Physiological and multi-omics analyses reveal osmotic effect, ionic function, and expression network underlying sperm activation and movement in giant grouper (Epinephelus lanceolatus)

Sperm activation and movement underpin reproductive success in marine teleosts, yet their mechanistic basis remains largely unresolved in coral reef fishes. Giant grouper ( Epinephelus lanceolatus ), the largest coral reef fish and a species of major aquaculture value, faces reproductive limitations due to the scarcity of mature functional males and suboptimal fertilization efficiency. In this study, we integrated sperm motility analysis with transcriptomic and proteomic profiling to characterize the physiological responses and candidate molecular processes associated with sperm activation and movement in giant grouper. Motility analyses showed that hyperosmotic exposure initiated sperm movement, while Na + , K + , and Ca 2+ activation media influenced motility performance. K + produced a relatively high proportion of rapidly motile sperm, suggesting an important role in motility regulation. Transcriptomics and proteomics identified 1235 differentially expressed genes and 1100 differentially expressed proteins, respectively. Multi-omics analyses revealed altered transcript and protein abundance associated with voltage-dependent Ca 2+ channels, HCN channels, and Na + /Ca 2+ -permeable receptors, suggesting the involvement of membrane potential dependent ionic signaling during the post-activation state. Expression patterns of genes and proteins associated with Insulin/IGF1-FOXO signaling suggested reduced involvement of this pathway after activation, while changes in molecules associated with pyruvate metabolism, the TCA cycle, lactate metabolism, and fatty acid degradation were consistent with increased involvement of mitochondrial oxidative metabolism. Our study provides physiological and molecular insights into sperm activation and movement in giant grouper and offers a basis for optimizing activation media and sperm utilization strategies in marine aquaculture.