This study was conducted to improve the production efficiency of in vitro produced (IVP) embryos in Korean Native cows. The optimal conditions and procedures for in vitro maturation(IVM), in vitro fertilization(IVF) and in vitro culture(IVC) of bovine follicular oocytes and IVP embryos were evaluated. Immature follicular oocytes were collected fiom the follicles of bovine ovaries obtained from abattoirs. The oocytes of Grade I and II for IVM were cocultured with monolayered bovine oviductal epithelial cells(BOEG) or granulosa cells in TCM-199 solution supplemented with follicle stimulating hormone, lutenizing hormone, estradiol-17 and heat inactivated fetal calf serum at 39 under 5% in air for 14 to 24 hours. Most of the oocytes(93%) matured to metaphase II in 24 hours. The cocultured IVM oocytes were fertilized in vitro at significantly(P<0.05) higher rate with BOEC(83.8%) and with granulosa cells(84.6%) than the non-cocultured IVM oocytes(73.6%). The IVM-IVF embryos developed to morula and blastocyst at significantly(P<0.05) higher rate in coculture with BOEC(41.2%) than with granulosa cells(23.1%) or conditioned medium(23.4%).

This study was carried out to determine the effect of cumulus cell attachment and various factors on in vitro maturation of pig foflicular oocytes. Oocytes with various configuration of cumulus cell mass were collected ftom ovaries of mature gilts by asperating with syringe equipped with needles of different gauges, follicle size and with or without cumulus cells. They were cultured in TCM-199 mediun containing FGS(fetal calf serum) for 30~48 hours in incubator with air containing 5% at 38.5. Mter orcein staining at in vitro maturation condition, GV, GVBD, anaphase, telophase and M II were observed. Results are surumarized as follows: 1. Recovery rates were 55.8, 55.5 and 34.4% when the cumulus-compacted oocytes were collected with 18, 21, 26 gauge needles of syringes, respectively. 2. 79% of oocytes with compacted cumulus cells were at GV stage and most of the oocytes with partially denuded and denuded cumulus cells were from GVBD to M- II stages. 3. Percentage of mature oocytes among those which are follicular diameter of 1~2, 3~6 and over 6 mm was 42.6, 53.2 and 60.8%, respectively. 4. Percentage of mature oocytes among those which are compacted, partially denuded and denuded was 60.5, 46.2 and 35.4% respectively. 5. Percentage of mature oocytes in co-cultured with monolayers of cumulus cells was higher (57.1%) than that found with oocytes cultured alone (53.4%).

In-vitro culture has provided new inforrnation on mechanisms of oocytes rnaturation and results obtained in vitro have led to new questions. In porcine, follicular and oocyte size have the crucial importance for the oocytes maturation. The addition of hormones to the culture medium was found to accelerate and facilitate meiotic maturation. The presence of some factors in serum trigger the resumption of meiosis and support the maturation of oocytes in vitro. The maturation rate of porcine oocytes was also increased by supplementation of porcine follicular fluid to the culture medium. The growth factors can stimulate nuclear maturation and enhances cytoplasnic maturation of oocytes by interaction with gonadotropins. The maturation-promoting factor brings about GVBD and the subsequent maturational events in oocytes. However, cAMP can block the spontaneous meiotic maturation of oocytes in culture. The understanding of these influences is a prerequisite to enhancing in vitro maturation of porcine oocytes.

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.

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.