수정란이식에 의한 우 ( 牛 ) 의 쌍태유기에 관한 연구 1 . 성선자극호르몬의 투여에 대한 난소반응에 영향을 미치는 요인

정길생,이훈택,정병현,유승환,나진수 ( K . S . Chung,H . T . Lee,B . H . Chung,S . H . Yoo,J . S . Na ) 한국축산학회 1983 한국축산학회지 Vol.25 No.3

These experiments were carried out to obtain the fundemental information essential for the induction of superovulation in cattle. The effects of parity, season and the source of gonadotropins on ovarian response to gonadotropins given for superovulation were investigated. The results obtained in these experiments were summarized as follows: 1. Of 43 donor cattles treated with PMSG to induce superovulation, 86.0% responded with 2 or more corpora lutea and 55.8% with 6 to 13 corpora lutea. 2. No differences in ovarian response to PMSG administration among breeds were observed. 3. Average numbers of corpus luteum per donor cattle following PMSG administration in heifers, 1-2, 3-4 and 5 or more calved cows were 6.4, 9.3, 6.4 and 5.3, respectively. 4. No seasonal variations in ovarian response to PMSG given to induce superovulation were observed. 5. There were no significant differences in numbers of corpora lutea between the donor cattles treated with PMSG and FSH, respectively.

수정란이식에 의한 우 ( 牛 ) 의 쌍태유기에 관한 연구 6 . 비외과적으로 이식한 신선 ( 新鮮 ) 및 동결 수정란의 분만성적

정길생,윤종삼,이훈택,유승환,김정익 ( K . S . Chung,J . S . Yoon,H . T . Lee,S . H . Yoo,C . I . Kim ) 한국축산학회 1983 한국축산학회지 Vol.25 No.5

These experiments were performed to develope a simple and reliable method of bovine embryo transfer under the field conditions. Of 42 synchronous (+12 hours) recipients, 37 and 5 were received single fresh and frozen embryo, respectively, by nonsurgical method. The results obtained in these experiments were summarized as follows: 1. Of 42 embryos transferred non-surgically, 20(47.6%) survived for more than 45 days after transfer. 2. Ten healthy calves (28.6%) were produced from 35 recipients which received single embryo, respectively. 3. Of 5 frozen embryos imported from Japan, and transferred non-surgically 3 developed to calving normally. 4. Embryos were transferred to recipients in estrus 12 hours later than donor (+12), the same time (0) or 12 hours earlier than donor (-12). The 0 group showed significantly higher (p$lt;0.05) calving rate (33.3%) than +12 group (25.0%) or -12 group (25.0%). 5. Calving rates of blastocyst (31.3%) or hatched blastocyst (30.0%) were significantly (p$lt;0.05) higher than that of morula (22.2%).

수정란이식에 의한 우 ( 牛 )의 쌍태유기에 관한 연구 2 . 다배란처리의 반복이 난소반응과 수정란의 발달에 미치는 영향

정길생,박흠대,노환철,Richard A . Carmichael ( K . S . Chung,H . D . Park,H . C . Rho,Richard A . Carmichael ) 한국축산학회 1983 한국축산학회지 Vol.25 No.4

These experiments were carried out to clarify the effect of repeated superovulation in cattle on ovarian response and in vivo development of fertilized ova. Each cattle was repeatedly superovulated five times with the mixture of FSH and LH, and the growth of follicle, ovulation rate, the recovery rate of ovulated ova and in vivo development of fertilized ova following repeated superovulation were investigated. The results obtained were summarized as follows: 1. The numbers of mature follicles per cow following 1st and 5th superovulation were 20.7±8.60 and 13.6±3.64, respectively, showing the significant decrease (P$lt;0.05) in number of follicle growth with repeated hormone treatment for superovulation. 2. The percentage of ruptured follicles to the mature follicles was ranged 80.0 to 95.2%. No significant differences due to the treatment order were observed. 3. The numbers of ova recovered by non-surgical technique was ranged 60.8 to 64.5% to the number of functional corpus luteum and no significant differences were observed among treatments. The same was true for the percentage of normal embryo, which was ranged 69.5 to 72.8%.

