Dynamic metabolomics of uterine fluid of laying hens: Key metabolites and pathways governing sperm motility and functional longevity.

The uterine fluid (UF) in hens constitutes a critical microenvironment for sperm entering the oviduct. The variation of metabolic components in the UF of two distinct physiological phases, plumping fluid (PF) and calcifying fluid (CF), may exert different effects on sperm storage, survival, and migration within the oviduct and thus influencing fertilization success. This concerns the efficiency of artificial insemination (AI) and in vitro sperm storage strategy. Therefore, this study aims to clarify this effect, and identify the underlying key metabolic components and mechanism. The sperm incubated with PF, CF, and PBS (control) for 6 h at 4°C was checked for sperm motility and other parameters including curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), and amplitude of lateral head displacement (ALH). Non-targeted metabolomics approach was employed to profile the metabolites present in the PF and CF. The key differential metabolite was supplemented to PBS at two concentration, 0.143 mM and 0.287 mM to incubate the sperm for 6 hours before the AI to confirm its potential effect. The results demonstrated that PF significantly enhanced sperm motility and key kinetics parameters (VSL, VAP, VCL) compared to CF and PBS during in vitro storage. Metabolomic analysis revealed profoundly distinct metabolic landscapes between PF and CF, which were enriched in pathways central to cellular energetics and homeostasis, including the tricarboxylic acid cycle (TCA cycle), lipid metabolism, and arginine biosynthesis and metabolism. Notably, the presence of sperm within the UF induced dynamic shifts in these metabolic profiles, particularly affecting lipid and fatty acid related metabolites. Consistent with the prominent enrichment of arginine-related metabolic pathways, the supplementation of L-arginine into PBS extender significantly prolonged sperm survival in vitro and improved subsequent in vivo fertility rate following AI. This study elucidates the intricate metabolic regulatory mechanisms within chicken UF that underpin sperm storage capacity and fertilization competence. It also highlights L-arginine as a potent additive for chicken semen extenders, offering a strategy to optimize chicken reproductive efficiency.