Melatonin is a well‐characterized antioxidant that has been successfully used to protect oocytes from reactive oxygen species during in vitro maturation (IVM), resulting in improved fertilization capacity and development ability. However, the mechanism via which melatonin improves oocyte fertilization capacity and development ability remains to be determined. Here, we studied the effects of melatonin on cytoplasmic maturation of bovine oocytes. In the present study, bovine oocytes were cultured in IVM medium supplemented with 0, 10^–7, 10^–9, and 10⁻¹¹ mol/L melatonin, and the cytoplasmic maturation parameters of MII oocytes after IVM were investigated, including redistribution of organelles (mitochondria, cortical granules [CGs], and endoplasmic reticulum [ER]), intracellular glutathione (GSH) and ATP levels, expression of endogenous antioxidant genes (Cat, Sod1, and GPx), and fertilization‐related events (IP3R1 distribution and expression of CD9 and Juno). Our results showed that melatonin significantly improved the cytoplasmic maturation of bovine oocytes by improving the normal distribution of organelles, increasing intracellular GSH and ATP levels, enhancing antioxidant gene expression levels, and modulating fertilization‐related events, all of which resulted in increased fertilization capacity and developmental ability. Meanwhile, melatonin also increased the mRNA and protein expression levels of the Tet1 gene and decreased the Dnmt1 gene mRNA and protein levels in bovine oocytes, indicating that melatonin regulates the expression of the detected genes via demethylation. These findings shed insights into the potential mechanisms by which melatonin improves oocyte quality during IVM.

Extreme temperature during summer may lead to heat stress in cattle and compromise their productivity. It also poses detrimental impacts on the developmental capacity of bovine budding oocytes, which halt their fertility. To mitigate the adverse effects of heat stress, it is necessary to investigate the mechanisms through which it affects the developmental capacity of oocytes. The primary goal of this study was to investigate the impact of heat stress on the epigenetic modifications in bovine oocytes and embryos, as well as on oocyte developmental capacity, reactive oxygen species, mitochondrial membrane potential, apoptosis, transzonal projections, and gene expression levels. Our results showed that heat stress significantly reduced the expression levels of the epigenetic modifications from histone H1, histone H2A, histone H2B, histone H4, DNA methylation, and DNA hydroxymethylation at all stages of the oocyte and embryo. Similarly, heat stress significantly reduced cleavage rate, blastocyst rate, oocyte mitochondrial-membrane potential level, adenosine-triphosphate (ATP) level, mitochondrial DNA copy number, and transzonal projection level. It was also found that heat stress affected mitochondrial distribution in oocytes and significantly increased reactive oxygen species, apoptosis levels and mitochondrial autophagy levels. Our findings suggest that heat stress significantly impacts the expression levels of genes related to oocyte developmental ability, the cytoskeleton, mitochondrial function, and epigenetic modification, lowering their competence during the summer season.

The aim of this study was to test the effects of five different concentrations (0, 10⁻³, 10⁻⁴, 10⁻⁵, and 10⁻⁶ M) of resveratrol (Res) supplementation in bull sperm washing and fertilisation medium on levels of reactive oxygen species (ROS), phosphatidylserine (PS) externalisation, mitochondrial membrane potential (Δψm), ATP and malondialdehyde (MDA), acrosomal integrity, blastocyst rate, and blastocyst quality after in vitro fertilisation (IVF). The results for sex-sorted sperm from three bulls showed: (1) ROS and MDA levels in 10⁻³ M and 10⁻⁴ M Res groups were significantly lower than those of controls (P < 0.05); (2) the percentage of viable sperm, percentage of sperm with high Δψm, and the ATP content in 10⁻³ M and 10⁻⁴ M Res groups were significantly higher than those of controls (P < 0.05); (3) the percentage of viable sperm with acrosomal integrity, and the blastocyst percentage and quality of the 10⁻⁴ M Res group were significantly higher than those of controls (P < 0.05). In conclusion, 10⁻⁴ M Res supplementation in washing and fertilisation medium of sex-sorted bull sperm significantly decreased ROS, PS externalisation, and MDA, and protected mitochondrial function and acrosomal integrity, thereby increasing blastocyst percentage and quality following IVF.

The quality and developmental capacity of oocytes derived from in vitro maturation (IVM) remain unsatisfactory, which greatly impairs the efficiency and application of embryo technologies. The present experiment was designed to investigate the effect of the supplementation of EGF, IGF-1, and Cx37 in an IVM medium on the maturation quality and development ability of bovine oocytes. The cytoplasmic maturation events of oocytes and the quality of in vitro fertilization (IVF) blastocysts were examined to investigate the relative mechanisms. Our results showed that the nuclear maturation and blastocyst development after the IVF of oocytes treated with 25 μg/mL Cx37 or the combination of 50 ng/mL EGF and 100 ng/mL IGF-1 were significantly increased compared to those of the control group (p < 0.05). Furthermore, the blastocyst rate, and blastocyst total cell number and survival rate after vitrification of the EGF+IGF-1+Cx37 group, were significantly higher than those of the control group (p < 0.05), but lower than those of the FSH+LH+EGF+IGF-1+Cx37 group (p < 0.05). The transzonal projection (TZP) intensity, glutathione (GSH) level, and mitochondrial function of the EGF+IGF-1+Cx37 group were significantly higher than that of the control group, and lower than those of the FSH+LH+EGF+IGF-1+Cx37 group, in contrast to the results of the reactive oxygen species (ROS) levels. In conclusion, our results showed that the supplementation of 50 ng/mL EGF, 100 ng/mL IGF-1, and 25 μg/mL Cx37 in the IVM of bovine oocytes significantly improved their quality and developmental ability by increasing the TZP, mitochondrial function, and GSH level.

Vitrification reduces the fertilisation capacity and developmental ability of mammalian oocytes; this effect is closely associated with an abnormal increase of cytoplasmic free calcium ions ([Ca²⁺]i). However, little information about the mechanism by which vitrification increases [Ca²⁺]i levels or a procedure to regulate [Ca²⁺]i levels in these oocytes is available. Vitrified bovine oocytes were used to analyse the effect of vitrification on [Ca²⁺]i, endoplasmic reticulum Ca²⁺ (ER Ca²⁺), and mitochondrial Ca²⁺ (mCa²⁺) levels. Our results showed that vitrification, especially with dimethyl sulfoxide (DMSO), can induce ER Ca²⁺ release into the cytoplasm, consequently increasing the [Ca²⁺]i and mCa²⁺ levels. Supplementing the cells with 10 μM 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM or BAPTA) significantly decreased the [Ca²⁺]i level and maintained the normal distribution of cortical granules in the vitrified bovine oocytes, increasing their fertilisation ability and cleavage rate after in vitro fertilisation (IVF). Treating vitrified bovine oocytes with 1 μM ruthenium red (RR) significantly inhibited the Ca²⁺ flux from the cytoplasm into mitochondria; maintained normal mCa²⁺ levels, mitochondrial membrane potential, and ATP content; and inhibited apoptosis. Treating vitrified oocytes with a combination of BAPTA and RR significantly improved embryo development and quality after IVF.