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Aberrant DNA methylation reduces the developmental competence of mammalian somatic cell nuclear transfer (SCNT) embryos. Thus, hypomethylation-associated drugs are beneficial for improving reprogramming efficiency. Therefore, in the present study we investigated the effect of zebularine, a relatively novel DNA methyltransferase inhibitor, on the developmental potential of ovine SCNT embryos. First, reduced overall DNA methylation patterns and gene-specific DNA methylation levels at the promoter regions of pluripotency genes (octamer-binding transcription factor 4 (Oct4), SRY (sex determining region Y)-box 2 (Sox2) and Nanog) were found in zebularine-treated cumulus cells. In addition, the DNA methylation levels in SCNT embryos derived from zebularine-treated cumulus cells were significantly reduced at the 2-, 4-, 8-cell, and blastocyst stages compared with their corresponding controls (P < 0.05). The blastocyst rate was significantly improved in SCNT embryos reconstructed by the cumulus donor cells treated with 5 nM zebularine for 12 h compared with the control group (25.4±1.6 vs 11.8±1.7%, P < 0.05). Moreover, the abundance of Oct4 and Sox2 mRNA was significantly increased during the preimplantation stages after zebularine treatment (P < 0.05). In conclusion, the results indicate that, in an ovine model, zebularine decreases overall DNA methylation levels in donor cumulus cells and reconstructed embryos, downregulates the DNA methylation profile in the promoter region of pluripotency genes in donor cells and ultimately elevates the expression of pluripotency genes in the reconstructed embryos, which can lead to improved development of SCNT embryos.

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Background: Fixed-time artificial insemination (FTAI) uses exogenous reproductive hormones to regulate the sexual cycle of sows and realize the mating of pigs at a fixed-time without estrus identification. There is a great difference in litter size following FTAI, which may be due to the ovarian status before treatment. However, the specific underlying mechanism remains unclear.Results: We selected replacement gilts (n=104) and divided them into four groups: CON (n=35), EST (n=16), pregnant mare serum gonadotrophin (PMSG)-15D (n=26), and PMSG-20D (n=27). The PMSG-15D and PMSG-20D groups were pretreated with PMSG for 15 and 20 days, respectively. The four groups were then subjected to the same FTAI treatment. Pretreatment with PMSG causes ovulation in the gilts, to artificially promote an estrus cycle. At 42 hours after ALT feeding stopped, The follicular diameter of PMSG-15D group (2.94±0.24 mm) was significantly higher (P<0.05) than the other groups.，and the proportion of gilts with follicular diameter greater than 3 mm in the PMSG-15D group was significantly higher than that in the CON group (37.50% vs 5.88%). The maximum follicular diameter before ovulation in the PMSG-15D group (6.28±0.23 mm) was significantly larger (P<0.05) than that of the PMSG-20D group (5.98±0.59 mm) and EST group (5.60±0.47 mm), indicating that the follicular development of the PMSG-15D group was better than that of the other groups. The ovulation time of the PMSG-15D group was concentrated on the 25th to 26th day of FTAI. The ovulation rate was as high as 94.5% in the optimal insemination window period and was more suitable for FTAI. Compared with the CON (52.70±12.71 h) and the PMSG-20D (47.88±13.98 h) groups, the GnRH to ovulation interval (42.33±5.87 h) was significantly shortened in the PMSG-15D group. The level of follicle-stimulating hormone of gilts in the PMSG-15D group was more uniform and significantly lower than that in the CON group (P<0.01) at 42 h after Altrenogest feeding. The pregnancy rate (80.77%), total litter size (10.44±2.83), and live litter size (10.00±2.61) of the PMSG-15D group were higher than those of the other groups.Conclusions: We feasibly optimized FTAI using PMSG pretreatments. When the ovaries of replacement gilts were in the luteal stage owing to PMSG pretreatment for 15 days, the follicular development and reproductive performance of replacement gilts were significantly improved.

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