외인성 estrogen이 성장과 유선발달 및 초발정일령에 미치는 영향을 6개월령 한우 암송아지에서 조사되었다. 10mgdml estrogen이 삽입된 송아지의 처리 후 90일 동안 무처리구와 다르지 않았다. 그러나 20mg의 estrogen이 삽입된 송아지의 증체량은 무처리구에 비하여 18%의 증가를 나타내었다(P<0.05). 처리 후 180일 동안의 증체량은 10mg 및 20mg의 estrogen 처리가 무처리에 비하여 각각 평균 6.7% 및 17.8% 증가하였지만 유의성은 없었다. 처리 후 90일 동안 유두 직경의 증가량은 estrogen 삽입량에 관계없이 처리구가 무처리구에 비하여 159%의 유의적 증가를 나타내었다(P<0.05). 처리후 180일 동안 유두 직경의 증가량도 10mg과 20mg의 estrogen 처리가 무처리에 비하여 각각 100% 및 128%의 유의적 증가를 나타내었다(P<0.05). 처리 후 90일 동안 유두 길이의 증가량은 10mg과 20mg의 estrogen 처리가 무처리에 비하여 각각 200% 및 295%의 유의적 증가를 나타내었다(P<0.05). 처리 후 180일 동안 유두 길이의 증가량도 10mg과 20mg의 estrogen 처리가 무처리에 비하여 각각 265% 및 325%의 유의적 증가를 나타내었다(P<0.05). 유두 용적의 증가량은 10mg과 20mg의 estrogen 처리가 무처리에 비하여 처리 후 90일 동안 각각 282% 및 246%의 유의적 증가를 나타내었다(P<0.05). 처리 후 180일 동안 유두 용적의 증가량도 10mg과 20mg의 estrogen 처리가 무처리에 비하여 각각 251% 및 244%의 유의적 증가를 나타내었다(P<0.05). 그러나 기간에 관계없이 10mg과 20mg의 estrogen 처리구 사이에 유두 직경, 길이 그리고 용적의 증가량에 있어서는 차이가 없었다. 10mg 및 20mg의 estrogen 처리는 초발정일령을 각각 20일 및 124일 단축시켰다. 전체적으로 이러한 결과는 송아지에 estradiol의 삽입은 성장과 유선 발달을 촉진시키고 초발정일령을 단축시킬수 있다는 것을 강력하게 지적한다.

To verify in vivo viability of IVF-derived bovine embryos, morula and blastocysts that developed from in vitro matured and fertilized ova were transferred to the uteri of recipient cows and normal calves were produced. To produce IVF-derived bovine morula or blastocysts, ova matured and fertilized in vitro were cultured in culture medium for 7~8 days at 39$^{\circ}C$ under the humicified atmosphere of 5% CO2. Two different culture systems, a co-culture system with TCM-199 and bovine epithelial cells (BOEC) and CR1aa without somatic cell support, were compared. Cleavage rates to 2~8 cell stage and developmental rates of IVF-derived bovine embryos to blastocyst stage were not different between co-culture system (51.3 and 14.0%) and CR1aa medium (60.4 and 22.1%), respectively. Embryos were classified into three grades by embryo quality and then one or two embryos in higher quality(A and B grades) were transferred to the uterus of recipients. In this study Korean Native calf was first born after transfer of IVF-derived embryos. Total four live calves were normally developed to term from IVF-derived bovine blastocysts and one female fetus was still-born approximatedly 8 months of gestation, but there was no pregnancy after transfer of morula. Therefore, normal calves could be produced after transfer of IVF-derived bovine embryos cultured in CR1aa medium without somatic cell support. In addition, our results suggest that in transfer of IVF-derived bovine embryos blastocyst stage is better than morula.

