산양의 발정주기의 생리에 관한 고찰

박충생 韓國受精卵移植學會 1983 한국동물생명공학회지 Vol.1 No.1

산양의 발정주기의 생리와 조절에 관한 최근 연구 보고들과 한국 재래산양의 대한 이들 연구 결과를 모아 고찰하였다. 온대지역에서 산양의 번식계절은 분명하나 한국재래산양의 경우는 비번식계절이 비교적 짧거나 계절적 무발정율이 낮은 편이며, 비번식계절에는 기능적 황체가 유지되지 않는다. 한국재래산양의 발정주기와 발정지속기간은 타품종과 비슷하며, 발정지속기간의 반복력은 매우 낮으며, 발정주기중의 progesterone과 estradiol-17의 소장을 규명하였다

Rat H-Y 항체에 의한 생쥐 Embryo 의 성 조절에 관한 연구 - 3 . H-Y 항혈청에 의한 BALB/C 생쥐 상실배의 성 판별

박충생,박희성,정장용 경상대학교 축산진흥연구소 1989 畜産振興硏究所報 Vol.16 No.1

Ethylene Glycol 을 이용한 유리화 동결시 배 발달단계별 생쥐배의 생존성

박충생,곽대오,공일근,정기화,박준규 경상대학교 축산진흥연구소 1993 畜産振興硏究所報 Vol.20 No.1

토끼에서 공핵란의 세포주기 조절과 수핵란의 세포질 상태에 따른 핵이식 수정란의 체외 발달과 복제동물의 생산

박충생,전병균,하란조,윤희준,곽대오,이효종,최상용 韓國受精卵移植學會 1997 한국동물생명공학회지 Vol.12 No.3

To improve the efficiency of production of cloned embryos and animals by nuclear transplantation in the rabbit, the effect of cell cycle of donor nuclei and type of recipient cytoplasm on the in vitro developmental potential and production efficiency of offspring was determined. The embryos of 16-cell stage were collected from the mated does at 48h post-hCG injection and they were synchronized to G phase of 32-cell stage. The oocytes collected at 14h post-hCG injection were freed from cumulus cells and then enucleated. One group of the enucleated cytoplasms was activated by electrical stimulation prior to injection of donor nucleus, and the other group was not pre-activated. The separated Gphase blastomeres of 32-cell stage embryos were injected into the perivitelline space of recipient cytoplasms. After culture for 20h post-hCG injection, the nuclear transplant oocytes were electrofused and activated by electrical stimulation and the fused nuclear transplant embryos were co-cultured for 120h and the nuclear transplant embryos developed to blastocyst stage were stained with Hoechst 33342 dye and their blastomeres were counted. Some of the nuclear transplant embryos developed in vitro to 2- to 4-cell stage were transferred into the oviducts of synchronized recipient does. The electrofusion rate was similar between the types of donor nuclei and recipient cytoplasms used. However, the nuclear transplant embryos using G phase donor nuclei were developed to blastocyst at higher rate(60.3%) than those using S phase ones(24.7%). Also, when non-preactivated oocytes were used as recipient cytplasms, the develop-mental rates of nuclear transplant embryos to blastocysts were significantly(P< 0.05) higher(57.1%) than those using preactivated ones(20.8%). The cell counts of nuclear transplant embryos developed to blastosyst stage were increased signficantly(P<0.05) more in the non-preactivated recipient cytoplasm(163.7 cells), as compared whit the preactivated recipient cytoplasm(85.4 cells), A total of 49 nuclear transplant embryos were tranferrid into 5 recipient does, of which two offsprings were produced from a foster mother 31 days after embryo transfer. these results showed that the blastomeres of G1 phase and non-preactivated oocytes might be utillzed efficiently as donor nuclei and recipient cytoplasms in the nuclear transplant procedure, thought the offspring production remained still low.

