마우스 핵이식란의 동결에 관한 연구

이병천,조충호,황우석,Lee, Byeong-chun,Jo, Choong-ho,Hwang, Woo-suk 대한수의학회 1993 大韓獸醫學會誌 Vol.33 No.1

The present study was carried out to investigate the developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulse at output voltage of 2.0 kV/cm for $100{\mu}$ sec to induce cell fusion. The recovery rate and developmental potency to blastocyst after freezing and thawing of nuclear transplanted 2-cell embryos was investigated. 1. The recovery rate of nuclear transplanted 2-cell embryos in normal morphology after freezing and thawing was significantly higher in rapid freezing(DMSO 4.5M) than in slow cooling(p<0.01). 2. When the recovered embryos in normal morphology were cultured in vitro, there were no significant differences in the developmental potency to blastocyst between the freezing methods and the concentrations of cryoprotectant. In summary, these experiments have proved that rapid freezing method(DMSO 4.5M) is effective in nuclear transplanted 2-cell mouse embryos. If improved micromanipulation techniques and freezing are combined, nuclear transplantation technique will contribute to the improvement of productivity in livestock animals.

핵이식 마우스 생산에 관한 연구

이병천,조충호,황우석,Lee, Byeong-chun,Jo, Choong-ho,Hwang, Woo-suk 대한수의학회 1993 大韓獸醫學會誌 Vol.33 No.1

The present study was carried out to investigate the best condition for nuclear-cytoplasm fusion and in vitro culture of nuclear transplanted embryos and to investigate the production of nuclear transplanted offsprings. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulses at output voltage of 1.0, 1.5 and 2.0 kV/cm for 100, 150 and $200{\mu}$ sec to induce cell fusion. 1. The culture of intact or zona cut 2-cell embryos in the medium supplemented with cytochalasin B($5{\mu}g/m{\ell}$) and colcemide($0.1{\mu}g/m{\ell}$)for 30 and 60 minutes did not affect the development to later stage. 2. The in vitro developmental rates of group A(a nucleus from one of the blastomeres was removed) and B(electrofusion of group A) were significantly lower than that of control group(p<0.01). 3. When nuclear transplanted embryos were submitted to electrofusion, the significantly higher fusion rates of 2-cell donor nuclei were achieved at the electric field strength of DC 1.5kV/cm for 100 and $150{\mu}$ sec, DC 2.0 kV/cm for $100{\sim}200{\mu}$ sec than DC 1.0 kV/cm for 100 and $150{\mu}$ sec(p<0.01). The significantly higher fusion rates of 4-cell donor nuclei were achieved at DC 2.0 kV/cm for 100 and $150{\mu}$ sec than DC 1.0kV/cm for $100{\sim}200{\mu}$ sec(p<0.01). These fusion rates in 8-cell donor nuclei were 88.7~99.3%. 4. The developmental potency to blastocyst in 2- and 4-cell donor nuclei was significantly higher in DC 1.0 and 2.0 kV/cm for $100{\sim}200{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group (p<0.01). The developmental potency to blastocyst in 8-cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}$ sec treated group than in DC 1.0 kV/cm for $100{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group(p<001). 5. The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 6. The success rate of nuclear injection into enucleated 2-cell embryos was significantly higher in 2-cell donor nuclei than in 4- or 8-cell donor nuclei(p<0.01). 7. The culture time taken for the nuclear transplanted 2-cell embryos to blastocyst stage was significantly longer in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 8. There was no significant difference in the developmental potency of nuclear transplanted embryos within the concentration of EGF at 0 to 15 ng per $m{\ell}$ of BMOC-3 solution. 9. The production rates of offspring after transfer of nuclear transplanted embryos to recipient mouse were significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01).

초음파유도에 의한 소 난포란의 채취에 관한 연구 I. 발정주기, 계절 및 bST처치 영향에 관하여

이병천,윤기영,김현일,노상호,이강남,황우석,Lee, Byeong-chun,Yoon, Ki-young,Kim, Hyun-il,Roh, Sang-ho,Lee, Kang-nam,Hwang, Woo-suk 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.4

Estrus cycle of cow, season and follicldar stimulating treatment, having an effect on the number of follicle, are investigated for the oocyte recovery rate in ovum pick-up(OPU). The number of follicle aspirated and oocyte collected on the different days of estrus cycle(D 4~5, D 9~10 and D 14~15) were not significantly different among the groups. The higher number of viable oocytes were produced on Jan-May(79.0%) than Jun-Aug(33.3%) by OPU in cow. The number of follicle and aspirated oocyte in cows treated FSH or PMSG combined with bovine somatotropin(bST) were 1.2~1.5 times higher than in cows treated alone follicular stimulating hormone(FSH) or pregnant mare serum gonadotropin(PMSG). In conclusion, OPU can be repeatedly practiced 2 or 3 times in an estrus cycle. In addition, the high environmental temperature is not good for ovarian function of cow and the bST co-treatment with FSH or PMSG is increasing the number of aspiratable follicle.

