The embryogenesis stimulating activity(ESA) had been shown in co-culture of embryos with bovine oviduct epithelial cell(BOEC) and culture in BOEG-conditioned medium. The present study was undertaken to purify and quantify the embryotropic proteins and to determine the optimum concentration of the embryotropic protein for the proper development of embryos. In BOEC-conditioned medium, five major bands of proteins were detected(66, 53, 40, 32 and 24 kDa) by SDS-PAGE. From these proteins, 288pg of protein that had a 32kDa molecular weight was purified by gel filtration column and perfusion chromatography ion-exchange column. When purified protein was supplemented to the in vitro culture media at various concentrations in protein-free media, 2.5g /ml supplement group showed significantly higher rates of embryo development into morula /blastocyst stages than other groups(p<0.05). In conclusion, we purified 32kDa protein from BOEC-conditioned medium and this protein showed optimum embryogenesis stimulating effect at 2.5g /ml.

The studies were carried out to investigate the effects of co-culture with cumulus cells and oviduct epithelial cells on the in vitro fertilization and cleavage rate of bovine follicular cocytes and to determine the optimum thawing temperature and equilibration time on in vitro developmental rate of frozen bovine embryos. The ovaries were obtained from slaughtered Korean native cows. The follicular oocytes were cultured in TGM-199 medium containing 10 IU /ml의 PM SG, 10 IU /ml의 hCG, ip g/ml의 -estradiol and 10% FCS for 24~48 hrs in incubator with 5% in air at 38.5. The bovine embryos following dehydration by cryoprotective agents and a various concentration of sucrose were directly plunged into liquld nitrogen and thawed in 3 water. Survival rate was defined as developmental rate on in vitro culture or FDA-test. The results are sunanarized as followes :1. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with cumulus cells in TCM499 medium were 75.0~76.8% and 17.3~27.6%, respect-ively. And in-vitro fertilization rates of cumulus-enclosed oocytes(55.4%)were significantly(p<0.05) higher than cumulus-denuded oocytes (23.1%). 2. The in vitro fertilization and in vitro developmental rates of bovine oocytes co-cultured with l l04cells /ml, 1 x l06cells /ml, lx l08cells /ml and 1 x l015cells /ml oviduct epithelial cells in TCM-199 medium were 74.5~77.8% and 15.7~21.20 respectively.3. The in-vitro fertilization and in vitro developmental rates of bovine oocytes cocultured in '1CM-199 media containing PMSG, hCG, PMSG+hCG. PMSG+-estradiol, hCG+-estradiol 0 to 40 hrs after insemination were 74.0~77.4% and l8.9~23.l%, re-spectiv ely.4.The survival rates of bovine embryos thawed after rapid freezing in the freezing medium containing a various concentration of sucrose added 1.5M and 2.OM glycerol,DMSO and propanediol were 23.5~31.4% and 20.6~34.l%, respectively. 5. The temperature thawed at 3 after rapid freezing of bovine embryos resulted in a significantly higher embryos survival rate than did at 2 and 35.6. The equilibration time on the survival rates of bovine embryos was attained after short period of time(2.5~5 min.) in the freezing medium higher than long period of time (10~20min.). (Key words : bovine embryos, co-culture, freezing, in vitro development)

Porcine follicular oocytes matured in culture were inseminated with frozen-thawed spermatozoa. When the oocytes were inseminated in the medium with oviductal epithelial cell monolayer, the penetration rates higher in those with (4.1, 31.7, 45.1, 54.5 and 69.4%) than without cells (0, 17.1, 34.8, 45.2 and 58.9%) at 4, 8, 12, 16 and 20 h after insemination. The proportions of polyspermy in penetrated oocytes in medium with or without cells increased with time of examine. In another experiment, the penetration rate was higher without (57.6%) than with (19.6~24.1%) preincubation of spermatozoa for 1~4 h in medium. However, when the oocytes were inseminated with spermatozoa preincubated for 1~2 h, the penetration rates significantly higher (P<0.05) in those with (65.6 and 55.9% for 1 and 2 h) than without (24.1 and 20.6% for 1 and 2 h) oviductal epithelial cell monolayer. On the other hand, the proportions of polyspermy decreased with time of spermatozoa preincubation. These results indicate the significant advantages of the spermatozoa preincubation with oviductal epithelial cell monolayer for 1 and 2 h to maintain penetration potential during in vitro fertilization in the porcine.

Bovine mammalian gland has biopotential in therapeutic protein production and could be used as a model in further lactation researches. In this study, we have isolated bovine mammary gland-derived epithelial cells (BMECs) from Korean Holstein dairy cattle and themselves show differential dynamic ability in in-vitro culture. BMECs enables to form lobulo-alveolar structure, express milk production related gene. Functional studies indicated that BMECs secret exogeneous antibacterial fragment-Bovine Lactoferricin B (bLfcin-B), which is inserted in PiggyBac system, and this bioactive fragment inhibits the growths of Escherichia coli and Staphylococcus aureus. These data demonstrated that BMECs open new scope in either bioactive fragment, heading to prevent the spread of mastitis or post-mastitis damage in dairy graze and could be an ideal bioreactor for antibacterial proteins.

Background : The objective of this study was to investigate antioxidant activities, inhibitory activities against heme induced colonic epithelial cell proliferations, anti-inflammatory activities and anthocyanin profiles in the anthocyanin rich fraction (ARFAM) from fruits of Aronia melanocarpa, where these are considered functional substances and available food coloring agents in Korea. Methods and Results : Anthocyanins were identified by reversed-phase C18 column chromatography and HPLC-DAD-ESI/MS analysis. To compare the antioxidative and anti-inflammatory capacity of Aronia melanocarpa berries, recognized for their high content of anthocyanins, isolation method was developed to obtain high-purity anthocyanins in the extract. Anthocyanin-rich fractions (ARFAM) enriched in anthocyanins were found to be potent strong inhibitory activity towards heme induced colonic epithelial cell proliferations are associated with an increased risk of colon cancer than acidic ethanol extract (AME). The immunomodulation properties were assessed in growth of both human B and T cells, its cytokines secretion such as IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-alpha). AME enhanced interleukin-6 and reduced tumor necrosis factor-a production, whereas ARFAM only had a effect in increasing of IL-6 expression. Conclusion : These results demonstrated that there was no major relationship between the antioxidative and immunomodulation capacities of AME and ARFAM.