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.

• Seung-A Cheong(Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea)
• Seong-Sung Kwak(Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea)
• Yubyeol Jeon(Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea)
• Junchul David Yoon(Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea)
• Sang-Hwan Hyun(Laboratory of Veterinary Embryology and Biotechnology, College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea)