Integrating Physiology and Multi-Omics Reveals Mechanisms Underlying Sperm Activation and Movement in Mandarin Fish (Siniperca chuatsi).

Sperm activation and movement are essential in freshwater fishes, where limited energy reserves and a short motility duration require rapid ATP-driven motility regulated by osmolality and ions. However, the physiological and molecular mechanisms underlying osmolality and ion regulation in mandarin fish (Siniperca chuatsi) remain unclear, and ions in aquaculture systems may exert additional effects. Here, we integrated physiology with transcriptomic and proteomic analyses to characterize the effects of osmolality and ions on sperm motility and identify key genes, proteins, and regulatory networks involved in sperm activation and movement. Low osmolality (<50 mOsm/kg) promoted sperm activation, with sperm motility progressively decreasing as osmolality increased. Although relatively high osmolality supported motility maintenance, the initial motility was comparatively low, indicating the limited energy reserves of freshwater fish spermatozoa. Na+ and K+ activation media enhanced motility, whereas Ca2+ supplementation suppressed activation. Ca2+ levels below a 1:500 molar ratio (<0.06 mM) did not significantly affect sperm motility, indicating the importance of limiting Ca2+ exposure during fertilization. Multi-omics analyses identified six differentially expressed genes, including grin3bb, cacna1c, and chrna7, and 35 differentially expressed proteins, including ATP1B, PRKCB, and CPT1A, as key molecules associated with Na+, K+, and Ca2+ signaling and energy metabolism. The calcium signaling pathway, cAMP signaling pathway, and oxidative phosphorylation were significantly enriched in KEGG enrichment analysis, while Gene Ontology (GO) enrichment analysis identified ion signaling and energy metabolism, including ion transport, the ATP metabolic process, and the glycolytic process. Our findings provide a putative regulatory network for sperm activation and movement in mandarin fish and a basis for optimizing artificial fertilization.