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.

소의 품종 , 배양액의 첨가제 및 발정주기가 난포란의 체외성숙 , 수정 및 발생에 미치는 영향

정구민(K . M . Chung),임경순(K . S . Im) 한국축산학회 1988 한국축산학회지 Vol.30 No.2

This experiment was conducted to investigate the effects of breeds, medium supplements and ovarian phases on the developmental pattern of bovine follicular oocytes. Ham`s F10 powder was dissolved in the 3rd demineralized and 5th distilled water and supplemented with 10% heat inactivated fetal bovine serum (HI-FBS) or 0.4% bovine serum albumin (BSA). The medium was equihlmated for 16±2 hours in 5% CO₂ incubator and adjusted in pH 7.4-7,6 and in 280±10 mOsm. The oocytes were recovered from small antral follicles (2 to 5㎜) and matured for 24 to 26 hours at 39℃. Matured oocytes were fertilized with ejaculated sperm (1-2 x 10^6/㎖) and cultured for 4 days at 38.5℃ in 5% CO₂ in air. The cleavage rate of oocytes was higher in Korean native cattle (KNC; 22.0%, 18/82) than ins Holsteins (4.4%, 4/93). Eleven among eighty two (13.2%) of KNC oocytes developed to morula stage, but none of Holstein oocytes cleaved up to 8 cells. Of the developed ova, rate of embryos with regular blastomeres was also higher in KNC (∼65%) than in Holsteins (∼33%). Holstein oocytes were fertilized with KNC sperm to clear the reason why the developmental potential of Holstein oocytes was extremely limited. Their cleavage rate was 16.7%o (4/24) and was higher than that of Holstein oocytes fertilized with Holstein sperm. Therefore the low potential in vitro of Holstein oocytes was due to oocytes and not to sperm. Cleavage rate of KNC oocytes was higher in FBS supplemented medium (22.0%, 18/82) than in BSA supplemented medium (14.8%, 4/27). The oocytesdeveloped to morula stage in FBS-medium, but non-cleaved up to 3 cells in B SA-medium. Of the developed ova, rate of embryos with regular blastomeres is higher in FBS-medium (∼65%) than in BSA-medium (∼33%). Of the uncleaved 1-cell ova, rate of fragmental ova was significantly higher in BSA-medium (33.3%) than in FBS-medium (14.6%). By contrast, the rate of deformed ova was significantly lower in BSA-medium (7.4%) than in FBS-medium (25.0%). The cleavage rate of oocytes was not different between luteal phase and follicular phase, but the rate of embryos with regular blastomeres was higher in luteal phase than in follicular phase. In conclusion, the rates of cleavage and normal development of follicular oocytes were higher in KNC than in Holsteins, in FBS-medium than in BSA-medium, and in luteal phase than in follicular phase. The cleavage rate of oocytes was related to the rate of normal development. Immature-follicular oocytes of KNC can normally mature and develop to morula stage in Ham`s F10 supplemented with 10% FBS but that of Holsteins can not.

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%).

혈액세포를 이용한 염색체 분리 분석에 관한 방법적 고찰

손시환,정구민,Sohn, S.H.,Chung, K.M. 대한생식의학회 1994 Clinical and Experimental Reproductive Medicine Vol.21 No.2

This study was carried out to develop the methodology of chromosome preparation from blood cultures in mammals which included human, mouse, cattle and pig. For karyotyping, 0.5-5.0ml of peripheral blood were collected and cultured. The satisfactory results were obtained from macroculture and microculture in all species. In culture, the patterns of cell growth were no difference among media except serum concentration and mitogen supplement. The presence of mitogen and fetal bovine serum in medium significantly affected the mitotic index. The optimal culture condition was 37$^{\circ}C$ for 3 days. And the concentration of colcemid and reincubation time also affected the chromosome morphology. In harvest, chromosome patterns were mainly affected on hypotonic treatment which included treated time and temperature, dropwise of fixative solution, and drying after slide preparation.

토끼 할구 융합배의 H-Y 항체에 의한 성감별에 관한 연구 1 . 초기배 절단에 의한 자토 생산

임경순(K . S . Im),최화식(H . S . Choi),정구민(K . M . Chung),양보석(B . S . Yang) 한국축산학회 1992 한국축산학회지 Vol.34 No.3

These experiments were carried out to examine development capacity of bisected blastomere by a simple hand method without micromanipulator from 4-cell to morula stage in rabbit. The results obtained from these experiments are as follows: 1) When rabbit embryos were bisected by hand method, both halves undamaged rate was 37.5(3/8). 65.5(33/52) and 65.2(43/66) in 4-8, 16-cell and morula stage, respectively. 2) When demi embryos were cultured, the percentage of bisected embryos developed into blastocysts was 75, 117 and 123% in 4-8, 16-cell and morula stage. respectively 3) When 30 demi-embryos from 16-cell and 70 demi-embryos from morula were transferred into 3 and 7 recipients, 3/30(10) and 6/70(8.9%) young obtained, respectively.

