We aimed to investigate the effect of melatonin on bovine frozen-thawed semen and its impact on fertilization outcome. Plasma membrane integrity, mitochondrial activity, acrosome integrity, and levels of intracellular reactive oxygen species (ROS) were measured in spermatozoa treated with different concentrations of melatonin. Melatonin-treated spermatozoa were then used for in vitro fertilization, followed by analysis of subsequent embryo development and the expression of apoptosis- and antioxidant-related genes. The results revealed that 10 and 10 M melatonin led to higher plasma membrane integrity, mitochondrial activity, and acrosome integrity, and significantly decreased intracellular ROS levels (P < 0.05). The blastocyst development rate of in vitro-produced bovine embryos originating from 10 M melatonin-treated spermatozoa was significantly higher, while the incidence of apoptotic nuclei in blastocysts was markedly lower than for embryos from any other group (P < 0.05). CASP3 and BAX mRNA abundance were significantly reduced whereas BCL2, XIAP, and CAT transcript abundance were significantly increased in embryos produced from spermatozoa treated with 10 M melatonin; GPX4 expression, however, was comparable in all treatment groups. Thus, 10 M melatonin can improve the quality of bovine frozen-thawed semen. These beneficial effects appear to influence preimplantation embryos, given the correlation with its anti-apoptotic and anti-oxidative properties. Mol. Reprod. Dev. 83: 993-1002, 2016 © 2016 Wiley Periodicals, Inc.

Preimplantation embryos are sensitive to oxidative stress-induced damage that can be caused by reactive oxygen species (ROS) originating from normal embryonic metabolism and/or the external surroundings. Paraquat (PQ), a commonly used pesticide and potent ROS generator, can induce embryotoxicity. The present study aimed to investigate the effects of melatonin on PQ-induced damage during embryonic development in bovine preimplantation embryos. PQ treatment significantly reduced the ability of bovine embryos to develop to the blastocyst stage, and the addition of melatonin markedly reversed the developmental failure caused by PQ (20.9% versus 14.3%). Apoptotic assay showed that melatonin pretreatment did not change the total cell number in blastocysts, but the incidence of apoptotic nuclei and the release of cytochrome c were significantly decreased. Using real-time quantitative polymerase chain reaction analysis, we found that melatonin pre-incubation significantly altered the expression levels of genes associated with redox signaling, particularly by attenuating the transcript level of Txnip and reinforcing the expression of Trx. Furthermore, melatonin pretreatment significantly reduced the expression of the pro-apoptotic caspase-3 and Bax, while the expression of the anti-apoptotic Bcl-2 and XIAP was unaffected. Western blot analysis showed that melatonin protected bovine embryos from PQ-induced damage in a p38-dependent manner, but extracellular signal-regulated kinase (ERK) and c-JUN N-terminal kinase (JNK) did not appear to be involved. Together, these results identify an underlying mechanism by which melatonin enhances the developmental potential of bovine preimplantation embryos under oxidative stress conditions.

The present study investigated the effects of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. Results showed that the nuclear and cytoplasmic maturation, characterized by first polar body extrusion, normal distribution of cortical granules and mitochondria, as well as increased mitochondrial membrane potential, were significantly improved in 10 mol/L melatonin-treated oocytes. Melatonin supplementation reduced intracellular reactive oxygen species level and enhanced glutathione production. Meanwhile, the presence of melatonin (10 mol/L) during oocyte maturation resulted in a decreased early apoptotic rate in oocytes. After in vitro fertilization, oocytes receiving melatonin supplementation exhibited a significantly higher blastocyst formation rate and yielded a markedly lower number of apoptotic cells. Mechanistic explorations showed that addition of 10 mol/L melatonin to in vitro maturation media significantly attenuated the transcript level of caspase-3, while the expressions of BCL-2, XIAP, CAT and HSP70 were significantly reinforced in the resultant embryos. Taken together, melatonin ameliorates bovine oocyte maturation potential, and the beneficial effects can affect subsequent embryonic development. The protective role of melatonin may be due to its anti-apoptotic and anti-oxidative activities.