This study was undertaken to access the effects of collection method, room temperature at oocyte recovery and culture media on the oocyte quality, fertilization and cleavage rates of in vitro matured and fertilized oocytes of Korean native goats. Ovaries obtained from a slaughterhouse were transported to the laboratory and were divided into 2 groups. One group of ovaries was maintained at 30 to 35 of the room temperature and another group was remained at 20 to during oocyte recovery. The oocytes were recovered by follicle aspiration, slicing and aspiration+slicing methods from 3 groups of follicles according to size; <2 mm, 2 to 6 mm and >6 mm. The matured oocytes were inseminated with buck epididymal spermatozoa at a concentration of 3~3.510 m1 and fertilization was identified when 2 pronuclei were present in the cytoplasm. Although the recovery rate per ovary obtained by the combination of follicle aspiration + slicing(19.62.2) method was higher than aspiration(11.71.1) and slicing(14.81.8) collection, optimal recovery according to oocyte grades resulted form ovarian slicing compared to aspiration or combined methods(P<0.05). However, no significant differences were found in the mean number(2.51.8; 3.33.3; 2.92.4) and the proportion of favorable oocytes(Grades I, II and III) recovered(31.6%, 36.0%, 36.4%,) according to follicle size(<2 mm; 2 to 6 mm; >6 mm). Fertilization rate was 60.0%, 67.7%, 70.6% and 56.4% and the proportion of embryos/zygotes was 11.1%, 7.1%, 5.0% and 2.8% in 20~/BO, 30~35/BO, 20~/TALP and 30~35 /groups, respectively.

This study was conducted to develop an improved method for oocyte pick-up(OPU) with finger-sensibility using ultrasound-guidance from ovarian follicles in Holstein heifers. Oocytes were aspirated from ovarian follicles of clear-outline (>2mm), obscure-outline and invisible( 2mm) on ultrasound images with 3 different vacuum pressure(40, 80, 120mmHg). Total number of oocytes recovered/follicles were 309/237(130.4%). 113/80(141.3%) and 107/74(144.6%) with 40, 80 and 120 mmHg of vacuum pressure, respectively. Mean number of oocytes recovered was higher in 2 OPU/week (18.35.3) than 1 OPU/week(14.54.1), but this difference was not statistical1y significant. The recovery rates were not affected by the number of OPU as 135.6%(282 oocytes/208 follicles) in 1~20 OPU, 137.7% (168/122) in 21~40 OPU and 148.4%(92/62) in 41~60 OPU, respectively. The proportions of good oocytes (Grades I) recovered were not significantly different by the number of OPU until 40 OPU(12.4% in 1~20 OPU vs 16.7% in 21~40 OPU). However, a significantly(P<0.05) lower recovery rate resulted from more than 40 OPU compared to less than 40 OPU(7.6%). These results imply that more fertilizable oocytes can be produced from invisible-immature follicles by transvaginal aspiration with finger-sensibility from Holstein heifers.

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 /mL), and higher rate of encleation was obtained at 7.5 and 15 /mL than at /mL. In Experiment 2, oocytes enucleated in 7.5 /mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 ) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 or 90 , but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.

The objective of this study was to identify a follicular fluid ingredient inhibiting the cumulus oocyte complex (COC) expansion. Thus, follicular fluid or liquid chromatographic fractions of follicular fluid was supplemented in COC culture medium. And COCs were incubated for 48 hours to investigate about cumulus expansion and also the first polar body extrusion. The results obtained were as follows; 1. The fluid of medium follicle significantly inhibited the COC expansion. 2. The fluid of large follicle inhibited the COC expansion. 3. Follicular fluid showed six major fractions at retention volumes (RVs) 1.83, 1.91, 2.15, 2.34, 2.53 and 2.74 ml after separation with Superose 12 column. Of the major fractions, fractions RV2.15, RV2.34, RV2.53 and RV2.74 inhibited both COC expansion and polar body extrusion. Especially, fractions of RV2.15 and RV2.53 significantly inhibited COC expansion, oocyte denudation and polar body extrusion. In conclusion, porcine follicular fluid contained a COC expansion inhibiting ingredient (CEI) that may be contained largely in fractions RV2.15 and RV2.53. And CEI may inhibit oocyte maturation by inhibition of oocyte denudation and extrusion of the first polar body.

