 ㎸/㎝로 전기자극을 주었을 때 융합율은 43.8%로서 1.30∼l.40 ㎸/㎝(26.7%)와 2.30∼2.39 ㎸/㎝ (34.8%)가 높게 나타났으며, 융합이 이루어진 핵이식란의 분할율은 82.9(1.30∼l.40 ㎸/㎝), 43.8(2.30∼2.39 ㎸/㎝) 및 51.8%(2.40∼2.46 ㎸/㎝)로서 전기자극의 세기에 따른 유의적(p＜0.05)인 차이는 없었다. 전기융합을 1회 실시하였을 때 in vivo 난자는 43.5%로서 in vitro 난자의 23.6%보다 유의적으로 높게 나타났으며, 2회 실시하였을 때는 55.7(in vivo) 및 39.2%(in vitro)로 in vivo에서 높게 나타났다. 3회 자극을 주어 전체 융합율은 in vivo가 66.1%로서 in vitro의 52.8%보다는 유의적으로 높게 나타났다.

본 연구는 이종간 핵이식란의 생산성 향상에 기여하기 위한 기초연구로서 핵이식 수정란의 융합과 활성화 과정에 있어서 수핵난자 및 전기적 융합조건이 핵이식 수정란의 융합 및 체외 발달에 미치는 요인들을 조사하기 위하여 실시하였다. 도축되어지는 소 및 돼지의 난소에서 난포란을 채취하여 TCM-199 및 NCSU-23에 혈청 및 호르몬을 첨가하여 39℃, 5% CO₂ 배양기내에서 24 및 48시간 체외성숙을 실시하여 수핵난자를 준비하고, 공여세포의 준비는 산양의 귀세포를 채취하여 0.25% Trypsin-EDTA의 처리로 세포를 분리, 배양하여 사용하였으며, 계대배양과 함께 세포는 TCM-199 ＋ 10% FBS ＋ 10% DMSO로 동결을 실시하였다. 핵이식은 성숙된 난자의 극체 및 전핵을 laser system으로 투명대를 drilling 하여 제거하고 준비된 공여세포를 핵이 제거된 난자에 주입하여 전기적 자극으로 융합을 실시하여 융합된 난자는 전기적 자극으로 활성화를 유도하였다. 활성화가 이루어진 복제 수정란은 수핵란이 소난자의 경우 monolayer가 형성된 10% FBS가 첨가된 TCM199 배양액에서 7∼9일 동안 체외배양하였으며, 수핵란이 돼지의 경우 10% FBS가 첨가된 NCSU-3 배양액으로 6∼8일 동안 체외배양을 실시하여 배반포기로 유도하였다. 본 연구에서 얻은 결과를 요약하면 다음과 같다. 전기자극의 세기를 1.95 kv/cm와 2.10 kv/cm로 주었을 때 수핵란이 소 난자의 경우 융합율은 47.7및 44.6%였으며, 분할율도 41.9 및 54.5%로써 차이가 없었다. 수핵란이 돼지 난자인 경우는 융합율은 51.3 및 46.1%로써 차이가 없었으며, 분할율도 75.0및 84.9%로써 차이가 없었다. 전기자극 시간을 30 또는 60μsec, 횟수는 1 또는 2회 주었을 때 수핵란이 소 난자의 경우 융합율은 30 μsec 1회(50.8%) 와 2회(31.0%) 간에 차이가 없었으나, 60μsec 1회(19.3%)가 가장 낮았다(P＜0.05). 융합란의 분할율은 30μsec 1회(53.3%)와 2회(50.0%) 간에 차이가 없었으나, 60μsec 1회(18.2%)보다 유의적(P＜0.05)으로 높게 나타났다. 돼지 난자의 경우 융합율은 30μsec 1회(48.1%), 2회(45.2%)및 60μsec 1회(48.6%)간에 차이가 없었으며, 분할율은 30μsec 1회(78.4%)와 60μsec 1회 (79.4%)간에 차이가 없었으나, 30μsec 2회(53.6%)보다 유의적(P＜0.05)으로 높게 나타났다. 이종간 핵이식란의 체외발달에 있어서 수핵란이 소 난자의 경우 상실배와 배반포기로의 발달율이 22.6%로써 단위발생란 30.6%와 차이가 없었으며, 돼지 난자의 경우는 이종간 핵이식란이 5.1%로써 단위발생란 37.4%보다 유의적(P＜0.05)으로 낮게 나타났다. 이상의 실험결과로 보아 산양의 체세포를 이용한 이종간 핵이식 복제수정란의 생산을 위하여 수핵란으로 소와 돼지를 사용하여 복제수정란의 발달을 확인할 수 있었으며, 이종간 핵이식에 있어서 수핵란, 공여세포, 융합, 활성화 및 배양조건 등 아직 초보 수준에 있으며, 앞으로 보다 많은 연구를 통하여 이러한 문제들이 해결되면 멸종위기 상태에 있는 동물들의 종 보존에도 활용이 가능할 것으로 생각된다.

