Effects of Melatonin Dose Regimens on Follicular Development and Oocyte Developmental Competence in FSH-Superovulated Ewes: Redox and Molecular Responses

Oxidative stress impairs gamete development and embryonic competence in female mammals. We compared four melatonin (MT) dose regimens (vehicle control and 20, 40, and 80 mg per injection) in FSH-superovulated ewes during in vivo ovum pick-up. The 40 mg regimen significantly reduced follicular atresia and increased high-quality oocytes and transferable blastocysts. Pregnancy and lambing rates after in vivo embryo transfer were numerically higher in the 40 mg group but did not reach statistical significance. The favorable responses observed with the 40 mg regimen were not maintained at the 80 mg dose. Oocyte transcriptomics and follicular fluid metabolomics revealed multilevel molecular responses associated with MT treatment. Systemically, 40 mg MT was associated with changes in serum reproductive hormones, including significant increases in MT, E2, and IGF-1. At the cellular and molecular levels, MT treatment was associated with changes in follicular development and oocyte quality, together with altered redox status and candidate changes in energy metabolism; MT1-related signaling was identified as a candidate pathway. MT treatment was associated with increased MT1 and Ki-67 expression in granulosa cells, an increased Bcl-2/Bax ratio, and reduced follicular fluid ROS and MDA levels, indicating improved redox homeostasis and granulosa cell proliferation. MT treatment was associated with altered redox status, including significantly increased GSH and reduced intracellular ROS, while ATP and mitochondrial superoxide showed non-significant changes. Hypoxia-inducible factor 1 (HIF-1) signaling and the tricarboxylic acid (TCA) cycle were identified as candidate pathways potentially associated with follicular energy–redox remodeling. These observations provide a basis for further investigation of MT dose regimens to mitigate disadvantages associated with FSH superovulation in ewes and other species.