Melatonin Supplementation Increases Oocyte Recovery and Quality Associated with Altered Follicular Steroidogenesis...

Ovum pick-up (OPU) enables high-frequency oocyte retrieval from selected dairy cow donors, facilitating favorable genetic progression. However, oocytes obtained via OPU and subjected to in vitro maturation frequently suffer from severe oxidative stress, mitochondrial dysfunction, and DNA damage, resulting in lower pregnancy rates compared to those of in vivo-derived embryos. Melatonin (MT), as a potent antioxidant, may exert beneficial effects on these OPU-retrieved oocytes. In the current study, by combining in vitro and in vivo experiments, the protective effects and potential molecular mechanisms of MT on disrupted oocyte quality and development caused by OPU were systemically investigated. By integrating oocyte quality assessment, follicular fluid metabolomics, and blastocyst transcriptomics, the results showed that subcutaneous administration of MT to cows prior to OPU significantly increased the number of high-quality Grade A oocytes and reduced the number of low-quality Grade D oocytes compared to the control. These improvements were accompanied by reduced local and systemic oxidative stress, enhanced glutathione metabolism, and lower serum progesterone concentrations at the measured time points, coinciding with a more favorable endocrine environment for follicular development. The results from untargeted metabolomics of the follicular fluid revealed that MT supplementation was associated with an altered follicular fluid microenvironment characterized by the upregulation of estrogen derivatives and glutathione-related pathways. Transcriptomic analysis of blastocysts showed significant upregulation of the rate-limiting genes steroidogenic acute regulatory protein (STAR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), suggesting a potential role in promoting the synthesis of reproductive hormones and maintaining the progress of embryo development. The in vitro study showed that MT supplementation at a concentration of 10^-7 mol/L in oocyte maturation media significantly improved cleavage rates, blastocyst rates, and total blastocyst cell numbers compared to the control. In vitro evaluations demonstrated that MT scavenged intracellular reactive oxygen species (ROS), while transcriptomic analysis indicated a reprogramming of the embryonic transcriptome. These transcriptomic changes were associated with the AMPK, FoxO, and autophagy pathways, as well as the modulation of endoplasmic reticulum stress and cellular senescence. The results from both the in vivo and in vitro studies suggested that MT supplementation was associated with a follicular microenvironment favorable for oocyte growth and protected embryos from oxidative damage. Therefore, MT supplementation effectively reduced the disrupted oocyte development caused by OPU and improved oocyte recovery and quality in general. These findings provide promising experimental evidence for the potential application of MT in dairy breeding and offer insights into the associated molecular pathways. However, further functional validation is required to fully elucidate the underlying mechanisms and establish its large-scale applicability.