This study was conducted to investigate the developmental ability of caprine embryos after somatic cell interspecies nuclear transfer. Donor cells were obtained from an ear-skin biopsy of a caprine, digested with 0.25% trypsin-EDTA in PBS, and primary fibroblast cultures were established in TCM-199 with 10% FBS. After maturation, expanded cumulus cells were removed by vigorous pipetting in the presence of 0.3% hyaluronidase. The matured oocytes were dipped in D-PBS plus 10% FBS＋7.5 ㎍/ml cytochalasin B and 0.05 M sucrose. The reconstructed oocytes were electrically fused with donor cells in 0.3 M mannitol fusion medium. After the electofusion, embryos were activated by electric stimulation. Interspecies nuclear transfer embryos with bovine cytoplasts were cultured in TCM-199 medium supplemented with 10% FBS including bovine oviduct epithelial cells for 7∼9 day. On the other hand, the NT embryos with porcine cytoplasts were cultured in NCSU-23 medium supplemented with 10% FBS for 6∼8 day at 39℃, 5% CO₂ in air. In caprine-bovine NT embryos, the cleavage(2-cell) rate was 36.8% in confluence and 43.8% in serum starvation. The developmental rate of morula- and blastocyst-stage embryos was 0.0% in confluence and 18.8% in serum starvation. In caprine-porcine NT embryos, the cleavage(2-cell) rate was 76.7% in confluence and 66.7% in serum starvation. The developmental rate of morula and blastocyst stage embryos was 3.3% in confluence and 3.0% in serum starvation, and no significant difference was observed in synchronization treatment between donor cells. In caprine-bovine NT embryos, the cleavage(2-cell) rate of cultured donor cells was 30.8% and 17.6% in 5∼9 and 10∼14 passage(P＜0.05). The developmental rate of morula and blastocyst stage embryos were significantly higher(P＜0.05) in 5∼9 passage(23.1%) than in 10∼14 passage(0.0%) of cultured donor cells. In caprine-porcine NT embryos, the cleavage rate was significantly higher(P＜0.05) in 5∼9 passage(86.7%) than in 10∼14 passage(50.0%) of cultured donor cells. The developmental rate of morula and blastocyst stage embryos were 3.3 and 0.0% in 5∼9 and 10∼14와 passage of cultured donor cells. In caprine-bovine NT embryos, the developmental rate of morula and blastocyst stage embryos were 22.6% in interspecies nuclear transfer, 33.9% in in vitro fertilization and 28.1% in parthenotes, which was no significant differed. The developmental rate of morula and blastocyst stage embryos with caprine-porcine NT embryos were lower(P＜0.05) in interspecies nuclear transfer(5.1%) than in vitro fertiltzation(26.9%) and parthenotes(37.4%).