This study was conducted to find out the effect of follicle size and oocyte type on in vitro maturation of poricine follicular oocytes. TCM-HEPEAS medium was used to basic medium, and the oocyte matured in vitro was stained with the Rapid staining method. The results obtained were summarized as follows; 1. The number of follicles an ovary was 20.5. The number of A-and B-typed oocytes an ovary was 2.34. The proportion of A-and b-types oocytes was 40% of the recovery oocytes. 2. Cumulus expanison indexes(CEI) by the follicle size were 1.62∼2.34(<2mm), 1.27∼2.28(2∼5mm) and 1.46∼2.75(>5mm). It was no differ to maturation rate by the follicle size. 3. The degree of oocyte maturation based on oocyte type did not differ for B-and C-typed oocyted but the index of oocyte type A was higher than that of b-and C-typed oocytes. 4. When follicluar oocytes were cultured for 42 hours, the proportion of the Met-II(second metaphase) stage were 22.5% (degree 1), 35.4%(degree 2) and 65.5% (degree 3).

도축장에서 채취한 돼지난자를 직경별로 간격으로 나누어, 난자의 크기에 따른 체외성숙과 발육능을 구명코자 체외성숙과 체외수정 후의 배 발달율을 조사하였다. 채취한 난자의 투명대를 제외한 평균직경은 이었으며, 직경별 분포는 < 105, 105~110, 110~115,115~120, 120~125, <이 각각 3.0, 11.0, 31.2, 41.5, 13.0 및 0.4%로 72.7%가 110부터 사이였다. 체외성숙율에 있어서 직경 미만난자는 66.7%인 반면

This study was to determine the effect of ionomycin and 6-dimethylaminopurine (6-DMAP) and/or elcetrical stimulation on the oocyte activation and production of rabbit nuclear transplant embryos. The oocytes were collected from the oviduct of superovulated rabbits at 14 h post hCG injection and cultured in TCM-199 containing 10% FBS until 19 h post hCG injection. To determine the optimum concentration and exposure time of 6-DMAP, some oocytes were activated with 5 M ionomycin for 5 min and then in 2.0 mM 6-DMAP for 0.5 to 3.0 h, or in 1.0 to 3.0 mM 6-DMAP for 2.0 h. Other control oocytes were stimulated electrically(3X, 1.25 kV/cm, 60 sec) in 0.3 M mannitol solution supplemented with 100 M CaCl and MgCl. The nuclear donor embryos of 8-cell stage were synchronized to G phase of 16-cell stage, and the recipient cytoplasms were obtained from removal of the first polar body and a portion of membrane bound cytoplasm of the oocytes collected at 15 h post hCG injection. A separated blastomere was injected into the perivitelline space of the enucleated oocytes. The oocytes injected with nucleus were cultured until 19 h post hCG and then electrofused and activated by electrical stimulation with or without ionomycin and 6-DMAP. These nuclear transplant embryos were cultured in TCM-199 containing 10% FBS in 39˚C, 5% CO2 incubator for 120 h. For the oncytes activated parthenogenetically with electrical stimulation with or with-out ionomycin and the various concentration of exposure time of 6-DMAP, the highest cleavage(92.3%) and development to blastocyst stage(41.0%) were resulted from the oocytes activated by ionomycin and 2.0 mM 6-DMAP for 2.0 h, which were found to be significantly(P<0.05) higher than the cleavage(45.2%) and developement to blastocyst stage(14.3%) from the oocytes activated with electrical stimulation. The significantly(P<0.05) more oocytes(71.4%) developed to 4 cell stage at 24 h post activation by ionomycin and 6-DMAP than those by electrical stimulation(18.9%). For the nuclear transplant embryos, the cleavage rate was similarly high in oocyte activation by electrical stimulation with(79.4%) or without ionomycin and 6-DMAP(70.5%). However, the embryo development to blastocyst stage was significantly(P<0.05) higher in oocyte activation by electrical stimulation with ionomycin and 6-DMAP(44.4%) than by electrical stimulation only(25.0%). The significantly(P<0.05) more nuclear transplant embryos(45.6%) developed to 4 cell stage at 18 h post activation by electrical stimulation with ionomycin and 6-DMAP than those by electrical stimulation only(10.6%). These results indicated that the supplemental oocyte activation by ionomycin and 6-DMAP with electrical stimulation enhanced and accelerated the preimplanted in vitro development of the rabbit nuclear transplant embryos.

