It is not easy for porcine embryos produced by in vitro systems to develop into blastocysts with high quality. To solve this problem, many researchers have developed novel culture methods. However, the formation of blastocysts with high quality is still low. In this study, we aimed to produce piglet following transfer of in vitro produced early embryos ( cell stage embryos) or morula and blastocyst. The cell stage embryos were transferred to five estrus-synchronized recipients (200 embryos per recipient). One of the five sows farrowed three piglets, which contain two live piglets and one dead piglet, 114 days after embryo transfer. However, two recipients transferred with morula and blastocysts did not farrow. Microsatellite analysis confirmed that the genomic DNA of two live piglets were not genetically identical to that of the recipient. These results indicate that it is possible to obtain piglets by transfer of early embryos produced by in vitro production (IVP) systems.

본 연구에서는 소의 체외 수정란에 대해 Y 염색체 특이 DNA 표지를 이용하여 FISH 기법으로서 수정란의 성감별을 수행하고 이를 이식하여 산전 성감별 개체 생산을 시도하였다. 본 연구에 이용된 Y-염색체 특이 DNA probe는 bovine male specific DNA로 알려진 btDYZ-1의 385bp 단편으로 제작하였고, Dig-PCR 방법으로 labeling하였다. 수정란의 성감별은 IVF로서 생산된 단계의 수정란을 공시하고 이들로부터 단일할구를 생검하여 분석하였다. 생검 방법은 투명대를 laser로 drilling하고 개의 할구를 squeezing하여 분리하였으며 demi-embryo는 1일간 재 배양하여 배반포기 단계에서 이식하였다. 우선, 본 probe의 신뢰성을 검정하기 위하여 생검된 할구는 FISH 기법으로 Y 염색체 존재 여부를 판단하고, 이의 demi-embryo는 재 배양하여 중기상을 유도하여 핵형분석한 후 비교 검토한 바 97.6%의 일치율을 나타내었다. 한편, 수정란에서 성감별을 위한 FISH의 적용 여부를 검토하고자 총 3937개의 공시란 중 3657개(93%)가 분석이 가능하였고, 이들 중 45.8%가 Y 염색체 특이적 접합 발현 양상이 나타났다. 본 결과를 토대로 경남 진주 인근 지역 농장의 15두의 소를 대상으로 성 감별 수정란을 이식한 결과, 7두가 임신한 것으로 확인되었고 이들 중 5마리가 출산하였다. 뿐만 아니라 출산된 송아지들은 모두 예견된 성과 100% 일치하는 것으로 나타났다. 따라서 본 연구에서 적용한 FISH 기법은 수정란의 성감별에 효율성이 높고 매우 높은 정확성을 나타냄에 따라 실질적으로 성감별 수정란의 대량생산이 가능할 것으로 사료되며, 농가차원에서 산업적 실용화가 될 수 있을 것으로 기대한다.twork descrition)를 통해 교통분석후의 제반 교통특성(교통량, 교통량/용량 비(比), 속도 등)을 교통망상에 표시할 수 있음으로서 의사결정에 보다 많은 도움을 줄 수 있을 것이다. 비트율의 증가와 화질 열화는 각각 최대 1.32%와 최대 0.11dB로 무시할 수 있을 정도로 작음을 확인 하였다.을 알 수 있었다. 현지관측에 비해 막대한 비용과 시간을 절약할 수 있는 위성영상해석방법을 이용한 방법은 해양수질파악이 가능할 것으로 판단되며, GIS를 이용하여 다양하고 복잡한 자료를 데이터베이스화함으로써 가시화하고, 이를 기초로 공간분석을 실시함으로써 환경요소별 공간분포에 대한 파악을 통해 수치모형실험을 이용한 각종 환경영향의 평가 및 예측을 위한 기초자료로 이용이 가능할 것으로 사료된다.염총량관리 기본계획 시 구축된 모형 매개변수를 바탕으로 분석을 수행하였다. 일차오차분석을 이용하여 수리매개변수와 수질매개변수의 수질항목별 상대적 기여도를 파악해 본 결과, 수리매개변수는 DO, BOD, 유기질소, 유기인 모든 항목에 일정 정도의 상대적 기여도를 가지고 있는 것을 알 수 있었다. 이로부터 수질 모형의 적용 시 수리 매개변수 또한 수질 매개변수의 추정 시와 같이 보다 세심한 주의를 기울여 추정할 필요가 있을 것으로 판단된다.변화와 기흉 발생과의 인과관계를 확인하고 좀 더 구체화하기 위한 연구가 필요할 것이다.게 이루어질 수 있을 것으로 기대된다.는 초과수익률이 상승하지만, 이후로는 감소하므로, 반전거래전략을 활용하는 경우 주식투자기간은 24개월이하의 중단기가 적합함을 발견하였다. 이상의 행태적 측면과 투자성과측면의 실증결과를 통하여 한국주식시장에 있어서 시장수익률을 평균적으로 초과할 수 있는 거래전략은 존재하므로 이러한 전략을 개발 및 활용할 수 있으며, 특히, 한국주식시장에 적합한 거래전략은 반전거래전략이고, 이 전략의 유용성은 투자자가 설정한 투자기간보다 더욱