This study was carried out to investigate the effects of concentration, kinds of cryoprotectants, equilibration time, optimum thawing temperature on the survival rate of rapidly frozen porcine embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 30, 35 or 37$^{\circ}C$ water bath, Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The high survival rate of porcine frozen embryos after rapidly thawed in freezing medium was attained 2.0M DMSO, 2.0M glycerol, 2.0M propanediol, 1.5M ethyleneglycol. 2. The high survival rate of porcine frozen embryos after rapidly thawed in freezing medium was obtained using single cryoprotectant(16.6~40.0%) than mixed cryoprotectants(12.5~33.3%). 3. The eqilibration time on the survival rate of rapidly thawed porcine frozen embryos was attained after short period of time(15.0~33.3%) in the freezing medium higher than long period of time(9.10~30.0%). 4. The thawing temperature on the survival rate of rapidly thawed porcine frozen embryos was attained at 3$0^{\circ}C$ of thawing temperature(33.3~40.6%) in the freezing medium higher than 25 or 37$^{\circ}C$ of thawing temperature.

This study was carried out to investigate the in-vitro fertilization rate survival rate of rapidly frozen porcine immature embryos. The porcine embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on vitro culture or FDA test. The results are summarized as follows : 1. The in vitro fertilization rate of all frozen immature oocytes(6.7~26.7%) was very low, 35.0% of unfrozen oocytes and the rate of immature oocytes was very higher than that of mature oocytes. 2. The survival rate of all frozen immature oocytes(10.3~25.0%, 13.3~30.0%) was very low, 45% of unfrozen oocytes and the rate of immature oocytes was slightly higher than that of mature oocytes.

This study carried out to investigate the effects of cryoprotectants in the medium on the survival rate of rapidly frozen porcine bisected demi-embryos. The porcine bisected demi-embryos following dehydration by cryoprotectants containing sucrose were directly plunged into liquid nitrogen and thawed in 3$0^{\circ}C$ water bath. Survival rate was defined as development rate on in vitro culture or FDA-test. The results are summarized as follows : 1. The survival rates of without-zona pellucida embryos and 2 blastomeres porcine embryos were 10.0 and 7.1%, respectively. The rate of unfrozen blastomeres (20.00%) was significantly higher than that of non-frozen oocytes. 2. The survival rates of with and without-zona pellucida of bisected porcine embryos by micromanipulator were 20.0 and 14.3%, respectively. 3. The developmental rates of with and without-zona pellucida of bisected poricine embryos by micromanipulator were 13.3 and 7.1%, respectively.

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

The objective of this study was to produce Korean native calves following transfer of in vitro matured, fertilized and cultured embryos into Holstein cows. The ovaries of Korean native cows or heifers were obtained from an abattoir and kept on 25 to 28$^{\circ}C$ and transported to laboratory within 2 hrs. The oocytes were matured in vitro (IVM) for 24 hrs in TCM-199 supplemented with 35$\mu\textrm{g}$/ml FSH, 10$\mu\textrm{g}$/ml LH, 1$\mu\textrm{g}$/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% CO2 in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (IVC) with bovine oviductal epithelial cells for 7 to 9 days. Late morulae and blastocysts produced in vitro were nonsurgically transferred to recipient cows by unilaterial. Recipients were monitored for estrus and for pegnancy by rectal plapation in 60 days after embryo transfer. One of them was pregnant to term and produced a female weighing 42.5kg at birth.

This study was carried out to test the effects of fluorescein diacetate(FDA 0${mu}ell$/ml, 0.5${mu}ell$/ml, 5${mu}ell$/ml, 10${mu}ell$/ml or 0${mu}ell$/ml, 5${mu}ell$/ml in PBS) treated before culture on the survival of vitrified mouse morulae in vitrification solution(20% glycerol＋glycerol＋10% ethylene glycol＋30% ficoll＋10% sucrose). The results were summarized as follows; 1. The survival rate of FDA-tested fresh mouse morulae after 24 hours culture was over 96%((P4.8) in the control or treatment groups with various levels of FDA. Because the rate of mouse morulae which developed to hatched blastocysts was higher with the various levels of FDA treatment(67%) than control(50%), it was considered that toxicity of FDA did not affect the survival of mouse morulae. 2. When mouse morulae were FDA-tested in FDA 0(control), 0.5, 5, and 10${mu}ell$/ml treatment after vitrification, the development rate to expanded blastocyst were 66, 82, 64 and 76%, and FDA scores were P4.2(84%), P4.7(94%), P4.2(84%) and P3.9(78%), respectively. There were no significant differences between control and FDA treatments, but there were significant difference between 0.5${mu}ell$/ml)94%) and 10${mu}ell$/ml(78%) treatment(P<0.01). 3. The survival rates of cultured mouse morulae according to FDA-scores(P0=non-fluorescence; P1~P5=according to their fluorescence) after vitrification were P5;92%, P4;67%, P3;42% and P2.P0;0%, respectively. 4. Implantation rates of morulae stage embryos cultured into early blastocysts and implanted into uterine hornes vitrification were 14 and 11% embryos treated control(0${mu}ell$/ml) and FDA 5${mu}ell$/ml and the normal fetus development was 2% embryos for both treatments. Results of this percent study indicated that toxicity of FDA does not affect not only the survival of fresh and vitrified mouse morulae but also the development rate and implantation of fetus after transfer as well. The development rates of mouse morulae with the FDA score of P5, P4 and P3 were 92, 67 and 42%, respectively, it was considered that FDA-test was fit for the judge of survival.