증례보고 : 진도견에서 발생한 난소기형종의 증례

신태영,이병천,김대용,권오경,황우석 (Tae Young Shin,Byeong Chun Lee,Dae Yong Kim,Oh Kyeong Kweon and Woo Suk Hwang) 한국임상수의학회 1996 한국임상수의학회지 Vol.13 No.1

컴퓨터 정액자동분석에 의한 동결융해 한우 정액의 운동특성 연구

이강남,이병천,김정태,박종임,신태영,황우석,Lee, Kang-nam,Lee, Byeong-chun,Kim, Jung-tae,Park, Jong-im,Shin, Tae-young,Hwang, Woo-suk 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.4

The aim of this experiments were to assess the time-interval change of motional characteristics in frozen-thawed semen of Korean native cattle (KNC) by using computer aided semen analysis (CASA) technology. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system (SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber (Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The measurement parameters in SIAS were as follows ; frame rate = 30 frames per sec, image capture = 1 sec, minimum motile speed = $10{\mu}m/s$, maximum countable sperm number = 400. Statistical analysis was done by Student t-test with use of the Sigma plot program on a IBM personal computer. The dancemean(DNM) and hyperactivated sperm(HYP) of frozen-thawed KNC semen kinematics were significantly decreased(p < 0.05) after 10 min of incubation at $37^{\circ}C$ water bath. But, wobble(WOB) of same sample semen was significantly increased(p < 0.05) after 10 min of incubation and significantly decrease(p < 0.05) after 60 min of same incubation. And, after 30 mim of incubation, significantly differences were found most of motion kinematics, motifity(MOT), curvilinear velocity(VCL), straight line velocity(VSL), average path velocity(VAP), amplitude of lateral head displacement(ALH), beat cross frequency(BCF), mean angular displacement(MAD), dance(DNC), on same sample semen. The DNM of KNC semen sample was variable kinematics after 30 min of incubation. Also, the linearity(LIN) and straightness(STR) was significantly decreased(p < 0.05) from 60 min of incubation. In conclusion, the AI within 30 min after thawing of frozen semen can be an effective method for obtaining high fertility rate in KNC reproductive program.

한우에서 Computer aided semen analysis(CASA) 기법을 이용한 수태율 예측에 관한 연구

이강남,이병천,황우석,Lee, Kang-nam,Lee, Byeong-chun,Hwang, Woo-suk 대한수의학회 1998 大韓獸醫學會誌 Vol.38 No.4

This experiment was conducted to predict the effects of motional characteristics on the fertility of Korean native cattle(KNC) by using CASA technology and in vitro fertilization system. Twenty-six KNC frozen semen straws were obtained from Korean KNC improvement department, livestock improvement main division, national livestock cooperatives federation in Korea. Specimens were allowed to thaw at $37^{\circ}C$ for 30 sec in water bath. Semen analysis was performed on semen image analysis system(SIAS, Medical supply, Korea) adjusted to the gate settings and used the semen droplet ($5{\mu}l$) placed on Makler counting chamber(Sefi medical instrument, Israel) prewarmed at $37^{\circ}C$. The same person used the same micropipette to fill the Makler counting chamber. A total of 150 or more of sperms were analysed in each specimen by a single trained person by scanning at least 5 to 10 fields. The oocytes collection, in vitro maturation, IVF, in vitro culture and determination of the cleavage rate were performed by the technique, as described by Hwang et al (1997). Statistical analysis was done by linear regression with use of the Sigma plot program on a IBM personal computer. The cleavage rate in vitro fertilized oocyte was significantly correlated(P<0.05) with MOT, VCL, VSL, VAP, ALH, BCF and MAD, but not CON, LIN, STR, WOB, DNM, DNC and HYP in regressional analysis. The results show that some kinematic characteristics of frozen-thawed semen by CASA can be predict the fertility in in vitro model system.