본 연구는 수정란이식의 현장 적용 측면을 중심으로, 한우 체외 수정란의 발생 단계, 일자 및 등급이 젖소 대리모에 이식 후 임신과 유산, 분만과 관련된 임신기간, 생시체중, 난산 및 쌍자율을 조사하였다. 또한 단자 및 쌍자 분만에 따른 임신기간, 생시체중, 성비 및 폐사율을 분석하였다. 발생 단계에 따른 임신율은 배반포 단계(64.4%), 유산율은 부화 배반포(HB) 단계(21.4%)가 유의하게 높았다(p<0.05). 발생 일령에 따른 임신율은 7일령

본 연구는 체내, 체외 소 배반포기 배의 GMP vitrification 후 활력도의 비교와 한우 수정란을 몽골 소에 수정란이식 후 산자생산 가능성을 조사하고자 실시하였다. 한우 수정란은 체외수정란 또는 과배란처리에 의한 체내수정란을 생산하여 GMP vitrification 방법으로 동결 후 몽고로 수송하였다. 수란우는 CIDR과 처리에 의하여 동기화를 유도하였다 체내수정란생산을 위하여 7두를 과배란처리하였다. 총 64개의 배반포기를 회수하였다. (pe

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol , to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.

The objective of this study was to improve the efficiency of bovine embryo transfer by transferring of Hanwoo embryos into Hanwoo or Holstein recipients. The cryopreserved or fresh in vitro produced(IVP) embryos were transferred into uterine horn contralaterally or ipsilaterally to the corpus luteum. The recipients were inseminated by artificially on the next day of estrus. The pregnancy was diagnosed by rectal palpation at 60∼90 days after transfer of the embryos. The pregnancy rate by transfer of one or two embryos was 78%(7/9) and 74%(31/42), respectively. The pregnancy rates according to the grade of corpus lutea of recipients was 75% (20/27) and 82.0%(18/22) at the grade of A and B, respectively. Ten(67.0%) of 15 Holstein recipients transferred with IVP Hanwoo embryos and 5(42.0%) of 12 Holstein recipients transferred with frozen IVP Hanwoo embryos were pregnant. The single and twin calving ratio in Hanwoos was 77.0%(10/13) and 23.0%(3.13) in the recipients transferred with IVP embryos and 64.0%(7/10) and 27.0%(3/10) in the recipients transferred with frozen IVP embryos, respectively. Twenty-four pregnant cows following transfer of IVP embryos, 21(88.0%) calved the normal calves, and 2(8.3%) aborted. When the frozen IVP embryos were transferred, 16 pregnant cows calved 14(88.0%) normal calves and 2(13.0%) aborted. In conclusion, when one or two IVP bovine embryos were transferred into recipients, the A and B grade of corpus luteum resulted in high pregnancy rates. For the production of twin calves, transfer of the IVP or frozen IVP embryos could be suitable.

Techniques for manipulation of spermatozoa and oocytes have been widely used for in vitro production(IVP) of Hanwoo. This study was conducted to examine the effects of theophylline and heparin on frozen-thawed Hanwoo sperm for enhancing the efficiency of IVP technique. Oocytes were inseminated with forzen bull semen treated with either theophylline or heparin for examining the effect of each substance on fertilization and subsequent development. More (P<0.05) oocytes formed pronucleus and develop to the morula and blastocyst stages after inseminated with sperm treated with heparin than after inseminated with sperm treated with theophylline. The pregnancy rate after embryo transfer was higher after heparin treatment than after theophylline treatment, but did not differ significantly. There was no significant difference of offspring delivery between two groups. In conculsion, theophylline and heparin can be used for enhancing the efficiency of IVP system for Hanwoo. Considering characteristics of these substance, theophylline may be useful in the artificial insemination system, which requires vigorous sperm motility. While, heparin supporting sperm viability in vitro can be effectively used for improving in vitro-fertilization system.

