Overproduction of reactive oxygen species (ROS) during sperm cryopreservation has a detrimental effect on sperm parameters. Therefore, the use of antioxidants in the sperm freezing extender can reduce ROS destructive effects. In this study, we investigated whether co-supplementation of melatonin and myo-inositol into the semen extender can improve the post-cryopreservation quality of goat spermatozoa. After the freeze-thawing process, sperm motility, viability, plasma membrane and acrosome intact morphology were improved in the combined myo-inositol and melatonin group compared to both individual and the control groups (p < .05). In addition, the mean of sperm ROS, DNA damage and lipid peroxidation were reduced in co-supplementation of myo-inositol and melatonin compared to their individual counterparts (p < .05).Therefore, the synergistic effects of myo-inositol and melatonin on the cryopreserved spermatozoa are highly likely mediated through the reduction in important factors involved in the sperm lipid peroxidation. Finally, we used the cryopreserved spermatozoa for in vitro production of embryos. Results showed that combined group of myo-inositol and melatonin improved the cleavage rate compared to both individual and control groups, although blastocyst rate was improved using both individual and combined groups. In conclusion, co-supplementation of melatonin and myo-inositol is a promising approach for the improvement of goat sperm cryopreservation.

SCNT embryos suffer from poor developmental competence (both in vitro and in vivo) due to various defects such as oxidative stress, incomplete epigenetic reprogramming, and flaws in telomere rejuvenation. It is very promising to ameliorate all these defects in SCNT embryos by supplementing the culture medium with a single compound. It has been demonstrated that melatonin, as a multitasking molecule, can improve the development of SCNT embryos, but its function during ovine SCNT embryos is unclear. We observed that supplementation of embryonic culture medium with 10 nM melatonin for 7 days accelerated the rate of blastocyst formation in ovine SCNT embryos. In addition, the quality of blastocysts increased in the melatonin-treated group compared with the SCNT control groups in terms of ICM, TE, total cell number, and mRNA expression of NANOG. Mechanistic studies in this study revealed that the melatonin-treated group had significantly lower ROS level, apoptotic cell ratio, and mRNA expression of CASPASE-3 and BAX/BCL2 ratio. In addition, melatonin promotes mitochondrial membrane potential and autophagy status (higher number of LC3B dots). Our results indicate that melatonin decreased the global level of 5mC and increased the level of H3K9ac in the treated blastocyst group compared with the blastocysts in the control group. More importantly, we demonstrated for the first time that melatonin treatment promoted telomere elongation in ovine SCNT embryos. This result offers the possibility of better development of ovine SCNT embryos after implantation. We concluded that melatonin can accelerate the reprogramming of telomere length in sheep SCNT embryos, in addition to its various beneficial effects such as increasing antioxidant capacity, reducing DNA damage, and improving the quality of derived blastocysts, all of which led to a higher in vitro development rate.

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Abstract. Sonic Hedgehog (SHH) is a signaling pathway mediated through a receptor system which seems to have effects on oocyte maturation and embryonic development. Purmorphamine is an SHH agonist that performs a crucial role in the regulation of the activity of SHH receptors and downstream transcription factors. The aim of this study was to analyze the effect of purmorphamine on the mRNA expression of SHH signaling downstream molecules (Patched1, Glioma-Associated Oncogene1, Smoothened, Histone Deacetylase1, Histone Deacetylase2 and Histone Deacetylase3) in ovine two-cell embryo. Ovaries were obtained from a slaughterhouse, and cumulus-oocyte complexes were aspirated and cultured in maturation media containing 0, 250 or 500 ng mL −1 purmorphamine. Then, oocytes were fertilized and cultured in a CR1 culture medium and after 24 h, two-cell embryos were collected for RNA extraction. Gene expression was evaluated by real-time polymerase chain reaction (PCR). Results indicated that in 250 ng mL −1 purmorphamine, Smo, Ptch1 and Hdac3 expression reduced, Hdac1 expression increased, and Gli1 and Hdac2 expression levels did not change. In 500 ng mL −1 purmorphamine, Gli1 and Smo transcripts increased, while Ptch1, Hdac2 and Hdac3 transcripts decreased. Regarding to the presence of SHH signaling molecules in two-cell embryos and their response to purmorphamine, it can be suggested that SHH signaling is probably active before embryonic genome activation in ovine embryos.