This study was performed to establish the effective culture condition for the establishment of clonal lines from porcine fetal fibroblast cells. Fibroblasts derived from a pig fetus (Day 50) were cultured and passaged two times before use. A single cell was seeded in 96-well plates, cultured in medium supplemented with different concentrations of FBS, catalase or β-mercaptoethanol (βME), and classified by cell size and morphology. Cells were passaged two times into 4-well dish before freezing. The establishment efficiencies were not different among different concentrations of FBS (0.3 to 5.1%). However, population doubling time (PDT) was significantly decreased by increasing the FBS concentration (P＜0.05). The establishment efficiency of βME-added group (10.4%) was significantly higher than those of catalase-added and control groups (3.5%, and 3.5%, respectively, p＜0.05), and PDT was significantly decreased (23.6 vs 28.1, and 25.5 h, respectively, p＜0.05). However, catalase did not show a positive effect on the establishment efficiency. Cell size and morphology did not affect the establishment efficiency and PDT of clonal lines. The result of present study shows that the establishment efficiency of clonal cell lines can be enhanced by the culture in media supplemented with 30% FBS and βME.

This experiment was carried out to evaluate the effect of early mouse embryonic development in vitro by co-culture with bovine and porcine oviductal epithelial cells (BOEC and POEC). The 2-cell embryos were collected from the oviducts of the superovulated and mated cultured in D-PBS /15% FCS at 48 hours after hCG injection. The in vitro developmental rate of blastocyst formation in the embryos were examined under the fllowing treatments; 1) TCM 199 added 15% HCS, 2) Ham's F-10 added 15% HCS, 3) MediCult IVF medium, 4) TCM 199 added 15% HCS + BOEC, 5) TCM 199 added 15% HCS + POEC, 6) Ham's F40 added 15% HCS + BOEC, 7) Ham's F-10 added 15% HCS + POEC,8) MediCult IVF medium + BOEC, 9) MediCult IVF medium + POEC. For a comparative study of in vitro development for 96 hours after hCG injection, were cultured with oviductal epithelial cell and media only. The obtained results were 2-cell embryos developed to the blastocyst stage in TCM 199, Ham's F-10 and MediCult IVF medium at the rates of 84.4,83.2 and 81.6%. respectively. The higher developmental rates(91~97%) of blastocyst formation was appeared when the embryos were co-cultured with a monolayer of bovine or porcine oviductal epithelial cells in TCM 199 or Ham's F-10 and MediCult IVF media. No significant difference in developmental rates was shown between bovine and porcine oviductal epithelial cells but significant difference in co-culture system in comparison between media only system and co-cultures. In conclusions, oviductal epithelial cells, BOEC and POEC, when co-culture with mouse early embryos improved the rates of development, blastocyst and hatching. Therefore, it is suggested that co-culture system using oviductal epithelial cells improve early embryonic developtnent in mouse.

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.

The studies on the carried out to investigate the effects of co-culture with uterine fluids and uterine epithelial cells on the in-vitro fertilization and developmental rate of porcine follicular oocytes. The ovaries were obtained from slaughtered swine. The follicular oocytes surrounded with cumulus cells were recovered by aspirating follicular fluids from the visible follicles of diameter 3~5 mm. The follicular oocytes were cultured in TCM-199 medium containing hormones and 10% ECS for 46~48 hrs in a incubator with 5% in air at 38.5 and then matured oocytes were again cultured for 12~18 hrs with motile capacitated sperm by preincubation of heparin. The results obtained in these experiments were summarized as follows ; 1.The in-vitro maturation and fertilization rate of porcine oocytes co-cultured with uterine fluids in TCM-199 medium were 68.0% arid 55.7%, the rates were higher than of control, 56.5% arid 38.7%. 2. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the fertilization rate was 60.3%, the rates were higher than that of control, 35.7%. 3. When the in-vitro fertilized oocytes were co-cultured with porcine uterine epithelial cells, the development rate to be blastocyst was 12.4%, the rates were higher than that of control, 9.2%(p<0.05).