초음파기기를 이용한 난포란의 채란 기술은 유전적으로 우수한 수정란의 다량확보가 가능함으로써 가축개량에 널리 응용할 수 있다. 그러나 초음파유도 난포란의 채란은 무엇보다도 회수율과 회수한 난포란의 질적 등급의 개선이 이루어져야 할 것이다. 본 연구는 등지방층두께, 일당증체량, 근내지방도 및 배최장근 단면적에 연관된 DNA marker가 검정된 한우로부터 FSH(FOLLTROPIN-V, Vetrepharm, Canada)를 투여하여 FSH 투여전.후의 포의 발달율과 등급별 회수율을 조사하였다. 공시동물의 발정주기에 관계없이 매주 무처리와 채란 12시간 전에 FSH를 각각 l00mg 과 200mg을 근육 주사하여 난포란의 채취를 유토하였다. 난소내 난포란의 채란은 SONOACE-600형 (Medison CO., 한국) 초음파기기를 사용하여 모니터 상에 검게 나타나는 난포를 17-gauge needle (Cook, Australia) 로써 70mmHg로 유지된 regulated vacuum pump를 이용하여 모두 채란하였으며, 소난포는 손가락 촉지를 이용하여 채란하였다. 난포의 크기는(large : 6mm, medium : 2-6mm, small 2mm) 무처리구에서 small(56.6%), medium(33.0%), large follicles(10.4%) 순으로 나타났으며, 전체 난포수는 106(8.23.6개) 였다. FSH 200mg 투여구에서는 medium(47.5%), small(32.5%), large follicles(20.0%) 순으로 나타났으며, 전체난포수는 40(10.02.2개) 였다. FSH l00mg 투여구에서는 medium(45.6%), small(43.5%), large follicles (10.9%) 순으로 나타났으며, 전체 난포는 92(7.72.5개) 였다. FSH 투여전 초음파 image 상으로 관찰한 난포수와 FSH 투여 후 초음파기기와 손가락 촉지를 이용하여 난포의 발달 및 난포란의 채란율은 200mg 투여 구에서는 투여 전에 4.30.6개였으나 투여 후에는 9.32.1개로 증가하였으며, 채란율은 75.0% 였다. l00mg 투여구에서도 투여 전후에 각각 5.31.2개 및 8.22.3개였으며, 채란율은 81.6%였다. 난포란의 채란율은 무처리구에서 88.7%(7.23.9개)로 나타났으며, 회수한 난포란의 등급은 각각 0.0%(GI), 5.3%(GII), 28.T%(GIII), 66.0%(GIV)로 나타났다. FSH 200mg 투여구의 채란율은 87.5%(8.82.6개)로 나타났으며, 등급별회수율은 GI(2.8%), GII(8.6%), GIII(34.3%), GIV(54.3%)으로 나타났다. 뿐만 아니라 FSH 100mg 투여구에서도 회수율은 89.1%(6.85.2개)였으며, 등급별 회수율은 각각 GI(8.5%), GII(13.4%), GIII(43.9%), GIV(34.2%)로 나타났다.

본 연구에서는 DNA marker가 검정된 한우로부터 생산한 체외수정란을 이식하여 육질 및 육량의 유전적 능력이 우수한 한우를 대량생산하여 고품질 한우 쇠고기 생산 시스템을 구축하기 위한 전단계로서 DNA marker 검정 한우로부터 초음파유도 난포란을 채란하여 체외수성 및 수정란의 체외 발달에 미치는 각종 요인들과 배반포기 수정란의 부화율 개선을 위하여 투명대를 laser로 drilling을 실시하여 부화율을 조사하였다. 초음파유래 체외수정란의 분할률

