Cryogenic Damage and Trehalose Protection in Culter alburnus Sperm: An Integrated Assessment of Quality, Physiology, and Protein Expression.

To address cryodamage in Culter alburnus sperm, this study evaluated the effects of trehalose supplementation in a conventional cryomedium (D-15 + 10% ethylene glycol). Six experimental groups were established: fresh sperm (G1), a conventional cryomedium (G2), groups supplemented with 10, 100, or 200 mmol/L trehalose (G3-G5), and a control group with extender only (G6). The group with 100 mmol/L trehalose (G4) was associated with improved post-thaw motility parameters (activation rate, movement time, and lifespan) and higher antioxidant (superoxide dismutase and catalase) and energy metabolism (ATPase, succinate dehydrogenase, lactate dehydrogenase) enzyme activities. Ultrastructural damage in G4 included partial plasma membrane rupture and mitochondrial swelling, while G6 exhibited additional damage features including membrane disintegration, mitochondrial disruption, and flagellar fracture. Proteomic analysis revealed that, compared to G1, G4 exhibited higher abundance of proteins (e.g., Histone H2A, cytochrome c oxidase, profilin) involved in structural integrity and energy homeostasis, whereas G6 showed signatures of oxidative stress and metabolic dysfunction (lower abundance of NADH dehydrogenase and higher abundance of calcium-transporting ATPase and glutathione S-transferase). In conclusion, 100 mmol/L trehalose was associated with improved cryopreservation outcomes, and the proteins identified provide a basis for further investigation. This approach offers a framework for refining germplasm conservation strategies in aquaculture.