From Molecular Perturbations to Functional Decline: Multi-Omics Reveals Sperm Cryodamage in Sichuan Bream (Sinibrama...

Sperm cryopreservation is pivotal for conserving fish germplasm, yet cryodamage-induced quality decline limits its application. This study focused on Sichuan bream (Sinibrama taeniatus), an endemic and economically important fish species in the upper Yangtze River. Based on an established cryopreservation protocol, we evaluated sperm quality using computer-assisted sperm analysis (CASA) and fertility assays, followed by a systematic assessment of structural and functional damage via flow cytometry (membrane integrity, mitochondrial potential, reactive oxygen species, and DNA fragmentation), enzymatic assays (energy metabolism and antioxidant enzymes), Western blotting, and ultrastructural observation. Finally, integrated proteomic and metabolomic analyses were employed to elucidate the underlying physiological mechanisms. The results demonstrated that freeze-thawing significantly impaired sperm motility, fertility, and ultrastructure, concurrently disrupting energy metabolism and the antioxidant system. Crucially, multi-omics revealed that these functional declines were linked to dysregulation in key pathways involving cytoskeleton organization, lipid metabolism, energy homeostasis, and oxidative stress, forming a coherent network from initial molecular perturbation to phenotypic dysfunction. This study provides a comprehensive characterization of sperm cryodamage in Sichuan bream, advancing the understanding of fish sperm cryobiology and informing targeted cryoprotection strategy development.