This study was carried out to improve a technique of cloned animal prodcution by preactivation of nuclear recipient oocytes with ionomycin and 6-dimethylaminopurine (6-DMAP) in rabbits. The oocytes were collected from the oviduct of superovulated rabbit at 19∼20 hours post hCG injection. The collected oocytes were preactivated and self-enucleated by treating 5 uM ionoycin for 5 min, and 2.0 mM 6-DMAP for two hours. Microsurgical removel of the chromation complex in the second polar bodies was effectively performd and single blastomere separated from 32-cell stage rabbit embryos was injected into the perivitelline space of the enculeated recipient oocyts. Follwoing electrofusion and in vitro culture for 18 hours, the nuclear transplant(NT) embryos were transferred into the uterine horns of naturally mated or synchronized recipient does. When 32 NT embryous reconstituted with preactivated oocytes were transferred to 2 recipient does, one foster doe delivered two offspring (6.3%), while not a offspring was delivered from three foster does which received 17 NT embryos reconstituted with non-preactivated oocytes. A total of 68 NT embryos reconstituted with preactivated oocytes were transferred into the uterine horns of 7 synchronized ecipient does. Among them, two recipients were pregnant and delivered three offspring(5.9%).

A combined technology of transvaginal ovum pick-up(OPU) system with in vitro-oocyte manipulation technique can be used for improving reproductive efficiency in the cattle. The objective of this study was to establish a newly-conceived breeding program using OPU in the pregnant cows. The OPU trial was performed in pregnant cows every 10 days from 40 through 90 days of artificial insemination (Al), and number of follicles in ovary, number of retrieved oocytes and embryo development following in vitro-fertilization, were evaluated. Reduced number of follicles in the ovaries of pregnant cows was firstly detected from 70 days after A' and a significant (P<0.05) decrease in the follicle number (5.4 follicles /donor) was found at 90 days than at 40, 50, 60 and 80 days after Al (8.0~9.2). A similar pattern was also observed in the number of oocytes retrieved by OPU apparatus during experimental period. When retrieved oocytes were matured and inseminated in vitro with frozen bull semen, development of the oocytes to the blastocyst stage was not significantly affected by the retrieval time. Four embryos (morula or blastocyst stage) derived from oocytes retrieved from pregnant cows were nonsurgically transferred to four recipient cows on day 7 of estrus cycle. For the first time in Korea, three of four transferred embryos developed to live calves with normal physiological parameters. In conclusion, an effective breeding program employing pregnant cow can be developed by use of OPU trial and in vitro culture techniques of oocytes ; OPU system could be repeated in pregnant cows with no risk of abortion and viable offsprings were borne after transfer to the recipients.

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%).

Production of calves after transfer of nuclear transplant embryos is the latest technology to be applied in commercial livestock breeding. The objective of this study was to establish an efficient procedure to produce offsprings from nuclear transplant embryos. The fusion rates (72.7% vs. 80.8%), cleavage rates (62.5% vs. 71.4%) and rates of development in vitro (12.0% vs. 15.2%) of nuclear transplant embryos were not significantly different between 30 and 40h maturation age of cytoplast. The in vivo and in vitro-derived embryos as nuclei donor were used in this system of bovine nuclear transplantation. Fusion rates of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (73.0 and 79.2%, respectively). The percentage of embryos reaching the morulae or blastocysts were 21.8% for in vivo-derived embryos and 11.9% for in vitro-derived embryos (p<0.01). Pregnancy rates after embryo transfer of nuclear transplant embryos were not significantly different between in vivo and in vitro-derived embryos (45.9 and 40.5%, respectively). However, calving rates after embryo transfer of nuclear transplant embryos were significantly higher in the in vivo-derived embryos than in vitro (p<0.01). Further research for age of cytoplast and use of in vitro-derived embryos as nuclei donor is required in this system. In conclusion, these results clearly show that the use of in vitro-derived oocytes as recipient cytoplast can improve the nuclear transplant system for genetic progress in cattle.

The purposes of this study were to produce cloned rabbit embryos and offsprings by nuclear transplantation(NT) using in vitro matured oocytes as nuclear recipient cytoplasm and to determine the effect of frozen nuclei donor embryos on the production efficiency of cloned embryos. The 8cell embryos were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline containing 10% fetal calf serum(FCS) at 40 hours after hGG injection. A portion of collected embryos were preserved at 4 for 24 hours and a portion of them were frozen by vitrification method. The embryos used for donor nuclei were synchronized in the phase of Gi /S transition. The in vitro matured oocytes were used as recipient cytoplasm following removing the nucleus and the first polar body. The synchronized blastomeres from fresh, cooled or frozen embryos were injected into the enucleated oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 sec at 1.0 W /cm in 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39 in a 5% incubator. Following in vitro culture of the NT embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The results obtained were summarized as follows: 1. The fusion rates of the blastomeres from fresh, cooled and frozen embryos with the in vitro matured and enucleated oocytes were 100, 95.8 and 64, 3%, respectively. 2. Development in vitro to blastocyst was significantly(p<0.05) different between the cloned embryos with the blastomeres from fresh, cooled or frozen embryos as 39.0, 20. 9 and 15.7%, respectively. 3. The mean numbers of cell cycle per day during in vitro culture of cloned embryos blastomeres from fresh, cooled or frozen embryos was 1.31, 1.29 and 1.16, respectively. 4. A total of 77 nuclear transplant embryos were transferred into 6 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer.