Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs) were collected from eighteen lambs which were randomly divided into control group (C) and melatonin group given an 18 mg melatonin implant subcutaneous (M). Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05), greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05). Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05). Furthermore, melatonin increased FI of FITC (global methylation level) on cumulus cells (p < 0.05). To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05) and higher methylation at two CpG sites of DNMT3a (p < 0.05), there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05). In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs.

A time-lapse live embryo monitoring system provides a powerful approach to recording dynamic developmental events of cultured embryos in detail. By obtaining continuous short-interval images, blastocyst formation can be predicted and embryos can be selected. The objective of this study was to investigate the morphokinetic parameters of fishing cat–domestic cat interspecies somatic cell nuclear transfer (iSCNT) embryos from one-cell to blastocyst stages, and in particular, the cleavage patterns of the first division in iSCNT and IVF embryos, as these play a central role in euploidy. Domestic cat in vitro fertilization (IVF) embryos were set up as controls. The results show that morula and blastocyst development rates were significantly lower in the iSCNT embryos compared to their IVF counterparts. All earlier time points of embryonic development before the onset of blastulation in the iSCNT embryos were significantly delayed when compared with their IVF counterparts. In iSCNT, normal embryos (defined as those that developed to the blastocyst stage) took a longer time to reach the morula stage, and these morulas were more likely to undergo compaction, compared to their arrested embryo counterparts. Direct cleavage in the first division is a morphological aberration, and was seen with greater prevalence in iSCNT embryos than control IVF embryos; these aberrant embryos displayed a significantly lower blastocyst development rate than embryos that had undergone normal cleavage. In conclusion, the morphokinetic parameters of fishing cat–domestic cat iSCNT embryos at early stages could be used to predict their potential for development to the blastocyst stage. A time-lapse imaging system is potentially a powerful tool for selecting early embryos with developmental potential for transfer, and hence, for improving feline iSCNT efficiency.