본 연구는 고품질육의 DNA marker가 규떵된 한우로부터 초음파유노 난포란의 연속적 채취를 통하여 능력이 우수한 한우 수정란온 대량생산하는 방법의 확립과 이를 한우농가에 응용하고자 초음파 난자채취기를 이용하여 등지방층두께, 일당증체량, 근내지방도 및배최장근 단면적에 연관된 DNA marker를 보유하고 있는 한우 5두로부터 개체및 난포수, 채취방법, 회수한 난포란의 등급 등을 조사하였다. 한우 5두의 개체별 난포수는 6, 10, 5, 4 및 11회

This study was undertaken to access the effects of collection method, room temperature at oocyte recovery and culture media on the oocyte quality, fertilization and cleavage rates of in vitro matured and fertilized oocytes of Korean native goats. Ovaries obtained from a slaughterhouse were transported to the laboratory and were divided into 2 groups. One group of ovaries was maintained at 30 to 35 of the room temperature and another group was remained at 20 to during oocyte recovery. The oocytes were recovered by follicle aspiration, slicing and aspiration+slicing methods from 3 groups of follicles according to size; <2 mm, 2 to 6 mm and >6 mm. The matured oocytes were inseminated with buck epididymal spermatozoa at a concentration of 3~3.510 m1 and fertilization was identified when 2 pronuclei were present in the cytoplasm. Although the recovery rate per ovary obtained by the combination of follicle aspiration + slicing(19.62.2) method was higher than aspiration(11.71.1) and slicing(14.81.8) collection, optimal recovery according to oocyte grades resulted form ovarian slicing compared to aspiration or combined methods(P<0.05). However, no significant differences were found in the mean number(2.51.8; 3.33.3; 2.92.4) and the proportion of favorable oocytes(Grades I, II and III) recovered(31.6%, 36.0%, 36.4%,) according to follicle size(<2 mm; 2 to 6 mm; >6 mm). Fertilization rate was 60.0%, 67.7%, 70.6% and 56.4% and the proportion of embryos/zygotes was 11.1%, 7.1%, 5.0% and 2.8% in 20~/BO, 30~35/BO, 20~/TALP and 30~35 /groups, respectively.

This study was conducted to develop an improved method for oocyte pick-up(OPU) with finger-sensibility using ultrasound-guidance from ovarian follicles in Holstein heifers. Oocytes were aspirated from ovarian follicles of clear-outline (>2mm), obscure-outline and invisible( 2mm) on ultrasound images with 3 different vacuum pressure(40, 80, 120mmHg). Total number of oocytes recovered/follicles were 309/237(130.4%). 113/80(141.3%) and 107/74(144.6%) with 40, 80 and 120 mmHg of vacuum pressure, respectively. Mean number of oocytes recovered was higher in 2 OPU/week (18.35.3) than 1 OPU/week(14.54.1), but this difference was not statistical1y significant. The recovery rates were not affected by the number of OPU as 135.6%(282 oocytes/208 follicles) in 1~20 OPU, 137.7% (168/122) in 21~40 OPU and 148.4%(92/62) in 41~60 OPU, respectively. The proportions of good oocytes (Grades I) recovered were not significantly different by the number of OPU until 40 OPU(12.4% in 1~20 OPU vs 16.7% in 21~40 OPU). However, a significantly(P<0.05) lower recovery rate resulted from more than 40 OPU compared to less than 40 OPU(7.6%). These results imply that more fertilizable oocytes can be produced from invisible-immature follicles by transvaginal aspiration with finger-sensibility from Holstein heifers.

The simulated DUPIC fuel provides a convenient way to investigate fuel properties and behaviours such as thermal conductivity, thermal expansion, fission gas release, leaching and so on. Several pellets simulating the composition and microstructure of the DUPIC fuel were fabricated from resintering powder through the OREOX process of the simulated spent fuel pellets, which were prepared from the mixture of stable forms of constituent nuclides. This study describes the powder treatment, OREOX, compaction and sintering to fabricate simulated DUPIC fuel using the simulated spent fuel. The homogeneity of additives in the powder was observed after attrition milling. The microstructure of the simulated spent fuel was in agreement with the previous studies. The densities and the grain size of simulated DUPIC fuel was pellets are higher than those of simulated spent fuel pellets. Small metallic precipitates and oxide precipitates were observed on matrix grain boundaries.

