The present study evaluated the influence of rumen inocula of different breeds on in vitro fermentation with forage and concentrate substrates. An in vitro was conducted under a 2×2 factorial arrangement with two breeds (Jersey and Holstein steers) and two feed substrates (forage and concentrate) as factors. Three Jersey and 3 Holstein steers were used for the source of in vitro inocula. Metataxonomic analysis of donor rumen fluids showed that Firmicutes was more abundant in Jersey, while Bacteroidetes in Holstein steers. In vitro ㏗ was lower in the fermented inocula of Jersey steers and in the concentrate substrate (p<0.05). After 24h, higher gas production, dry matter, and neutral detergent fiber degradability, and total volatile fatty acids concentration were noted in concentrate substrate (p<0.05). After 24h, inocula of Jersey steers had higher methane and ammonia-nitrogen (p<0.05). After 24h, fermented inocula of Holstein steers produced higher propionate (p<0.05). Conversely, in vitro butyrate production was higher in the fermented inocula from Jersey steers (p=0.072) and in those with concentrate substrate (p<0.05). After 24h, the total bacterial population (log10 c opies) was h igher in t he fermented inocula received from Jersey steers and in the concentrate substrate whereas, Fibrobacter succinogenes and Ruminococcus flavefaciens population were higher (p<0.05) only in the concentrate substrate. Overall results suggest that rumen inocula of different donors influence in vitro fermentation either with forage or concentrate substrates.

For useful research animal to study human’s disease and for xenotransplantation donor, pig was studied to improve the quality of in vitro production (IVP). But, still the developmental ability of in vitro porcine embryos is still lower than in vivo embryos. Using a antioxidant is one of the strategy to overcome the drawback of in vitro producted embryos by protecting the oocyte from free radicals during in vitro maturation (IVM). Resveratrol, one of the plant-derived polyphenol antioxidants, have been used as effective antioxidants. Therefore, resveratrol treatment during IVM of porcine oocytes is expected to improve efficiency of the IVP by reducing free radical accumulation. In this study, we designed control (no treated) and resveratrol treatment groups (0, 2 and 4uM), evaluated maturation rate, cleavage rate, blastocyst formation rate and total cell number. Additionally GSH and ROS accumulation levels were measured via staining oocytes. In the results, maturation rate had not shown significant difference among the groups. However, in further development, not only the results of cleavage rate (0uM : 84.64±2.65 vs 4uM : 93.67±2.36, p<0.05) and blastocyst formation rate (0uM : 6.39± 0.90, vs 4uM : 13.67±2.32, P<0.05) were significantly increased in 4uM resveratrol treated group, and result of total cell number (0uM : 22.47±0.76 vs 2, 4uM : 30.35±1.76, 27.65±1.23, P<0.05) also shown significant difference in 2, 4uM resveratrol groups with control. GSH accumulated levels of matured oocytes in resvetrol treated groups were significantly higher than control. Meanwhile ROS levels of treated groups were significantly reduced [GSH (0uM : 142±10.49 vs 2, 4uM : 163.2±3.29, 169.7±0.94, P<0.05), ROS (0uM : 170.2±7.76 vs 2, 4uM : 118.6±7.90, 130±7.07, P<0.05)]. From these results, we conclude the treatment of resveratol improved further development of porcine embryos by regulating intracellular GSH, ROS levels during porcine IVM. Therefore, exogenous antioxidants such as a resveratol can be supportive substances for obtaining the improved quality of IVP.

The present study was conducted to develop a simple method for porcine oocyte maturation without CO2 regulation. In experiment 1, we evaluated that the effect of CO2 non-supplement on porcine oocyte maturation. Cumulusoocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three groups (Control, tube-CO2, and tube-non-CO2). For control, COCs were cultured in 4-well multidish in a CO2 incubator. For tube-CO2, COCs were cultured in a round-bottom tube in a CO2 incubator, and for tube-non-CO2, COCs were cultured in a round-bottom tube sealed tightly without CO2 supplement in a dry incubator. The proportion of oocytes reached to metaphase II (M-II) was not significantly different among three groups (87.9% to 91.4%). In experiment 2, we evaluated the effect of CO2 non-supplement during oocyte maturation on development of embryos. Oocytes with a polar body were divided into two groups (Control and tube-non-CO2) and applied 1.1 kV/cm or 1.2 kV/cm voltages for parthenogenetic activation. After activation, embryos were cultured for 6 days and examined the development. The proportion of embryos cleaved was not significantly different among treatment (86.3% to 91.5%). The proportion of embryo reached to blastocyst stage was not significantly different among treatment (13.9% to 25.2%). The cell number of blastocysts was not significantly different among treatment (29.0 to 32.4). In conclusion, oocytes cultured in a dry incubator without CO2 supplement have enough competence to development after parthenogenetic activation. These results would be useful for transporting oocytes or embryos a long distance.

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

Correlations between cumulus cells and germinal vesicle (GV) chromatin configuration were examined in porcine oocytes. Cumulus-oocyte complexes (COCs) were collected from 2～6 mm follicles and divided into three categories according to cumulus cell morphology. "A" group was compacted COCs with more than three cumulus cell layers. "B" group was COCs with less cumulus cell layers than "A" group. "C" group was COCs with one or less layer of cumulus cells. Cumulus cells were removed 0.1% hyaluronidase, and denuded oocytes were stained with Hoechst 33342. GV chromatin configuration was classified into GV-Con and GV-Dis. GV-Con meant that a nucleus was surrounded by condensed chromatin in a ring. GV-Dis meant that filamentous chromatin clumps were distributed in nucleus. The proportion (80.2%) of GV-Con in "A" group was significantly higher than "B" (62.0%) or "C" (44.9%). The proportion (55.1%) of GV-Dis in "C" group was significantly higher than "A" (19.8%) or "B" (38.0%). The meiotic competence of COCs was examined after 44 h culture. The proportion (90.0%) of oocytes reaching to metaphase II (M-II) in "A" group was significantly higher than "B" (76.5%) or "C" (45.5%). In conclusion, oocytes with good quality cumulus cell layers are synchronized early GV stage, and early GV stage is important for meiotic competence in pigs.

Pig organ is thought to be the most suitable nonhuman organ for xenotransplanstation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of a(1,3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute rejection. Many efforts have made characterization of GGTA1 in structure and function, improvement in the technique of DNA transfection of somatic cells and advancement of the pig NT, a specific modification has been made to one copy of the GGTAl gene by Missouri group in 2002 To date because homozygousity of the genetic modification has been achieved in this gene, the role of gala(1,3) gal specific natural antibody in HAR and the efficacy of xenotransplantation in a nonhuman primate model will be addressed. Of other genes are found to be involved in rejection of pig donors by primates, the technology will be available to modify those genes so that rejection can be overcome.