중금속 이온이(Cd2+, Hg2+, Cu2+, Pb2+) 산개구리 여포난자의 성숙에 미치는 영향을 알아보기 위해 배양액에 일정 농도의 이온들을 첨가한 후 여포난자들을 일정시간 배양하였다. 여포난자의 성숙을 유도하기 위하여 FPH(Frog pituitary homogenate: 0.1p.e./ml)를 사용하였으며 여포난자의 성숙율은 난자의 핵막 붕괴율로부터 구하였다. 실험결과 Cd2+은 0.1ppm의 농도부터 여포난자의 성숙을 억제하였으며 Hg2+과 Cu2+는 1ppm부터, Pb2+는 5ppm에서 현저히 억제효과를 나타내기 시작하였다. 이들 중금속 이온 작용의 가역성을 조사하기 위해 3시간 동안 여포난자들을 중금속 이온에 노출시킨 후 보통 배양액으로 옮겨 계속 배양을 해 본 결과 Cd2+은 1ppm에서 가역성을 나타내었으나 2.5ppm에서는 비가역적인 손상을 주었다. Hg2+, Cu2+, Pb2+의 효과는 2.5ppm에서는 비가역성을 나타냈다. 위 결과로부터 개구리 여포난자의 배양계는 환경오염물질의 독성 검정에 요긴하게 사용할 수 있을것으로 생각되었다.

This study was carried out to determine the effect of bovine follicular fluid(bFF), hormones, and fetal bovine serum(FBS) supplemented in the medium on the in vitro fertilization and development of bovine embryos. The ovaries were obtained from a local abattoir and placed in physiological saline kept at 30~32˚C and brought to the laboratory within 3~4 hours. The oocytes and follicular fluid were collected by aspiration from visible follicles, and the oocytes of grades I on the basis of the morphology of cumulus cells attached and the homogeneity of cytoplasmic granules were selected and used for maturation. The basal media used for oocyte maturation, fertilization and embryo development in vitro were Ham' F-10, TALP and TCM-199, respectively. The hormones supplemented in maturation medium were consisted of 35 pg /ml FSH, 10 pg /ml LH and 1 pg/mi estradiol-l7. The bFF collected from 5~9 mm follicles was centrifuged, filtered and inactivated by heat-treatment at 56˚C for 30 min. FBS also was inactivated with the same method and kept at -20˚C until use. The embryos were co-cultured with the monolayer of bovine oviductal epithelial cells at 39˚C under 5% in air for 9 days. The results obtained were summarized as follows: The fertilization rate of oocytes was found 87.4% from 10% FBS and hormones treatment for IVM, and 37.1% of these TVF embryos were developed to blastocyst stage in 10% FBS groups. Compared with this control system, the fertilization rate was decreased significantly(P<0.05) in the maturation without either FBS or hormones. These IVF embryos were developed to morula stage at the similar rate, but to blastocyst at significantly(P<0.05) lower rate in the embryo culture with or without FBS supplementation. The fertilization rate(82.9%) in hormones and 10% inactivated bFF was similar with 10% FBS and hormone groups(87.4%), but decreased significantly(P<0.05) in 20 or 30% bFF (61.0 or 66.0%), respectively. In vitro developmental competence to blastocyst stage in 10% FBS and 20% inactivated bFF(37.1% and 31.4%) was higher than in 10 or 30% inactivated bFF(20.0 or 19.2%) or 10, 20 and 30% fresh bFF(19.1, 21.0 and 17.5%) The results indicated that the in vitro fertillzation and development rate of the embryos should be improved in 10% FBS or 20% inactivated culture system and 20% inactivated bFF might be available economically for bovine oocyte maturation and embryo culture instead of fetal bovine serum.

