The present study was undertaken to examine the effect of Scriptaid treatment on histone acetylation, DNA methylation, expression of genes related to histone acetylation, and development of buffalo somatic cell nuclear transfer (SCNT) embryos. Treatment of buffalo SCNT embryos with 500 nM Scriptaid for 24 h resulted in a significant increase in the blastocyst formation rate (28.2% vs. 13.6%, p<0.05). Meanwhile, treatment of buffalo SCNT embryos with Scriptaid also resulted in higher acetylation levels of H3K18 and lower methylation levels of global DNA at the blastocyst stage, which was similar to fertilized counterparts. The expression levels of CBP, p300, HAT1, Dnmt1, and Dnmt3a in SCNT embryos treated with Scriptaid were significantly lower than the control group at the eight-cell stage (p<0.05), but the expression of HAT1 and Dnmt1a was higher than the control group at the blastocyst stage (p<0.05). When 96 blastocysts developed from Scriptaid-treated SCNT embryos were transferred into 48 recipients, 11 recipients (22.9%) became pregnant, whereas only one recipient (11.1%) became pregnant following transfer of 18 blastocysts developed from untreated SCNT embryos into nine recipients. These results indicate that treatment of buffalo SCNT embryos with Scriptaid can improve their developmental competence, and this action is mediated by resulting in a similar histone acetylation level and global DNA methylation level compared to in vitro-fertilized embryos through regulating the expression pattern of genes related to histone acetylation and DNA methylation.

The DNA methylation of imprinted genes is an important way to regulate epigenetic reprogramming of donor cells in somatic cell nuclear transfer (SCNT). However, the effects of sexual distinction on the DNA methylation of imprinted genes in cloned animals have seldom been reported. In this study, we analysed the DNA methylation status of three imprinted genes (Xist, IGF2 and H19) from liveborn cloned buffaloes (L group, three female and three male), stillborn cloned buffaloes (S group, three female and three male) and natural reproduction buffaloes (N group, three female and three male), using bisulphite sequencing polymerase chain reaction (BS-PCR). The expression levels of these imprinted genes were also investigated by quantitative real-time PCR (QRT-PCR). The DNA methylation levels of H19 were not significantly different among the groups. However, the Xist in female and IGF2 in male of the S group were found to be significantly hypomethylated in comparison with the same sexual buffaloes in L group and N group (p < .05). Furthermore, the expression levels of Xist, IGF2 and H19 in the stillborn female cloned buffaloes of S group were significantly higher than that of the female buffaloes in the L group and N group (p < .05). The expression levels of IGF2 and H19 in the stillborn male cloned buffaloes in the S group were significantly higher than that of the male buffaloes in the L group and N group (p < .05). These results indicate that Xist may be associated with the viability of female cloned buffaloes, and IGF2 may also be related to the viability of male cloned buffaloes.

Somatic cell nuclear transfer (SCNT) holds vast potential in agriculture. However, its applications are still limited by its low efficiency. Histone 3 lysine 9 trimethylation (H3K9me3) was identified as an epigenetic barrier for this. Histone demethylase KDM4D could regulate the level of H3K9me3. However, its effects on buffalo SCNT embryos are still unclear. Thus, we performed this study to explore the effects and underlying mechanism of KDM4D on buffalo SCNT embryos. The results revealed that compared with the IVF embryos, the expression level of KDM4D in SCNT embryos was significantly lower at 8- and 16-cell stage, while the level of H3K9me3 in SCNT embryos was significantly higher at 2-cell, 8-cell, and blastocyst stage. Microinjection of KDM4D mRNA could promote the developmental ability of buffalo SCNT embryos. Furthermore, the expression level of ZGA-related genes such as ZSCAN5B, SNAI1, eIF-3a, and TRC at the 8-cell stage was significantly increased. Meanwhile, the pluripotency-related genes like POU5F1, SOX2, and NANOG were also significantly promoted at the blastocyst stage. The results were reversed after KDM4D was inhibited. Altogether, these results revealed that KDM4D could correct the H3K9me3 level, increase the expression level of ZGA and pluripotency-related genes, and finally, promote the developmental competence of buffalo SCNT embryos.

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