Research On The Isolation And Cultivation Of Porcine Parthenogenetic Embryonic Stem Cells And Oocytes Maturation In Vitro

This study has taken abandoned porcine ovarian from slaughterhouse as the original materials to research oocytes in vitro maturation and the cultivation of parthenogenetic embryonic stem cells. We used brilliant cresyl blue(BCB) to select the oocytes and by adding db-cAMP to achieve the synchronous mature between nuclear and cytoplast to improve the quality of in vitro matured oocytes. Then, we used parthenogenetic blastocysts in subsequent experiments to study the isolated and cultured of parthenogenetic embryonic stem cells. The main results were as follows:1. In this study, different concentrations of BCB(13?26?39 and 52 ?mol/L) were used to stain oocytes for 90 min, and then compared the numbers of BCB+ and BCBrespectively. After IVM, oocytes maturation rates and developmental competence of early embryos were evaluated. The results of the test indicated that, the percentages of staining rate and maturation rate from oocytes staining by 26 ?mol/L BCB for 90 min were higher than that of other groups. The cleavage and blastocyst rate of parthenogenetic activation and SCN embryos in BCB+ group were significantly higher than that of BCB- group(P<0.05). Reconstructed embryos stained by BCB were transferred to five surrogates, and six cloned piglets were obtained from one of the two pregnant pigs.2. As the concentration of db-cAMP increased(0?0.5?1.0?1.5 and 2 mmol/L), the maturation rate of oocytes reduced successively. But the blastocyst rate of 1 mmol/L group(27.03%) was significant higher than others(P<0.05); When oocytes were co-cultured with pig cumulus cell monolayer, the maturation rate?cleavage rate and blastocyst rate(86.00%?89.04%?28.36%) were significantly improved. 0.4 mmol/L db-cAMP co-cultured with the monolayer cumulus cell had no significant difference in maturation rate and cleavage rate(85.78% and 88.95%), but the blastocyst rate(35.14%) were significantly improved than others. 3. The embryonic age, feeding layer and treatment of blastocysts were studied on the cultivation of parthenogenetic embryonic stem cells. The test was divided into three groups. the blastocysts with 8 day shown the highest attachment rate(25.00%); ? ?Take the blastocyst with 8 days to remove the zona pellucida and placed on the feeder layer made by mouse fetal fibroblast cells, the adherent rate was 18.52%, while pig fetal fibroblasts and uterine epithelial cells were no adherent blastocyst; Taking the ?blastocyst with 8 days after parthenogenetic activation and divided into four groups including whole blastocyst, hatched blastocyst group, removed the zona pellucida and isolated inner cell mass. The adherent rate of isolated inner cell mass was significantly higher than other groups(P<0.05), and we get six outgrowth in subsequent culture.