The present study was carried out to produce in vitro fertilized embryos with immature follicular oocytes collected by transvaginal aspiration from Holstein cows. A simple aspiration apparatus consists of two stainless steel tubes, an inner tube (needle holder; 1.2cmdiameter, 55cm long) and an outer tube (1.5cm diameter, 4Scm long), and a hand-operated vacuum pump was used. Under epidural anesthesia, the needle guide was passed into the vagina of the cow to a point next to the cervix. An ovary was placed against the wall of the vagina over the end of the aspiration needle by rectal manipulation. As the needlepassed into the ovary, an assistant was asked to apply vacuum(l00mrnHg) and the ovary was manipulated back and forth in all directions over the needle. When all sites of the ovary was aspirated, the needle was withdrawn and the needle guide was moved to the other side of ovary and the procedure was repeated. When the oocyte aspiration procedure was finished, collected fluid was transported to laboratory. Oocytes surrounded with at least 1 layer of cumulus cells were matured, fertilized and cultured in vitro. The results were as follows; Ninety seven oocytes were collected by transvaginal aspiration from seventeen Holstein cows(5.7 /head). The number of oocytes surrounded with at least 1 layer of cumulus cells were 60(61.9%). Following in vitro maturation, fertilization and culture, the cleavage and development rate to morula+blastocyst were 83.3% and 30.0%, respectively. From this study, transferable in vitro fertilized embryos could be produced with imma- ture follicular oocytes collected by transvaginal aspiration from Holstein cows using a simple aspiration apparatus

The present study was conducted to investigate the influence of embryo cell stage at 18h post-fusion and oocyte preactivation on sebsequent in vitro developmental potential in the nuclear transplant rabbit embryos. The embryos of 16-cell stage were collected and synchronized to G phase of 32-cell stage. The recipient cytoplasms were obtained by removing the first polar body and chromosome rnass from the oocytes collected by non-dis-ruptive microsurgery procedure. The separated G phase blastomeres of 32-cell stage were injected into non-preactivated recipient cytoplasms. Otherwise, the enucleated recipient cytoplasms were preactivated by electrical stimulation at 18h post-hCG injection and the separated G phase blastomeres of 32-cell stage were injected. Mter culture until 20h post-hOG injection, the nuclear transplant oocytes were electrofused by electrical stimulation. The fused nuclear transplant embryos were classified into 3~4-cell, 2-cell and 1-cell stage at 18 hrs post-fusion and cultured until the embryos reached blastocyst stage. The developmental rate to blastocyst stage was significantly (P <0.05) higher in all the reconstituted embryos of 3~4-cell stage(58.0%) than in 2 and icell stage. The developmental rate to blastocyst stage in the embryos of 3~4-cell stage at 18 hrs post-fusion was significantly (P<0.05) higher in the reconstituted without oocyte preactivation(77.8%) than in the oocyte-preactivated embryos (33.3%). These results indicated that the higher rate of in the in vitro development to blastocyst stage might be obtained form the embryos which were reconstituted with nuclear donor of G phase and non-preactivated oocyte, and developed more rapidly for 18 hrs post-fusion.