30마리의 암양에게 surgical embryo collection과정을 통해 oviduct와 uterine horn에 trauma를 생성시켰다. 수술시 절개부위를 봉합하기 직전 노출된 abdominal tissue에 irrigigant로 10% dexamethasone을 사용하였다. 처리군에게는 17mg의 colchicine(1ml/ewe)를 투여했으며, 대조군에게는 1.0ml의 placebo를 처리하였다. 처음 17mg/im의 colchicine를 처리한 15마리 양은 colchicine독성증세를 2∼5일 내에 보였기 때문에 본 연구에서 제외되었다. 17mg에서의 colchicine독성 때문에 colchicine수준은 8, 4 그리고 2mg으로 낮추어졌다. 8mg group에 있던 또 하나의 양은 5일째에 독성증세를 보였기 때문에 나머지 양들은 4와 2mg의 수준으로 이틀에 한번씩 처리되었다. 두 번째 laparotomy는 첫 번째 처리로부터 9주후에 실시되었다. 두 번째 laparotomy후 처리군은 4mg의 colchicine을 이틀에 한번씩 14일 동안 처리되었는데 아무런 독성증세를 나타내지 않았다. 세 번째 laparotomy는 마지막 처리 5주후에 실시되었고 adhesion score로 계산하였다. Adhesion grading은 0∼4의 분포에 근거하여, 4는 가장 심한 adhesion을 나타낸다. 두 번째 laparotomy 결과 adhesion grading( 3)은 두 group 사이에 큰 차리가 없었다(P>0.05). 세 번째 laparotomy결과는 처리군에서 약간 낮은 수치를 보였지만, 통계적으로 두 grouprks에는 큰 차이는 없었다(P>0.05). 10마리의 양(5마리는 대조구, 5마리는 처리구)들은 처리후 5일경에 bone marrow analysis를 통해 cytogenetically분석되었다. 두 grouprks 염색체수와 구조에 있어서 차이가 없었다(p>0.05).

These experiments were carried out to investigate the effects of human follicular fluid (hFF) as a protein supplement on development of mammalian embryo as well as to find out ways toward effective use of hFF. The developmental rates of mouse embryos to the blastocyst and implantation stages were significantly higher in T6 ＋hFF than T6＋hFCS. Classified hFF according to the maturity of contained oocytes (M-hFF and Im-hFF), and compared the rates of development of mouse embryo cultured in M-hFF or Im-hFF to culture medium T6. Total protein, albumin and estradiol concentrations were higher in M-hFF than Im-hFF (P<0.05). The developmental rates of mouse embryos to the blastocyst and hatching blastocyst stages cultured in Im-hFF were significantly lower than those in M-hFF and the basic medium. In accordance of the results of human IVF, hFF has been divided into 4 groups. The developmental rates of mouse embryos to the blastocyst stage in presense of hFF from pregnant patients, who have good grade embryos, were significantly higher than those in hFF from patients who have poor grade embryos or were not pregnant. In addition, the rates of development of human embryo were compared in presense of BSA, hFF or hFCS. The developmental rates of human embryos cultured in Ham's F10＋hFF were significantly higher than those in the Ham's F10＋BSA. These results suggests that the culture system using hFF could improve the development ability of mammalian embryos and the viability of blastocysts cultured in vitro.