계대세포를 이용한 소 수정란의 체외배양 체계에 관한 연구

주석천,이병천,이원유,최윤석,황우석,Joo, Suk-chun,Lee, Byeong-chun,Lee, Won-yu,Choi, Yun-seok,Hwang, Woo-suk 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.3

To overcome the difficulties of collecting and culture of primary cell from genital tract on embryonic development, the present study was carried out to investigate the critical effect of cell lines, such as BRL and Vero cell and its conditioned medium on the development of early Korean native cattle embryos in vitro. Oocytes collected from slaughterhouse ovaries were matured in TCM199 containing FSH, estradiol-$17{\beta}$ and FBS with granulosa cell monolayer for 24 hours and then fertilized in vitro using frozen-thawed, heparin-treated spermatozoa in TALP for 30 hours. And then early embryos (1-2cell) were cultured in TCM199 containing 10% FBS with BOEC, Granulosa, BRL, Vera cell monolayers and conditioned medium for 2~3 days. Development to morulae and blastocysts were recorded, also examined the number of blastomeres presented a valuable parameter for the evaluation of embryonic development. The early cleavage rates of in vitro fertilized embryos co-cultured, there was no differences between primary cell and cell lines(p<0.05). The rate of development to the later stage, coculture of BRL cell was significantly higher than that of the primary cell(p<0.05). The rates of development to morula and blastocyst were significantly higher in vero cell than BRL, Granulosa, Oviduct epithelial cell conditioned medium. In the result of effect of serum on development of early bovine embryos, the use of media containing serum were significantly higher than the use of not containing one on development of early and later stage of embryos. The result of number of blastomeres in blastocysts, there is no differences between primary cell and cell lines. The blastocysts from coculture were higher than from conditioned medium in blastomere cells. In summary, these experments have proved that the culture system in TCM199 with BRL, Vero cell monolayers is effective on in vitro development of early bovine embryos, In addition, it is effective to development of bovine embryos that containing serum in conditioned medium, or in co-culture rather than in conditioned medium alone. The use of cell lines opponent to primary cells is effective in bovine embryo culture.

과배란 처치시 우세난포 조절에 의한 한우 수정란 생산성 향상에 관한 연구 II. 과배란 처치 시기에 따른 난소반응

이동원,이병천,황우석,Lee, Dong-won,Lee, Byeong-chun,Hwang, Woo-suk 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.4

This study was investigated the ovarian response following superovulation treatment at different day of diestrus. The criterion for the presence or absence of a dominant follicle based on their morphological examination. Dominant follicle was puntured 48 hrs before the oneset of superovulation treatment by ultrasonography guided aspiration needle. Superovulation was induced by subcutaneous administration of FSH twice a day for 4 day in a decreasing regimen. There was no significant different between presence of dominont follicle and progesterone concentration/diameter of corpus luteum in HanWoo. Number of corpus luteum of donor after superovulation treatment was not significantly different in FSH administration at day 9, 11 and day 13 of estrus($14.5{\pm}4.5$, $15.5{\pm}5.6$ and $11.0{\pm}5.5$, respectively). But, the diameter of CL was significantly correlate(R2 = 0.757) with progesterone levels on day of superovulatory induction. After 7 days of artificial insemination, the embryos at 7 days were collected by uterine flushing after dominant follicle aspiration and superovulation treatment, and evaluated their quality by morphological criteria. Fifty five embryos with excellent, good and fair grade were transferred into 24 recipient cows. Seventeen offsprings, 1 of triplet, 4 of twins and 6 of singlet, were yield from 10 recipient cow. In conclusion, the present study showed that 1) dominant follicle can be determined by ultrasonography with rectal palpation by morphological evaluations, 2) superovulation response after follicular aspiration was not differ at day 9, 11 and 13 of estrus, 3) dominant follicle did not affect to progesterone concentration and diameter of CL, and 4) diameter of CL was significantly correlate to the level of progesterone concentrations in HanWoo.

유전자감식에 의한 개에서의 친자감별

이항,채영진,이병천,Lee, Hang,Chae, Young-Jin,Lee, Byeong-Chun 한국임상수의학회 1998 한국임상수의학회지 Vol.15 No.2

The biological father of two Golden Retriever puppies was determined between two proposed stud dogs by using microsatellite DNA analysis. DNA was obtained from all the relevant dogs by buccal swabbing and three loci of tetranucleotide repeat microsatellite were PCR-amplifiedl and analyzed by polyacrylamide gel electrophoresis and silver staining. One of the two proposed stud dogs was assigned as the biological father of the puppies by the genotyping. The result demonstrated that the microsatellite DNA analysis is a simple, efficient method of paternity test in dogs.

핵이식을 이용한 복제송아지 생산에 관한 연구 II. 효율적인 복제수정란 생산을 위한 난자의 활성화, 공여핵의 세포주기조절 및 적정 배양조건

황우석,노상호,이병천,Hwang, Woo-suk,Roh, Sang-ho,Lee, Byeong-chun 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.3

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.