This study was undertaken to investigate effects of granulosa cells on mejotic maturation of porcine oocytes in vitro. The results obtained in this study were summarized as follows : The germinal vesicle breakdown(GVBD) rates were 91.5, 93.3 and 96.6%, respectively, when the cumulus oocy:e cornplexes(COC) in the TCM-199 medium with sodium bicarbonate, Na pyruvate, penicillin G, streptomycin sulfate and 10% FCS were cultured in the condition of FSH(0.02 Au/ml), LH(10 g/ml) and FSH + LH added. And when the COC were co-cultured with granulosa cell (5 106 cells /ml) in the condition of FSH, LH and FSH + LH added, GVBD rates were 94.3, 92.9 and 98.9%, respectively. However, when the COC were cultured in the condition of hormone free and co-cultured with granulosa cells in the condition of hormone free, the GVBD rates were 40.4 and 86.3%, respectively. The GVBD rates were 41.0, 62.7, 84.6, 88.1 and 93.6%, respectively, when the COC were co-cultured with granulosa cells that the concentrations are 0 cells /ml, 1 106 cells /ml, 5:: 106 cells /ml, 1 107 cells /ml and 5 107 cells /ml.

In vitro development of parthenogenetic embryo was examined after ethanol treatment of follicular oocytes matured in vitro for 42, 48, 54 and 60h in the pig. The follicular oocytes were matured in TCM 199 containing 15% FCS and gonadotrophins in an atmosphere of 39 5% . The cumulus-free oocytes were activated by 10% ethanol treatment in M2+4mg /ml BSA for 10 min. The ethanol-activated oocytes were washed and further cultured in TCM199+20%FCS containing granulosa cell monolayer. Maturation rates at 42, 48, 54 and 60h of IVM were 75.0, 86.5, 81.6 and 87.9%, respectively. Thus the oocytes maturated in vitro for longer periods did not improve nuclear maturation much. Pronuclear formation rates at 18h post-activation in ethanol-activated oocytes were 21.9, 25.0, 47.4 and 32.6%. The cytoplasmic maturation leading to pronuclear formation upon activation increased when the I VM period was extended from 42 to 54h. When the activated oocytes were cultured for 96~120h to analyse early development of the activated oocytes, the rates of embryonic development upto 5~8 cell were 5.3, 5.8, 12.0 and 11.7% among the cultured embryos. The result indicate that earlier development of activated porcine occyte is dependent on the duration of oocyte maturation, and that better development could be obtained from the oocyte matured for 54h.

This study was carried out to investigate the effects of gonadotropins added during maturation of porcine oocytes on the in vitro maturation(IVM), in vitro fertilization(IVF) and developmental potential of embryos. The follicular oocytes were cultured in TCM-199 medium containing different combination of gonadotropins(5g /ml FSR or 1OIU /ml PMSG and 1Og /ml LH or 1OIU /ml hCG), 10% FCS and 10% PFF for 36~48h in a incubator with 5% in Air at 39 and then matured oocytes were again cultured to 120h after IVF for 6~7h with heparin(100g /m')-treated sperm. When the oocytes were matured for 42brs in the medium containing FSH+LH, FSH+hCG, PMSG+LH or PMSG+hCG, the JVF rate of each treatment was 50.0%, 52.9%, 66.7% and 70.0%, respectively. The highest CEI (cumulus cell expansion index) was obtained from PMSG+hCG-added medium and the highest polyspermic penetration resulted from FSH+LH-added medium. The cleavage of IVF oocytes derived from hormone added IVM was significantly(P<0.05) promoted by PMSG+hCG and the cleavage rate after 36-h, 42-h and 48-h maturation aws 53.0%, 56.7% and 45.6%, respectively. The highest developmental potential resulted from the oocytes derived from PMSG+LH -added IVM.

The objective of this study was to develop an effective in vitro production system capable of obtaining more porcine embryos from immature oocytes. These experiments were thus conducted to examine the effect of oocytes type and maturation time on the in vitro maturation(IVM) and fertilization(IVF) of oocytes and the in vitro development (IVD)of IVF embryos. 1. The degree of oocyte maturation based on cumulus expansion index(GEI) did not differ for A- and B-typed oocytes but the index of oocyte type C was lower(P<0.05) than that of other oocyte types. 2. When the oocytes of type A and B were matured for 36, 42 and 48hrs, the GEl was not different between the 36- and 42-h maturation but the GEl after 48hrs was greatly lower(P<0.05) than that of other maturation times. 3. The highest cleavage rate(48.6%) of IVF oocytes was obtained from A typed oocytes and 42-h maturation but the developmental potential based on cleavage index was the highest when B-typed oocytes were matured for 42hrs.

