This study aimed at investigating whether a porcine follicular fluid (pFF) supplementation positively affects the characteristics of donor cells and the developmental competence of porcine cloned embryos. Ear fibroblast cells (donor cell) from an Massachusetts General Hospital miniature pig were cultured in different culture methods: (1) Dulbecco's modified Eagle's medium (DMEM)+10% FBS (Control); (2) DMEM+0.5% FBS (SS); and (3) DMEM+10% FBS+10% pFF (pFF) for 72 h. In each conditioned medium, the concentrations of 4 amino acids (Thr, Glu, Pro, and Val) in the pFF group were significantly different from those in the control group (p<0.05 or p<0.01). The proliferation of the cells cultured in the SS group was significantly lower than that of the other treatment groups (p<0.01). The population of apoptotic and necrotic cells in the SS group was significantly higher than that of either the control or the pFF group (p<0.01). The number of embryos that cleaved (p<0.05) and developed into blastocysts (p<0.01) in the SS group was significantly lower than that of either the control or the pFF group. Compared to other groups, the blastocysts produced from the donor cells in the pFF group had higher total cells and lower apoptotic cells (p<0.05). It can be concluded that pFF supplementation in the donor cell culture medium positively affects cell death, cell cycle and quality of the cloned blastocyst.

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.