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.

한우정자의 일반성장 , 보존성 및 내동성에 관한 연구

임경순,서국성,김중계,설동섭,이용빈 ( Kyung S . Im,Kug S .Suh,Jung K . Kim,Dong S . Sul,Yong B . Lee ) 한국축산학회 1975 한국축산학회지 Vol.17 No.3

Semen were collected from 20 Korean bulls raised in Kyunggi Province with artificial vagina and quality, preservability in liquid state with and without glycerol and freezability were investigated. The results obtained in this study were summarized as follows: 1. Volume of semen, initial sperm motility index, number of sperm per ㎖ of semen, abnormality, pH and mounting times till ejaculation was 4.4㎖, 83.7, 10.3×10^8, 13.6%, 6.4 and 2.2, respectively. 2. In the case of liquid semen stored at 5℃ for 7 days, sperm motility index of diluted semen with glycerol was higher than that of diluted semen without glycerol. 3. Freeze-thawed semen which was stored at 5℃ for 6 days decreased slightly its sperm motility index during 1 and 2 days, but rapidly thereafter. 4. Forty-five percentage of the bull ejaculated showed more than 41 sperm motility index after freeze-thawing.

Prostaglandin F2α 근주 ( 筋注 ) 에 의한 홀스타인 젖소의 발정동기화에 관한 연구

임경순,박충생 ( K . S . Im,C . S . Park ) 한국축산학회 1983 한국축산학회지 Vol.25 No.1

For induction of estrus from the respeat breeders and silent heat cows, 25 or 50 ㎎ of PGF₂α was intramuscularly injected and symptoms of estrus such as vaginal swollen, mucus state, existence of mounting and receptive standing conception rate and return intervals were observed. The results were as follows: 1. Eleven (78.5%) of 14 cows treated showed estrus and ten (90.9%) of the eleven observed estrus on day 3 after treatment. 2. Estrus was induced in five of the seven cows with silent heat and in six of the seven cows with repeat heat. 3. Ten cows in heat following the treatment showed the same vaginal redness and swollen cervix as those of natural heating cows. 4. A considerable amount of mucus in vigina was observed in the four cows. 5. Only three cows showed the sexual behavior of mounting and receptive standing during heat. 6. Three cows were conceived at the induced estrus and calved these after. 7. Four cows showed return intervals of 18-25 days. This investigation was supported by grant of PGF₂α from The Upjohn Company, Kalamz-zoo, Michican, U.S.A.. I sincerely thank Dr. S.H. Langford, Pacific region technical manager for his effort of granting PGF₂α and his deep consideration of this work.

돈정액의 동결보존에 관한 연구 1 . 보존액의 조성및 동결조건이 융해후 돈정자의 생존성에 미치는 영향

임경순,정장용 ( K . S . Im,J . Y . Chung ) 한국축산학회 1978 한국축산학회지 Vol.20 No.6

In this experiment effects of straw-size, glycerol equilibration time, thawing temperature, centrifugation of semen, kinds of diluent and addition of ethylene diaminetetraacetic acid 2 Na (E.D.T.A), egg yolk, skim milk and sugar on motility and livability of frozen boar semen were studied. The results obtained from this studies were as follows: 1. The highest post-thawing livability was obtained from the diluted semen exposed to glycerol for 4 hours and from the semen thawed at 40℃ rather than at 10℃. 2. The boar semen in 0.5㎖ straw showed higher post-thawing livability than the semen in 1㎖ straw. 3. 0.2 percent of E.D.T.A and 10 to 20% of egg yolk in the diluent showed higher post-thawing livability between 10 and 20% of egg yolk in diluent. 4. The basic egg yolk tris buffer was modified in this experiment as follows: Trisarminomethane 3.078g, citric acid 1.780g, glucose 0.3g, fructose 0.3g, Glycine 0.48g, catalase 0.02g, E.D.T.A. (Na₂) 0.2g and egg yolk 20㎖ were adjusted to 100㎖ distilled water. One mg of streptomycin arid 1,000 I.U. of penicillin per ㎖ were added to this diluent. 5. The boar semen in skim milk diluent without glycine showed higher post-thawing livability than the semen in skim milk with glycine. 6. The highest post-thawing livability was obtained from the semen diluted with diluent containing 5 to 7% of skim milk and 6% of glucose. 7. The semen diluted with skim milk extender containing 3% of glucose and fructose respectively showed highest post-thawing livability. 8. The basic skim milk diluent was modified in this experiment as follows: skim milk 5g, glucose 3g, and fructose 3g were adjusted to 100㎖ distilled water.

