Melatonin synergizes with constant FSH stimulation to improve oocyte yield and in vitro embryo production in dairy goats.

Ovum Pick-Up (OPU) enables efficient, repeated oocyte retrieval from elite dairy goats, making it a critical technology for accelerating genetic progress through in vitro embryo production (IVEP) in dairy goat breeding. However, the limited quality and quantity of retrieved oocytes often restrict its overall efficiency. Melatonin (MT) has been known to regulate mammalian reproductive physiology and the ovarian microenvironment, but its specific effects across different follicle-stimulating hormone (FSH) superovulation protocols to enhance oocyte yield and quality remain unclear. This study investigated the effects of MT supplementation (20 mg/dose, total 120 mg/goat across 6 injections) on oocyte recovery in dairy goats subjected to three different FSH injection protocols (incremental, constant, and decremental). Following in vitro fertilization, embryo development and pregnancy rates after embryo transfer (ET) were subsequently evaluated. MT supplementation significantly increased the total number of recovered oocytes (P < 0.05), with the constant FSH protocol yielding optimal outcomes. MT increased blastocyst rates (P < 0.05), although the improvement of cleavage and fresh ET pregnancy rates were not significant. MT elevated both estrogen and progesterone levels in the serum of dairy goats (P < 0.05). Further researches to determine the mechanism of melatonin effects was performed by evaluation of serum hormones and oxidative stress related molecule, follicular fluid metabolomics, and ovarian transcriptomics. Multi-omics analysis revealed that MT significantly altered the follicular fluid metabolic profile, particularly affecting antioxidant and steroid hormone metabolism (Epigallocatechin, Quercetin 3-O-Xylosyl-Rutinoside, Hexahydrocurcumin, Protocatechuic Aldehyde, Isodityrosine, Pregnanediol, 16-Ketoestradiol), and regulated antioxidant and steroid hormone-related genes (GSTM1, MGST2, RXFP1, 3βHSD, HSD17B7, and PAQR7). Furthermore, ovarian redox homeostasis was enhanced by increasing glutathione peroxidase (GSH-Px) activity and reducing malondialdehyde (MDA) levels (P < 0.05). Overall, MT exerts multidimensional beneficial effects on ovarian microenvironment to enhance oocyte developmental competence. Combining a constant FSH protocol with MT achieves optimal superovulation efficiency, providing a robust strategy for advancing overall IVEP efficiency in dairy goat.