투명대 존재/부재 햄스터 난자의 동결보존;1-단계 평형과 2-단계 융해의 효과

정구민,방명걸,김석현,신창재,김정구,문신용,이진용,장윤석,Chung, K.M.,Pang, M.G.,Kim, S.H.,Shin, C.J.,Kim, J.G.,Moon, S.Y.,Lee, J.Y.,Chang, Y.S. 대한생식의학회 1992 Clinical and Experimental Reproductive Medicine Vol.19 No.2

The present experiments were focussed to modify a short slow-cooling protocol used for freezing of early stage embryo(Testart et al., 1986) and also to apply the modified method for the cryopreservation of hamster oocytes with Zona or without. The protocol was modified by changing the 4-step equilibration into 1-step and the 1-step thawing into 2-step. The oocytes were added in 1.5M PROH and 0.1M Sucrose, seeded at $-7^{\circ}C$, slow cooled($0.3^{\circ}C$/min) to $-30^{\circ}C$ before plunging to $-196^{\circ}C$. The oocytes were thawed at $23-25^{\circ}C$ air(20sec/150sec) and/or 33-35 water(10sec). The survival of the frozen-thawed oocytes was determined by morphologic criteria and their fertilizing ability was also estimated by Sperm Penetration Assay(SPA) system(Chang et al, 1990) using fertile men semen sample. One-step equilibration showed slightly higher survival rate(83.9% vs. 71.0%) and fertilization rate(83.9% vs. 71.0%) compared with four-step(p>0.05). And two-step thawing(air & water exposing) of oocytes frozen after 1-step equilibration showed significantly higher survival rate(96.3%) than one-step thawing at air(85.2%) or water(65.0%) only(p<0.05). Therefore, by the modified method(l-step equilibration & 2-step thawing), Zona-intact(ZI) and Zona-free(ZF) oocytes were frozen and thawed. ZI-oocytes showed significantly higher survival rate(95.4%, 308/323 vs. 67.6%, 240/355) than ZF-oocytes(P<0.01). But the survival of ZF-oocytes was as high as ZI-oocytes in fourteen of twenty-four replicates. ZI-oocytes was also significantly higher fertilization rate($92.4{\pm}8.9%$ vs. $63.7{\pm}18.5%$) and higher mean number of penetrated sperm($6.2{\pm}4.2$ vs. $3.9{\\pm}3.3$) than ZF-oocytes, but not higher than control(fresh oocytes;$99.3{\pm}2.4%$, $8.4{\pm}4.2$)(P<0.001). Conclusively, this modified method will contribute to freeze effectively the hamster oocytes for simplifing of the logical consideration of performing SPA and also to freeze the human and other animal oocytes.

생쥐 2-세포배아에 의한 시험관아기 배양용 대아제대혈청의 절적평가에 관한 연구

문신용,신창재,정구민,오선경,방명걸,장윤석,Moon, S.Y.,Shin, C.J.,Chung, K.M.,Oh, S.K.,Pang, M.G.,Chang, Y.S. 대한생식의학회 1989 Clinical and Experimental Reproductive Medicine Vol.16 No.2

The purpose of this study was to examine the qualitative variation of human fetal-cord sera (HCS) and to accept the sera in human lVF-ET program. One hundred and sixteenth RCS were tested with 1772 2-cell embryos of F1 (C57BL x CBA) virgin mice, Ten to sixteenth embryos were cultured in m-KRB medium with a aliquot of each serum (10%, v/v) or with bovine serum albumin(O.4%, w/v) as a control medium. Embryonic development were recorded at every 24hr for 4 days by such events as cellular compaction, cavitation, and hatching. In the control groups of eight assays, 98.1%(106/ 108) of 2-ce1l embryos developed above expanded blastocyst and the embryonic development was unified through the tests. But the developmental pattern in medium with each serum was various. Namely, the sera that supported development of 100% 2-cell embryos to above morula, early blastocyst, expanded blastocyst and hatching blastocyst was 45,7%(53/116) , 35.3%(41/116), 15.5%08/116.) and 6.9-%(8/116), respectively. And the sera that supported development of above 80% 2-cell embryos to the each embryonic stage was 92.2% (107/116), 83.6%(97/116), 63.8%(74/116) and 36.2%(42/116), respectively. Meanwhile two kinds of toxic pattern to the embryonic development were observed in some sera. The first pattern is that some sera arrested development of most embryos in pre- or post-stage of morula or blastocyst. The second pattern is that some sera promoted or arrested a part of embryos in the same dish. The ability of serum was depended on the batch of serum. Finally we could accept 69%(80/116) of the tested sera for human IVF-ET program. The base line for acceptance was the ability that supported above 80% 2-ce1l embryos to blastocyst. But some deterious sera were contained in this range. We cut off about 10% of the sera (83.6% , 97/116) that passed the baseline. This final percent of sera was similar to that of grade N of this study.

