Mammalian gametes and embryos are particularly vulnerable to oxidative stress-induced damage, which is mainly caused by reactive oxygen species (ROS) originating from normal metabolism and/or the external environment.  Several researchers have implicated the role of oxidative stress in the activation of apoptosis, causing peroxidative damage to sperms/oocytes and inducing embryo fragmentation, arrest, or demise.  Melatonin is a tryptophan derivative that is known for its powerful free radical-scavenging activity and broad-spectrum antioxidant property.  Numerous studies have shown that melatonin and its metabolic derivatives can sequentially detoxify ROS in an antioxidant cascade, and modulate various antioxidant enzymes via its receptors to prevent radical-mediated damage.  The identification of melatonin receptors in cumulus/granulosa cells, oocytes, and epididymal tissues implies that melatonin has protective actions on gametes and embryos.  Enriching the semen extender or culture medium with melatonin significantly benefits sperm characteristics, improves oocyte maturation potential and quality, and enhances the developmental competence of preimplantation embryos.  Certainly, further comparative studies are needed to show the unique antioxidant role and the advantage of melatonin in this field.  This review summarizes the harmful effects of ROS and the beneficial role of melatonin against oxidative damage of gametes and embryos. 

The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres (groups of at least 30 at the eight-cell stage) were separated into eight segments (to regard an eight-cell embryo as a tangerine, a blastomere as one segment) and one, two and four segments (blastomeres) were cultured respectively in microwells on the bottom of the four-well dish (Nunc, Denmark) with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics (cocultured with nothing, intact embryos, bovine cumulus cells (bCCs), intact embryos & bCCs) were applied to the group of four segments (blastomeres). Finally, diameter and inner cell mass (ICM):trophectoderm (TE) cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group (P<0.05). In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo & bCCs (P<0.05). Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo (P<0.05), more important quality indicator of the blastocyst diameter and ICM:TE cell ratio was similar between our experimental group and the control group (P>0.05). Thus, these results suggest that combined with intact embryos & bCCs coculture system, culturing four isolated segments (blastomeres) per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.