유리화 및 완만동결법에 의한 토끼 전핵배의 동결보존 후 배발달율

박충생,강다원,하란조,공일근,최상용,이효종 韓國受精卵移植學會 1997 한국동물생명공학회지 Vol.12 No.2

This study was carried out to investigate the effect of vitrification and slow freezing methods on the post-thaw developmental rate of rabbit zygotes. After exposing rabbit zygotes in EFS solution for 0.5, 1, 2, 3 and S min at room temperature, they were washed with 0.5 M sucrose solution, D-PBS and TCM-199 and then cultured in TCM-199 plus 10% FBS with bovine oviduct epithelial cells(BOEC) to examine whether the cryoprotectant induced injury during the various exposure periods. The embryo development rates to hatched blastocyst after exposing in EFS solution for 3 and 5 min(40.0 and 16.7%) were significantly lower than in 0.5, 1 and 2 min(63.0, 72.0 and 54.5%), respectively. The post-thaw development rates to hatched blastocyst were significantly(P<0.05) higher in in vivo morula with intact mucin coat(85.2%) and mucin seperated morula(77.8%) than those of in vitro morula(58.5%) and zygote(5.9%), hut no difference was shown between in vitro morulae and mucin separated morula. The cryoprotectant dilution procedures showed no effects on the post-thaw development rates to hatched blastocyst under the present culture conditions. The post-thaw development to hatched blastocyst in the rabbit zygotes was not significantly different between the slow freezing(12.8%) and vitrification(5.9%). These results indicated that the rabbit frozen zygotes could he successfully developed in vitro to hatched blastocysts, though their developmental rate was very low, compared with morula stage embryos, in either vitrification or slow freezing procedure under the present conditions.

토끼의 난포발육 처리 후 난포란 체외 성숙 시 핵의 발달과정

박충생,이경미,전병균,강태영,이효종,최상용 韓國受精卵移植學會 1995 한국동물생명공학회지 Vol.10 No.1

In order to determine the optimum condition and timing for in vitro maturation of oocytes to metaphase of meiosis II (M II), the immatured follicular oocytes were recovered by puncturing the large(1.0~1.5 mm in diameter) and small(<1.0 mm in diameter) follicles in the ovaries of rabbits treated intramuscularly with a single dose of 100 TU PMSG 68 hours previously. The follicular oocytes were classified into three grades by the attachment of cumulus cells. The Grade I and II follicular oocytes from large follicles were cultured in BO-DM medium with 10% FCS, 35 g /nl of FSH, 10 g /ml of LH and 1 g /ml of estradiol-17 at 39t in a 5% incubator for 11 to 23 hours. In 3 hours interval during the culture period, the oocytes were harvested and their cumulus cells were removed with hyaluronidase. The denuded oocytes were stained with Hoechst 33342 dye and their meiotic status and extrusion of the first polar body (PB) were examined under a fluorescence microscope. Also the fragmentation of the first PB and the distance between the first PB and nucleus were examined. The results obtained were as follows: 1. The mean recovery rate of follicular oocytes from the large and small follicles was 59. 9 and 31.3%, respectively. The mean number of oocytes recovered per rabbit and the Grade I percentage were 14.6 and 94.4% in large follicles, but 2.1 and 61.1% in small follicles, respectively. All the parameters examined were different significantly (p<0.05) between both the folliclular size. 2. Most of the follicular oocytes(86.8%) were matured in vitro to M II phase in 14 hours in Grade I oocytes, but the significantly(p<0.05) less oocytes(45.5%) were matured in Grade II oocytes. 3. The first PB was extruded in most of the oocytes(94.7%) in 14 hours of culture with the fragmentation rate of 29.6%, but the fragmentation rate of the first PB increased significantly (p<0.05) as the culture period for maturation was longer to 20 hours(63.5%). 4. The distance between the first PB and nucleus was increased linearly (p<0.05) as the maturation time passed from 14(7.1rn) to 23 hours(58.4m). 5. From the above results it was concluded that the optimum time for in vitro maturation culture might be 14 hours in the follicular oocytes from rabbit primed with PMSG for 68 hours, expecially when these follicular oocytes were used for recipient cytoplasms in embryo cloning.