Experiments were conducted to assess the effect of quality and viability of bovine blastocysts derived from in-vitro culture(IVC) of in vitro matured and fertilized(IVM-IVF) oocytes during their transport 2 hours. Follicular oocytes were collected form ovaries obtained at a slaughterhouse and were cultured for 24 hours in TCM-199. The IVM oocytes were fertilized in vitro with caudal epididymis spermatozoa. Fertilized oocytes were cultured for 7 to 9 days, and embryos that developed to the blastocyst stage were used for the experiment. The blastocysts, packed in straws with storage medium that consisted TCM-199 with HEPES equilibratd in air and supplemented with 10% FCS were transported at 39~(2.0 h). The quality of blastocysts was assessed and ranked as A(excel-lent), B(Good), fair or poor after transportation. The percentages of A and B grade blastocysts after transport duration for < 1 hours(97.7%) were similar to the result from transport duration for 1~2 hours (92.9%) and 2~3 hours(89.6%), but significantly(P<0.05) higher than transpot duration for 3~4 hours(76.3%). The percentages of A and B grade blastocysts after transport duration for two hours from developed blastocyst at 7day(100%) and 8day(85.0%) were higher 9day(96.6%) and >9day (40.0%). And early to expanded blastocyst produced in vitro were transferred to recipient cow by additional embryos at 7 and 8th day after AI. Three of them were pregnant to term and produced four twin calves, and two calves was premature birth. The gestation lengths of male to female and female to female twin were 282 and 281 days, respectively. And birth weight of twin calves were male to female(22.Skg) and female to female twin(20.3Okg), respectively.

The present study was carried out to develop a cloning technology of mouse embryos by nuclear transplantation with electrofusion and to produce cloned offsprings by transfer of reconstituted embryos. A single nucleus from two- and eight-cell embryos was transplanted into the enucleated two-cell embryos by rnicromanipulation. The fusion of nucleus with recipient cytoplasm and the subsequent development of reconstituted embryos in vitro as well as in vivo to term were examined to determine the optimal electrofusion parameters for nuclear transplantation in mouse embryos. The successful enucleation of donor embryos was 84.9 and 83.3% in two- and eight-cell stage, respectively, and the successful injection of nucleus from two- and eight-cell donor embryos into the perivitelline space of enucleated two-cell embryos were 85.1 and 84.7%, respectively. No significant differences were found in enucleation or injection rate between the cell stages of donor embryos. When the blastomeres of intact two-cell mouse embryos were electrofused in 0.3 M mannitol medium(100 sec., 3 pulses), the fusion rate was similarly 93.2, 92.2 and 92.0% in 1.0, 1.5 and 2.0 kV /crn, respectively, but in vitro development to blastocyst of the fused two-cell embryos was significantly(P<0.05) lower in 2.0 kV/cm (63.4%) than in 1.0 kV/cm (91.7%) or 1.5 kV/cm (82.4%). The development in vitro to eight-cell stage of the reconstituted embryos with nucleus from two-cell stage(45.5%) was significantly(P<0.05) higher than that from eight-cell stage blastomeres (16.7%). The number of blastomeres of the intact embryos at blastocyst stage was 50i0.6 and 552.4 in in vitro and in vivo cultured mouse embryos, respectively, but significantly(P<0.05) decreased to 350.7 in nuclear transplanted blastocyst embryos. The conception rate of mice following embryo transfer was 32.1% in the reconstituted two-cell embryos using two-cell donor nuclei, which was comparable to the fresh two-cell embryos(40.6%). However, the rate of development in vivo to term following embryo transfer of the reconstituted two-cell embryos using two-cell donor nuclei (23.5%) was significantly(P<0.05) lower compared with the percentage of two-cell fresh embryos(31.5%).