H-Y 항체에 의한 토끼배의 성조절에 관한 연구 1 . 배의 발달과 형광 발현에 의한 자 웅 수정란의 분리

이창규(C . K . Lee),정구민(K . M . Chung),김수헌(S . H . Kim),임경순(K . S . Im) 한국축산학회 1990 한국축산학회지 Vol.32 No.7

Antisera to histocompatibility (H-Y) antigen were used to immunologically presume the sex of rabbit embryos. H-Y antisera were prepared in inbred SD female rat by repeated immunization of spleen cells from males of same strain. The titre of H-Y antibody in antiserum was examined by mouse sperm cytotoxicity and biological tests. Experiments applied delaying ability of development of embryos in H-Y antiserum and binding ability of FITC labelled second antibody. After culture, embryos were observed their morphological characteristics under phase contrast microscope and detected fluorescence on embryos under fluorescence microscope. After detection of fluorescence, embryos were transfered to normal medium and observed their morphological characteristics. 1. When rabbit morula were treated with H-Y antiserum only, the rate of developed and delayed embryos was 47.2 and 52.8% respectively, and the rate of non-fluorescing and fluorescing embryos was 51.4 and 48.6%, respectively. 2. When rabbit morula were cultured in H-Y antiserum followed by complement, the rate of non-fluorescing and fluorescing embryos was 53.6 and 46.4%. respectively. 3. After detection of fluorescence, the embryos were cultured in normal medium. When embryos were treated with H-Y antiserum only, the rate of arrested and developed embryos was 20.8 and 79.2% respectively. However, when embryos were treated with H-Y antiserum followed by complement, the rate of arrested and developed embryos was 42.9 and 57.1% respectively.

소에 있어서 비외과적 방법에 의한 수정란의 채란 기술개발에 관한 연구

임경순,이용빈,정구민 ( K . S . Im,Y . B . Lee,K . M . Chung ) 한국축산학회 1983 한국축산학회지 Vol.25 No.3

These experiments were conducted to develop embryo collection by nonsurgical methods and. establish basis of embryo transfer in bovine. The recovery rate of embryos between natural and superovulated cows, the ovarian response following 3 to 4 times repeated uses of 2000 IU PMSG with some intervals, cleavage stage of embryo and expansion of uterine cervix with laminaria were studied with korean native cows. The results obtained were as follows: 1. Laminaria No. 1 was expanded into 3.2 times in diameter within 3 hrs and 4 times in diameter within 4 hrs when it was set in warm water at 38℃ and uterine cervix, respectively. After removing it from uterine cervix, foley catheters (18-22FR) were easily inserted into uterus without rectal manipulation. 2. Average numbers of corpus lutea on 9-12 days following estrus induced with 2000 IU PMSG in I, II, III and IV replications taking for 60±15, 90±15 and 150 days intervals among replications were 6.0, 10.3, 7.3 and 7.0, respectively. Accordingly, appropriate interval between replications is thought to be 3 to 4 months and can be shorter or extended according to variation of estrus cycle in the individual. 3. Ovarian response following PMSG injection was greater in fall than in summer and winter. 4. The cows showed standing estrus at average 71±2 hrs following PMSG injection and 85% of the cows showed the estrus between 3 to 4 days following PMSG injection. 5. 92.6% of the flushing fluid was recovered by flushing and 24 eggs (34.3%) among 70 corpus lutea were collected from the superovulated cows. 6. The recovery rates of embryos from I. II, III and IV replications were 50, 3.6, 8.6 and 100%, respectively. The shorter the interval between replications was, the lower the recovery rate of embryo was. 7. Six embryos (100%) were collected from the six cows in natural ovulation by nonsurgical method. 8. Cleavage stages of embryos recovered respectively on 5,7,8,10 and 15 days following estrus ware 16-cell, morula, tight morula, early hatching blastocyst and late hatching blastocyst, respectively. 9. Abnormal, non-fertilized and broken ova among 30 ova recovered were 3(10%), 2(6.7%) and 2(6.7%), respectively. 10. In case of cows in natural ovultion, embryo was not recovered in 1S fractions obtained only by flushing, but mostly recovered in 2nd to 5th factions obtained by flushing with massage of uterus. 11. The embryo recovered on the same day following estrus by PMSG showed 1½±½ difference in cleavage stage between Holstein and Korean native cows and also showed 1±½ difference between the natural and superovulated cows.

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.

건고추의 생산과 유통과정 및 품질조사

황재문(Hwang J. M),정구민(K. M. Chung) 한국원예학회 1995 한국원예학회 학술발표요지 Vol.13 No.2

경북지역 재래종고추의 생육특성 및 건고추 품질평가

황재문(Hwang J. M),정구민(K. M. Chung) 한국원예학회 1995 한국원예학회 학술발표요지 Vol.13 No.2