생쥐배의 발생단계별 미세분할 , 배양 및 이식에 관한 연구

박충생,강대진,이효종,박희성 경상대학교 축산진흥연구소 1989 畜産振興硏究所報 Vol.16 No.1

These experiments were carried out to determine the effect of cell stage in embryo bisection on the subsequent in vitro and in vivo development in mouse. The embryos of ICR mouse were microsurgically bisected at 2-cell, 4-cell, 8-cell, morula and blastocyst stage using microsurgical blade attached a micromanipulator. These demi-embryos without zona pellucida were cultured up to blastocyst stage and transferred to pseudopregnant mice, and the development of these demi-embryos was compared with the results of intact embryos of the corresponding cell stagee. The successful rate of mouse embryo bisection at 4-cell stage(59.0%) was significantly(P$lt;0.05) lower than those at 8-cell(75.6%), 2-cell(80.7%) or morula stage(84.8%), and highest at blastocyst stage(95.7%). When the bisected embryos without any damage from microsurgery were cultured in vitro up to blastocyst the in vitro development of demi-embroys bisected at morula to blastocyst was 91.6 to 95.3%, which was similar to the culture result of intact embryos of corresponding stage. However, the in vitro development of demi-embryos bisected at 2-to 8-cell stage was significantly(P$lt;0.05) lower. The post-transfer implantation rate of demi-embryos developed in vitro to eu-blastocyst were 19.6 and 25.4% in demi-embryos bisected at morula and blastocyst stage, respectively and not significantly(P$lt;0.05) different from the result of intact embryos of the same stage. However, the implantation rates of demi-embryos bisected at 2-or 8-cell stage were significantly(P$lt;0.05) lower than the result from the intact embryos of the corresponding stage.

토기에서 공핵란의 발달단계가 할구주입, 전기융합 및 핵이식 수정란의 체외발달에 미치는 영향

박충생,전병균,이효종,최철민,최상용 韓國受精卵移植學會 1994 한국동물생명공학회지 Vol.9 No.2

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39 in a 5% incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

계절 및 연도가 납유량 , 유지율 및 불합격율에 미치는 효과

박충생,강대진,김철욱 경상대학교 축산진흥연구소 1984 畜産振興硏究所報 Vol.11 No.1

This study was conducted to determine the effect of season, year and season×year interaction on the amount of collected milk, fat content and rate of rejected milk in Holstein bow based on the data collected from a survey during 4 years from January 1979 to December 1982 in the western Gyeongnam area. The least square method was used for the analyses and the results obtained are summarized as follows. Average daily amount of milk collected per herd and fat contents were 14.48kg and 3.44 in the overall observations analyzed. Average daily amount of milk collected per herd decreased in spring and fat content decreased in summer. Season and season×year interaction significantly influenced on average daily amount of milk cellected per herd and all the factors analyzed on fat content. (P$lt;.05) Season, year and season×year interaction significantly influenced on the rate of milk rejected in milk plant. (P$lt;.01) The rate of rejected milk was 1.47% in the overall observations analyzed. The amount of rejected milk was more in summer than in the others. The rates of rejected milk due to adification, specific gravity and miscellaneousness of the overall rate of rejected milk were 69.60%, 14.33% and 16.07%. The amount of rejected milk due to adification tended to be much in winter and specific gravity in spring.

핵이식에 의한 복제동물 생산의 최근 연구 성과

박충생 韓國受精卵移植學會 1993 한국동물생명공학회지 Vol.8 No.1

Nuclear transplantation techinque has been found to be the most potential and efficient method for producing a large number of genetically identical animals from a single embryo. The technical development of nuclear transplantation in mammals and its application to the production of cloned animals were reviewed. For the efficient and successful production of cloned embryos by nuclear transplantation, selection and micromanipulation of recipient eggs or embryos as capacious recipient cytoplasm, and benefitial preparation of multiple totipotent embryonic cells as donor nuclei, and also fusion technique are very critical. Recent works approaching to these critical points were introduced and discussed.