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 g/ml FSH, 10 g/ml LH, 1 g/ml estradiol-17 and granulosa cells at 39 under 5% in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

The objective of this study was to produce calves derived from in vitro fertilization of in vitro matured follicular oocytes. Oocytes aspirated from small antral folicles of ovaries obtained at a local slaughter house were matured and fertilized in vitro. At l8hrs after insemination with Korean native cattle semen, oocytes were co-cultured for 6~7 days by utilizing co-culture system with bovine oviduct epithelial cell. After co-culture, good or excellent quality late morulae or early blastocysts were selected by morphological criteria under stereo microscope. Selected embryos were transferred to recipients on day 6 or 7 (estrus = day 0). Recipients were monitored by observation for estrus and rectal palpation after 60 days from embryo transfer. One of them went to term with the birth of a calf. This case is the first production of calf derived from in vitro fertilization in Korea.

소 난자의 시험관내 수정시 이성체인 1, 2 propanediol과 1, 3 propanediol과의 동결보호제의 이용에 대한 독성효과를 조사하였다. 프랑스에 있는 도축장의 난소에서 채취한 미성숙 난자 212개를 시험관내 성숙과 heparin(10g/ml)첨가에 의한 수정 및 배양을 실시하였다. 소 난자의 시험관내 성숙과 수정시 propanediol의 독성효과를 시험한 결과 1,3 propanediol은 80%의 정상 난할분할율과 5.7%의 낮은 다핵분

The nudear changes of bovine oocytes during 24 hrs. of culture for mejotic maturation were examined. Bovine oocytes were collected from small(<2 mm), medium(2~6 mm) and large(>6mm) follicles and classified into three grades by their morphological characteristics. A total of 242 oocytes collected were obtained:from 184 small, 157 medium and 1 large follicles, respectively and were classified into 95 grade I, 155 grade H and 92 grade III oocytes. All the bovine oocytes collected and graded were washed with a basal medium and incubated in groups of 10 for 24 hrs in 5% and 39. The basal medium used was composed of TCM-199 supplemented with sodium bicarbonate, sodium pyruvate, streptomycin, penicillin G and 10% FCS. The oocytes were cultured in drops of 50,l basal medium supplemented with 35g /ml FSH, 10g /ml LH and 1g /ml estradiol-17. The oocytes were fixed and examined on their chromosomal status by 1% acetorcein staining in the interval of 3 hrs. Most of the grade I oocytes developed to germinal vesicule stage at 0 to 3 hrs., germinal vesicle breakdown at 6 hrs., metaphase I at 9 to 15 hrs., anaphase I and telophase I at 18 hrs., and metaphase II and the first polar body at 24 hrs. after culture for meiotic maturation. However, it was found that compared to grade I oocytes, grade H and W oocytes reached earlier to germinal vesicle breakdown and most of them developed earlier to M II stage at 21 hrs. after culture.

돼지난포란의 안정된 체외배양 체계를 위하여 도살장에서 재취된 난소로부터 난포 및 난자난구세포체의 혀태적인 선정기준을 설정하고 이의 이론적 배경을 확립하였다. 난소를 난포상태 및 분포나 황체 존재 여부에 따라 A,B 및 C 의 세 가지 형태로 분류하고 각 type의 난소에서 직경 3-5mm인 난포로부터 난포란을 회수하였다. 회수된 난포란을 난구세포 부착상태에 따라 Good, Fair 및 Poor 의 세 가지 형태로 분류하여 각각을 호르몬이 첨가된 M16+

This research was conducted to investigate the interrelationship among methods of injection of PMSG-hCG to the number of ovulated eggs, percentage of matured oocytes and in vitro fertilization using out-bred ICR mice. The results obtained are as follows, 1) The optimurn dose was 5 IU for both PMSG and hCG, while the number of ovulated eggs was 428, percentage of M II was 73% and in vitro fertilization rate was 81 %. 2) The optimum injection interval of PMSG-hCG was 48 hours, while the number of ovulated eggs was 48 8, percentage of M II was 80% and in vitro fertilization rate was 81%. 3) The optimum time for collecting eggs was between 16 and 18 hours after hCG injection, while the numbers of ovulated eggs were 448, 427 and 437 in 14,16 and 18 hours after hCG injection respectively, and percentages of M II were 79 and 81 %, and in vitro fertilization rates were 81 and 80% in 16 and 18 hours after hCG injection, respectively. 4) The repeat of superovulation decreased with the number of ovulated eggs, percentage of M II and in vitro fertilization rate, than in control. But it was recovered by increasing the repeat interval.

본 연구는 소의 난포란의 체외성숙과 체외수정에 영향을 미치는 요인을 구명하기 위하여 미숙 난포란을 채취하여 형태적 분류를 통해 우수한 란을 공시한 후 난포의 크기, 정액의 형태, 수정능득법, 혈청, 호르몬, 난포액, 난구세포등을 첨가한 TCM-199 배양액에서 배양하면서 체외성숙 및 수정율을 조사하였는 바 그 결과는 다음과 같다. 1. 소 난포란을 채취하여 배양을 통해 형태적 분류를 했을때 A형란은 61.4%, B형란은 12.1%, C형란은 19.2%,