미성숙 및 성숙미경산우의 다배란 처리가 난소반응 , 수정란 생산과 수태에 미치는 영향

김종국(J . G . Kim),정구민(K . M . Chung),임경순(K . S . Im),이용빈(Y . B . Lee) 한국축산학회 1988 한국축산학회지 Vol.30 No.5

This experiment was carried out to investigate the effects of superovulation on the ovarian response and non-surgical recovery in immature and mature heifers. The possibility of nonsurgical recovery from the immature heifers and pragnancy of embryos transferred to mature recipients was also investigated. Total 8 heifers (S heads of Holstein, 2 heads Korean Native Cattle (KNC) and 1 head of Angus × Hereford crossbred) were devided into 2 groups by their age. Immature heifers (Group I) were 7-10 months old and mature heifers (Group II) were 12-18 months old. Heifers of Group I and Group II were superovulated with muscular injection of 1,000 or 2,000 IU Pregnant Mare`s Serum Gonadotropin (PMSG) on day 10-12 of estrous cycle followed by muscular injection of 25㎎ PGF₂α 48h later. Heifers were artifically inseminated or copulated 2 to 3 times with the interval of 12h while they were in heat. The embryos were collected non-surgically on day 6 of estrous cycle. The results obtained from this study were summarized as follows: 1. The average number of corpus lutea (CL) by the superovulation treatment was much greater in Group II (9.2) than in Group I (3.2). 2. The average number of unruptured follicles by superovulation treatment was much grater in Group II (6.8) than Group I (1.0). 3. The interval to onset estrus after PGF₂α injection was longer in Group I (44.2) than in Group II (31.8h). 4. There was no difference in the duration of estrus between Group I (32.0h) and Group II (37.5h). 5. It was possible to flush the uterus of 7 month old KNC heifers non-surgically. 6. The average number of embryos recovered was much greater in Group II (6.0) than in Group I (2.3) and the recovery rate of embryos in Group I (73.6%) was similar to that Group II (87.3%). More flushing fluid was recovered in Group I (93.0%) than in Group II (87.3%). 7. Twelve of 14 embryos recovered (87.5%) were normal in Group I, while 17 of 54 embryos (31.5%) were normal in Group II. 8. All of the normal embryos (12/12) were morula in Group I, while 82.4% (14/17) of the normal embryos were monala and 17.6%o (3/17) were blastocysts in Gooup II. 9. The estrous cycle after superovulation treatment tended to become longer with the average of 24.8 days in Group I and 24 days in Group II. Some donor heifers had cystic ovaries. 10. Six embryos were transferred to 4 recipients non-surgically. One recipient who had received 2 morula that were recovered from a 10 month old KNC-2 became pregnant and delivered a male Korean native calf of 27 Kg.

발정혈청 및 양수가 소 난포란의 체외성숙에 미치는 영향

임경순(K . S . Im),김형선(H . S . Kim),정구민(G . M . Chung),박연진(Y . J . Park),김현종(H . J . Kim),박광욱(K . W . Park) 한국축산학회 1993 한국축산학회지 Vol.35 No.3

These experiments were carried out to investigate the effect of addition of estrous cow serum(ECS), amniotic fluid(AF) and AF + FCS on maturation of bovine oocytes in vitro. The oocytes with cumulus cells and unfragmented cytoplasm were used. The oocytes were matured in vitro for 24 hr in a CO₂ incubator with 5% CO₂ in air at 38.5℃. The medium used for maturation was TCM 199 and Ham`s F10 supplemented with hormones and antibiotics. The results obtained are as follows : I. When the oocytes were cultured in TCM (99 medium, maturation rate of oocyte in FCS 10%. ECS 5, 10, 15 and 20% was 70.0, 74.4, 75.0, 78.1 and 58.8%, respectively. There were no significant (p$gt;0.05) difference among the treatments. 2. When the oocytes were cultured in Ham`s F10, maturation rate of oocyte in AF 0, 10, 50 and 100% was 63.0, 87.3, 63.6 and 7.8%, respectively. AF 10% showed significantly (p$lt;0.05) higher maturation rate than others. 3. When the oocytes were cultured in Ham`s F10, maturation rate of oocyte FCS + AF showed significantly (p$lt;0.05) higher maturation rate(76.9%) than FCS only (50.8%).