Porcine embryonic stem cells (pESCs) have great potential for application in translational biomedical research, including xenotransplantation and disease models. Obtaining high-quality blastocysts is the most important factor in the isolation and colonization of primary ESCs and the establishment of ESC lines. In pigs, in vitro-derived blastocysts have a limited cell number compared to in vivo-derived blastocysts and show an indefinite inner cell mass, which may result in failure to establish pESC lines. In the present study, the effects of resveratrol (RES), granulocyte-macrophage colony stimulating factor (GM-CSF) and β-mercaptoethanol (β-ME) on the quality of blastocysts and the efficiency of colony derivation were investigated for the establishment of ESCs. A novel culturing system was developed in which 2 µM RES was added to the oocyte in vitro maturation (IVM) medium, and 10 ng/ml pGM-CSF and 10 µM β-ME were added to embryo in vitro culture (IVC) medium. This novel system showed significantly more parthenogenetic activation (PA) blastocysts (54.5 ± 1.8% vs. 43.4 ± 1.2%; P<0.05) and in vitro fertilization (IVF) blastocysts (36.9 ± 3.3% vs. 26.2 ± 2.9%; P<0.06) at day seven as compared with that in the control system. The PA and IVF blastocysts from the novel system showed a significantly greater hatching rate (P<0.05) and greater cell numbers (55.1 ± 2.0 vs. 45.6 ± 2.0; P<0.05 and 78.9 ± 6.8 vs. 58.5 ± 7.2; P<0.06, for PA and IVF, respectively) at day seven compared to that in the control system. After seeding on feeder cells, the PA blastocysts produced by the novel system showed a significantly increased rate of attachment (28.8 ± 3.9% vs. 17.2 ± 2.4%; P<0.062). Finally, two putative pESC lines from PA embryos produced by the novel system and one by the control system were established. In conclusion, the novel system improved blastocyst quality and increased the derivation efficiency of putative pESC lines from porcine PA and IVF embryos produced in vitro.

Interspecies somatic cell nuclear transfer (iSCNT) could be a useful method for embryo research of wildlife animals or endangered species. Because it is hard to obtain the oocytes or embryos of wildlife animals, its embryo research is not progressing well. Therefore, iSCNT is one of the alternative ways for wildlife animal embryo research and conservation of their genetic source. Until now, iSCNT has been applied to conservation of wildlife animals including guar, mouflon, banteng and African wildcat. The domestic pig oocytes have been used for iSCNT of other species such as tiger, sheep and dog and they successfully developed to the blastocyst stage. According to this concept, we performed wild-captured Korean raccoon (Nyctereutes procyonoides koreensis) iSCNT using porcine oocytes matured in vitro. Raccoon fibroblasts from ear skin samples of male raccoon were used as donor cells in 3 to 5 passages. The donor cells were cultured in DMEM supplemented with 15% FBS. Enucleated porcine oocytes were fused with raccoon fibroblasts by electrofusion. The iSCNT embryos were cultured in PZM-3 at 39°C for 7 days in an atmosphere of 5% CO2 and 5% O2. A total of 158 iSCNT embryos were cultured. More than 77% of the raccoon somatic cells successfully fused with the porcine oocytes; 68.5% of the iSCNT raccoon embryos developed to the 2- to 8-cell stage at Day 2 (1-cell: 9.7%, 2-cell: 14.4%, 4-cell: 34.1%, 6-cell: 12.7%, 8-cell: 7.3%, fragmented: 21.8%). This is similar to porcine SCNT results that 62.5% of the SCNT porcine embryos developed (1-cell: 8.0%, 2-cell: 4.2%, 4-cell: 23.6%, 6-cell: 13.6%, 8-cell: 23.8%, fragmented: 26.8%). But no embryos were further developed to blastocyst stage at Day 7 in iSCNT. In fragmentation evaluation in iSCNT embryos using by Hoechst stain at Day 2, two-cell stage embryos and four-cell stage embryos showed the normal numbers of nucleus. However, 6-cell stage embryos showed 4 to 5 nuclei and 8-cell stage embryos also showed 5 to 6 nuclei. Almost iSCNT embryos showed the developmental block at 4-cell stage embryos. This result was probably caused by an incomplete reprogrammed raccoon cell after iSCNT. Therefore, we treated with trichostatin A (TSA), a histone deacetylase inhibitor that has been used to enhance nuclear reprogramming following SCNT. Ninety-seven iSCNT raccoon-pig embryos were treated with 5 nM TSA during 15 h before being cultured in PZM-3. The TSA-treated iSCNT embryos showed similar developmental status to non-treated embryos (1-cell: 13.5%, 2-cell: 12.5%, 4-cell: 35.0%, 6-cell: 10.1%, 8-cell: 6.3%, fragmented: 22.5%). No embryos were further developed to blastocyst stage at day 7. Our results showed that 4-cell stage embryos of raccoon-porcine iSCNT embryos may be produced by iSCNT methods, but they were unable to support complete reprogramming of raccoon-porcine iSCNT embryos. This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. 007133022011), Rural Development Administration, Republic of Korea.

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Previous studies have demonstrated that treatment of cloned embryos with trichostatin A (TSA) or scriptaid, inhibitors of class I and II histone deacetylases (HDAC), significantly enhanced their developmental competence. In the present study, we investigated the effects of sirtinol, an inhibitor of class III HDAC, on the embryonic development of porcine cloned embryos. Data were analyzed with SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) using Duncan’s multiple range test and all experiments were replicated at least 5 times. In experiment 1, 648 parthenotes were divided into 4 groups (0-, 6-, 12-, and 24-h sirtinol treatment after activation) to investigate optimal treatment time using 100 µM sirtinol. The cleavage rate of the 24-h treatment group (81.3%) was significantly (P < 0.05) decreased compared with the 12-h treatment group (88.4%) but there was no difference compared with the control (86.9%) and 6-h treatment groups (86.9%). The parthenotes treated with sirtinol for 12 h after activation had a significantly higher blastocyst formation rate and total cell number in blastocysts (50.5% and 66.9, respectively) than the control (39.4% and 54.1, respectively). In experiment 2, 806 cloned embryos were divided into 5 groups (0, 50, 100, 150, and 200 µM sirtinol treatment for 12 h after activation) to investigate optimal concentration. There was no significant difference in cleavage rate. The rate of blastocyst formation and total cell number in blastocysts were significantly (P < 0.05) improved by treatment with 150 µM sirtinol for 12 h after activation (28.8% and 51.0, respectively) compared with the control (17.5% and 37.1, respectively). The total cell number in blastocysts was also significantly increased in 50 and 200 µM groups (47.9 and 48.4, respectively) compared with the control (37.1). In experiment 3, we examined the effects of 150 µM sirtinol treatment for 12 h after activation with or without 5 nM TSA on in vitro embryonic development after somatic cell nuclear transfer. The rate of blastocyst formation was significantly improved in sirtinol-treated and TSA-treated groups (30.9 and 31.3%, respectively) but not in the sirtinol with TSA group (27.6%) compared with the control (21.7%). The total cell number in blastocysts was significantly increased by treatment with sirtinol and TSA together (73.9) compared with the control (49.0) but there was no difference in only sirtinol- (59.8) or TSA- (59.2) treated groups. There was no significant difference in cleavage rate among groups. Our results suggest that sirtinol improves the embryonic development of porcine cloned embryos and sirtinol with TSA synergistically increases the blastocyst quality. This work was supported by a grant from the Next-Generation BioGreen 21 program (no. PJ008121012011), Rural Development Administration, Republic of Korea.