생쥐배의 핵치환에 관한 연구 1 . 핵치환 생쥐배의 시험관내 발달

정병민(H . M . Chung),박용석(Y . S . Park),정길생(K . S . Chung),이경광(K . K . Lee) 한국축산학회 1988 한국축산학회지 Vol.30 No.8

These experiments were carried out to develop new techniques necessary for elevating utilization efficiency of high quality embryos and production of cloned animals by nuclear transplantation. Pronuclei removed from the mouse embryos of pronuclear stage by micromanipulation procedures were transplanted to enucleated mouse embryos of same development stage by similiar micromanipulation procedures. Fusion of transplanted pronuclei to the cytoplasm of recipient embryos was mediated by Sendai Vitus. Of 194 pairs of pronuclei transplanted, 126 (64.9%) pairs were fused to the cytoplasm of enucleated recipient embryos. The number of embryos developed to 2-cell embryos, morula and blastocyst after the fusion of transplanted nuclei were 108 (85.7%), 94 (74.6%) and 88 (69.8%), respectively.

염색체 분석에 의한 생쥐 분할란의 성감별

백청순,한용만,이경광,정길생,김종배,고대환 ( C . S . Baik,Y . M . Han,K . K . Lee,K . K . Chung,J . B . Kim,D . W . Ko ) 한국축산학회 1986 한국축산학회지 Vol.28 No.11

These experiments were carried out to obtain basic information necessary to produce the offsprings which were sexed by chromosomal analysis prior to transfer in the mouse. Morula stage embryos were obtained at 3 clays post-coitum. Embryos were mechanically bisected by a glass microneedle and then the pairs of demi-embryos were cultured in BMOC-3 medium under the humidified atmosphere of 5% CO₂ at 37℃ for 22-26 hours. To increase the appearance of metaphase, bisected embryos normally developed to the blastocyst were cultured in MOC-3 medium containing colcemid (0.04㎍/㎖) under the same condition for 3 hours. One of both demi-embryos was sexed by chromosomal analysis and the other was transferred to the uterine horn of a foster mother. The results obtained in these experiments were summarized as follows; 1. Of 241 pairs of demi-embryos cultured for 22-26 hours, 162 pairs (67.2%) were normally developed to the blastocysts. In the control groups, the percentage of intact and zona-free embryos normally developed to the blastocysts was 96.5% and 93.9%, respectively. 2. The mean Mitotic Index of 121 demi-embryos subjected to chromosomal analysis was 11.3±13.2%r and 18 demi-embryos (14.9%) were sexed. 3. Of 18 demi-embryos transferred to recipients after sexing, 2 offsprings were born and their sex was corresponded to the sex determined by chromosomal analysis.

Cryopreservation of Human Multi-Pronuclear (PN) Zygote by Ultra-Rapid Freezing

김은영,이봉경,남화경,이금실,윤산현,박세필,정길생,임진호,Kim, E.Y.,Yi, B.K.,Nam, H.K.,Lee, K.S.,Yoon, S.H.,Park, S.P.,Chung, K.S.,Lim, J.H. The Korean Society for Reproductive Medicine 1998 Clinical and Experimental Reproductive Medicine Vol.25 No.2