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.

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%).

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.

생쥐와 흰쥐배의 세적 분리에 관한 연구

오성종,임경순,이용빈 ( S . J . Oh,K . S . Im,Y . B . Lee ) 한국축산학회 1983 한국축산학회지 Vol.25 No.4

This study was carried out to investigate the cloning of mouse and rat embryos. Three-hundred females of DDY and ICR strain mice and fifty females of Wistar strain rats were superovulated by intramuscle injection of PMS and HCG. Two-, four-, and eight-cell embryos were dichotomized in vitro by fine glass, needle with hand following the papaya protease digestion of zona pellucida. The results obtained were summarized as follows; 1. The mice were superovulated by injections of 5 IU of PMS and HCG. One, two, and four to eight-cell embryos were mostly obtained from the oviduct at 18 to 24, 48 and 72 hours after injection of HCG. In rat, at 48 hours after HCG, two-cell embryo was 66%, but at 72 hours, four-cell embryo, 44% and eight-cell embryo, 40%, respectively. 2. The percentage of mouse and rat showed superovulation after PMS and HCG treatment was 44 and 30%, respectively. The average number of embryos ovulated per head was 13.7 ± 4.6 and 25.9 ± 8.3 in mouse and rat. There was highly significant differences in the ovulation response and the number of ova ovulated per head between breeds. 3. The rate of dichotomized two-cell embryo in control, zona softened and removed was 0, 48 and 95% in mouse and 0, 44 and 58% in rat, respectively. Rate of the embryos dichotomized without damaged blastomere was 40 and 93% in zona softened and removed, respectively. 4. The rate of dichotomized four-cell mouse embryo was 0, 64 and 96% in control, zona softened and removed and the rate of embryo dichotomized with out damaged blastomeres was 36 and 46%, respectively. The rate of dichotomized four-cell rat embryo in control, zona softened and removed was also 0, 56 and 63%, respectively. The dichotomized rate of embryo was lower in rat than that in mouse. 5. The dichotomized rate of 8-cell mouse embryos was 60 and 82% in zona softened and removed and showed lower dichotomized rate than that of two- and four-cell embryos. In eight-cell mouse embryo, the rate of embryo dichotomized with out damaged blastomees was 17 and 34% in zona softened and removed.

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.

동해방지제 , 식빙 , 동결속도 및 보존기간이 생쥐 초기배의 생존성에 미치는 영향

진동일,임경순,오봉국,이용빈 ( D . I . Jin,K . S . Im,B . K . Ohh,Y . B . Lee ) 한국축산학회 1986 한국축산학회지 Vol.28 No.7

To study the factors affecting cryopreservation of mouse embryos, 6-12 week old ICR mice were used. Embryos were dehydrated in IPBS medium containing DMSO or Glycerol by 3 step procedure and cooled to -50℃ at different cooling rates (1.0, 3.0, 5.0, 7.0 and 10.0℃/min) and plunged into liquid nitrogen (-196℃) and stored for 12hrs to 30 days. Embryos were thawed in air to room temperature at rate of about 20℃/min and rehydrated by 3 step procedure. When the embryos were dehydrated in the medium containing cryoprotectants, then survival rates were decreased compared to embryos which did not dehydrated, and same trend was noted when the ice nucaeation of embryos was induced at -5℃. The survival rate of embryos dehydrated or cooled to -5℃ showed no difference between 8-cell and morula stage embryos. The survival rate of embryos was best (45%) when the cooling rate was 1℃/min, but it was decreased when the cooling rate was increased. Embryos could be stored up for 15-30 days without significant decrease in their survival rate when they were cooled at 1℃/min to -50℃ and stored in -196℃.