Melatonin alleviates the heat-stress-induced decline in sperm quality in dairy goats by regulating amino acid metabolism

Heat stress severely impairs male reproductive performance in dairy goats by disrupting testicular function, antioxidant defense, and metabolic homeostasis. This study investigated the protective mechanisms of melatonin using both natural and artificial heat stress models in dairy goats and mice. In goats, we found that heat stress significantly reduced sperm's motility, viability, and density while increasing the levels of oxidative stress markers (reactive oxygen species [ROS], malondialdehyde [MDA]) and pro-inflammatory cytokines (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α). Pre-treatment with melatonin implants restored serum melatonin levels, enhanced antioxidant capacity (glutathione [GSH], total antioxidan capacity [T-AOC]), and ameliorated inflammatory responses, ultimately improving sperm quality. Untargeted metabolomic profiling uncovered that heat stress induced systemic metabolic dysregulation, with significant enrichment in the arginine and tryptophan pathways. Melatonin supplementation partly corrected these disruptions and restored the serum levels of both amino acids. In the murine model, melatonin significantly alleviated the testicular damage induced by heat stress by reducing apoptosis, preserving the integrity of the blood–testis barrier (BTB) through the upregulation of occludin and ZO-1, and improving the histological architecture of the seminiferous tubules and epididymis. Further, we observed that melatonin increased the expression of key rate-limiting enzymes involved in arginine (ARG1) and tryptophan (TPH1, KYNU) metabolism and activated steroidogenic genes (Star, Cyp11a1), restoring testosterone production under heat stress conditions. Supplementation with arginine or tryptophan alone also alleviated heat stress-induced sperm abnormalities and testicular injury, confirming the functional relevance of these pathways. These findings demonstrate that melatonin protects against heat stress-induced male reproductive dysfunction by modulating oxidative stress, inflammation, amino acid metabolism, and steroidogenesis. Targeting arginine and tryptophan metabolism may represent an effective strategy for improving livestock's fertility under thermal stress.