본 연구는 인간 전핵기 수정란의 동결시 election microscope grid론 사용하는 초급속 동결방법이 유용하는지 여부를 조사하고자 실시하였다. 본 실험에서는 인간 IVF 시술시 생성되는 다-전핵기 (>2PN) 수정란을 정상 2PN 수정란 대신 사용하였으며, 또한 이들은 3PN과 $\geq4PN$ 수정란으로 나누어 전핵의 수에 따른 냉해와 융해 후 체외 배발달율을 조사하였다. 동해제는 30% ethylene glycol, 18% ficoll, 0.5 M sucrose와 10% FBS 등이 D-PBS에 첨가되어 제작된 EFS30을 사용하였다. 본 연구에서 얻어진 결과는 다음과 같다. 초급속동결-융해 후, 인간 다-전핵기 수정란의 생존율은 85.5%였다. 대조군과 동결군의 난할율을 진핵의 수에 따가 니누어 비교하였을 때, 각 군간에는 유의한 차이를 나타내지 않았다 (3PN; 81.3%와 85.4%, $\geq4PN$; 90.0%와 95.7%). 또한, 융해 후 체외발달율을 조사하였던바, $\geq4PN$수정란에서는 동결군 (4.5%)의 체외발달이 대조군 (44.4%)보다 유의하게 낮게 나타났던 반면, 3PN 수정란에서는 동결군 (22.0%)의 결과가 대조군 (38.5%)에 비해 유의한 차이를 나타내지 않았다. (p<0.05). 따라서 인간 다-전핵기 수정란은 EM grid와 EFS30 동결액을 이용한 초급속 동결로 배발달을 유도할 수 있다는 것을 알 수 있었다. The objective of this study was to test whether the developmental capacity of human multi-pronuclear (PN) zygotes after ultra-rapid freezing using EM grid can be maintained. For this experiment, multi-PN zygotes which produced in human IVF program were used as an alternatives of normal 2PN zygotes, and they were separated into 3PN or $\geq4PN$ zygotes to compare their in vitro development and cryoinjury according to PN number. As freezing solution, EFS30 which consisted of 30% ethylene glycol, 18% bcoll, 0.5 M sucrose and 10% FBS added D-PBS was used. The result obtained in this experiment was summarized as follows; When the multi..PN zygotes were ultrarapidly frozen and thawed, the high mean percentages (85.5%) were survived. Also when the cleavage rates between control and freezing group were compared with PN number, there were not significantly different in each group (3PN; 81.3% & 85.4% and $\geq4PN$; 90.0% & 95.7%). When the in vitro development rates after thawing were examined, freezing 3PN group (22.0%) was not differed to control 3PN group (38.5%), although the development result of freezing $\geq4PN$ group (45%) was significantly lower than that of control $\geq4PN$ group (44.4%) (p<0.05). These results demonstrate that developmental capacity of human zygote can be obtained by ultra-rapid freezing method using EM grid and EFS30.

우 동결수정란의 산업적 이용에 관한 연구

노환철(H . C . Rho),정광업(K . E . Chung),신규용(G . Y . Shin),정병현(B . H . Chung),백운화(U . H . Pek),정길생(K . S . Chung) 한국축산학회 1988 한국축산학회지 Vol.30 No.3

To establish the industrial utilization of frozen bovine embryo, bovine embryos were frozen to -196℃, storaged, thawed and transferred. The results obtained from these experiments are summarized as follows : 1. Superovulation was induced from 27 Holstein donor cows. The average numbers of corpus luteum per donor cow was 10.3. The egg recovery rate was 56.3%, and the average number of recovered embryo was 4.6 per donor cow, 2. The pregnancy rates of frozen embryos by embryonic stages such as late morula, early blastocyst and blastocyst were 37.5%, 50.0%o and 42.9%, respectively, and those of fresh embryos were 55.0℃, 64.3% and 69.2%, respectively. 3. When fresh and frozen embryos were transferred to recipients whose estrus were naturally induced, the pregnancy rates were 53.3% and 36.8%, respectively. However, the pregnancy rates were 65.7% and 66.0%, respectively when fresh and frozen embryos were transferred to recipients whose estrus were artificially induced by PGF₂α. 4. When fresh and frozen embryos were transferred into tip of uterine horn, the pregnancy rates were 77.4% and 72.4%, respectively. On the other hand, pregnancy rates were 31.3% and 21.4%, respectively when fresh and frozen embryos were transferred into the bottom of uterine horn.

생쥐에 있어서 Ethanol 처리에 의한 단위발생의 유기

한용만(Y . M . Han),백청순(C . S . Baik),이경아(K . A . Lee),이경광(K . K . Lee),정길생(K . S . Chung) 한국축산학회 1987 한국축산학회지 Vol.29 No.9

