Porcine blastocyst’s quality derived from in vitro is inferior to in vivo derived blastocysts. In this study, to improve in vitro derived blastocyst’s quality and then establish porcine ESCs (pESCs), we treated in vitro fertilized (IVF) embryos and parthenogenetic activated (PA) embryos with three chemicals: porcine granulocyte-macrophage colony stimulating factor (pGM-CSF), resveratrol (RES) and β-mercaptoethanol (β-ME). The control group was produced using M199 media in in vitro maturation (IVM) and porcine zygote medium-3 (PZM3) in in vitro culture (IVC). The treatment group is produced using M199 with 2 μM RES in IVM and PZM5 with 10 ng/mL pGM-CSF, 2 μM RES and 10 μM β-ME in IVC. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. In total, 1210 embryos in PA and 612 embryos in IVF evaluated. As results, we observed overall blastocyst quality was increased. The blastocyst formation rates were significantly higher (p<0.05) in the treatment groups (54.5%) compared to the control group (43.4%) in PA and hatched blastocysts rates in day 6 and 7 were also increased significantly. Total cell numbers of blastocyst were significantly higher (p<0.05) in the treatment group (55.1) compared to the control group (45.6). In IVF, hatched blastocysts rates in day 7 were increased significantly, too. After seeding porcine blastocyst, the attachment rates were higher in the treatment group (36.2% in IVF and 32.2% in PA) than the control group (26.6% in IVF and 19.5% in PA). Also, colonization rates and cell line derivation rates were higher in treatment group than control group. Colonization rates of control group were 10.8% in IVF and 2.4% in PA, but treatment group were 17.75% in IVF, and 13.1% in PA. And we investigated the correlation between state of blastocysts and attachment rate. The highest attachment rate is in hatched blastocyst (78.35±15.74 %). So, the novel system increased quality of porcine blastocysts produced from in vitro, subsequently increased attachment rates. The cell line derivation rates were 4.2% (IVF) and 2.4% (PA) in control group. In treatment group, they were 10.0% (IVF) and 7.2% (PA). We established 3 cell lines from PA blastocysts (1 cell line in control group and 2 cell lines in treatment group). All cell line has alkaline phosphatase activity and express pluri-potent markers. In conclusion, the novel system of IVM and IVC (the treatment of RES during IVM and RES, β-ME, and pGM-CSF during IVC) increased quality of porcine blastocysts produced from in vitro, subsequently increased derivation rates of porcine putative ESCs.

In this study, we examined the effects of porcine granulocyte-macrophage colonystimulating factor (pGM-CSF) on in vitro development of porcine embryos produced by somatic cell nuclear transfer (SCNT) at first time. The objective of present study was to verify effects of pGM-CSF on SCNT-derived blastocyst formation and evaluate gene expressions and qualities of the blastocyst formed after pGM-CSF treatment. Data were analyzed with SPSS 17.0 using Duncan’s multiple range test. A total 522 cloned embryos in 6 replicates were treated with 10 ng/ml concentration of pGM-CSF during in vitro culture (IVC). It was demonstrated that treatment of 10 ng/ml pGM-CSF could increase blastocyst formation and total cell number in blastocyst significantly (p<0.05) compared to the control (12.3% and 41.4 vs. 9.0% and 34.7, respectively). However, there was no any effect on cleavage rate. It was found that the number of cells in the inner cell mass (ICM) and trophectoderm (TE) were significantly increased compared to the control (4.4 and 31.9, respectively) when cloned embryos were cultured with 10 ng/ml pGM-CSF (6.0 and 43.0, respectively). It was also found that treatment of 10 ng/ml pGM-CSF significantly (p<0.05) increased POU5F1 and Cdx2 mRNA expressions in blastocysts. In addition, Bcl-2 mRNA expression was found to be significantly (p< 0.05) up-regulated in blastocysts in the pGM-CSF supplemented group compared to the control. In conclusion, these results suggest that pGM-CSF may improve the quality and developmental viability of porcine cloned embryos by enhancing nuclear reprogramming via regulating transcription factors expression.