These experiments were carried out to obtain the basic informations and to establish the best conditions needed for the activation of mouse eggs by ethanol treatment. The influence of ethanol concentration and the age of eggs, time from hCG injection to recovery, on activation frequency of eggs was investigated, To activate the mouse eggs parthenogenetically, cumulus masses including eggs were released into a fresh solution of ethanol for 5min and then rinsed out 5-6 times with ethanol-free medium. After incubation of cumulus masses in the drops of medium for 5 hrs at 37℃ in the humidified atmosphere of 5% CO₂ in air, the adherent cumulus cells were removed with hyaluronidase (100unit/㎖). The types of parthenogenetic eggs were morphologically classified into haploid (H), immediate cleavage (IC) and diploid (D) under a inverted microscope. These parthenogenetic eggs were then incubated into the drops of mKRB medium for 96 hrs under the same conditions as above. The results obtained in these experiments were summarized as follows : 1. ICR female mice (1) When the eggs were treated with a variety of ethanol concentrations, 0 to 11%, the highest parthenogenetic activation rate (94.4%) was observed at 7% ethanol concentration. (2) Activation frequency was comparatively high when the eggs were recovered from the oviducts in more than 18 hrs after hCG injection. 2. (C₃H × C57BL) F₁ hybrid female mice (1) Of 341 F₁ eggs treated with 7% ethanol for 5 min at 18 hrs after hCG injection, 317 eggs (93.0%) were activated and a majority of parthenogenetic eggs were haploids (71.9%). (2) After 96 hrs of in vitro culture, the developmental rate of haploid, immediate cleavage and diploid eggs to morphologically normal morula or blastocyst stage was 16.7, 66.7 and 72.3%, respectively. (3) Of 18 haploid eggs recovered from the uteri on the 4th day of pregnancy after transfer to the oviducts of recipients, 12 eggs (60.0%) were normally developed to morula or blastocyst stage.

생쥐 수정란의 양분에 의한 일란성 쌍태의 생산

김남형,정길생,노환철,백운화,이경광 ( N . H . Kim,K . S . Chung,H . C . Rho,U . H . Pek,K . K . Lee ) 한국축산학회 1986 한국축산학회지 Vol.28 No.8

These experiments were carried out to produce monozygotic twin by bisection of mouse morula. The morula was bisected by microglass needle without any aid of microinstrument or by lateral incision using sharp blade attached to micromanipulator. Bisected dermi-embryos were cultured and transferred to pseudopregnant recipient mouse. The results obtained in these experiments were summarized as follows: 1. Of 285 decompacted morulae bisected by microglass needle, 180 embryos were divided into two demi-embryos, respectively, without any visible damage. 2. Total 180 pairs of demi-embryos separated from decompacted morula and subjected to in vitro culture were resulted in 85(47.2%) pairs of eu-blastocyst, 40(22.2%) pairs of eu-blastocyst and pseudo-blastocyst, 35(19.4%) pairs of pseudo-blastocyst and 20(11.1%) pairs of trophectodermal vesicle and degeneration. 3. Of 255 intact morulae bisected by microblade, 100(39.2%) embryos were divided into two demi-embryos, respectively, without damage. However, 95(37.3%) embryos were bisected into one normal and one damaged demi-embryos, respectively. The percentage of demi-embryos developed to blastocyst after in vitro culture with and without zona pellucida were 71.4 and 64.9% respectively. 4. Total 15 twins were produced following transfer of the 38 pairs of eu-blastocysts developed from demi-embryos to 25 recipients. However, none of pseudo-blastocyst gave birth to young, 5. The percentage of eu-blastocyst cultured with or without zona pellucida and developed to live young following transfer were 11.1 and 9.3% respectively.

X- 정자와 Y- 정자의 분리에 관한 연구 2 . Percoll 중층원심분리법에 의한 인간정자의 분리

엄기붕(K . B . Oum),이주영(J . Y . Lee),고대환(D . H . Ko),정길생(K . S . Chung) 한국축산학회 1988 한국축산학회지 Vol.30 No.5

These experiments were carried out to develop new techniques for in vitro separation of X-and Y-bearing spermatozoa. One ㎖ of washed human sperm suspension was loaded on the isotonic discontinuous Percoll density gradient, and then it was centrifuged at 250×G for 25 min. After centrifugation, spermatozoa were fractionated according to Percoll density gradient. Spermatozoa included in each fraction were subjected to the estimation of motility, morphological abnormality, F-body test, and recovery rate of spermatozoa was also investigated. The results obtained in these experiments were summarized as follows: 1. Following centrifugation of discontinuous Percoll density gradient, population of spermatozoa increased progressively from low density to high density. The highest concentration of spermatozoa was observed in 7th fraction which included 20% of spermatozoa. 2. High percentage of motile spermatozoa was observed at high Percoll concentration and the highest percentage was obtained at 6th fraction. 3. Following Percoll centrifugation, percentage of X-sperm increased from 53.2% (control) to 74.1% (7th fraction). 4. Following centrifugation, sperm abnormality was increased at low Percoll gradient and decreased at high Percoll gradient. The lowest abnormality (15.0%) and the highest abnormality (46.0%) were observed at 7th fraction and 1st fraction, respectively.