High-Efficiency Cryopreservation of Silver Pomfret Sperm: Protocol Development and Cryodamage Assessment.

The silver pomfret (Pampus argenteus), widely distributed across the Indo-West Pacific and prevalent in China's coastal waters, has experienced significant resource decline due to anthropogenic impacts such as habitat alteration and overfishing, which disrupt its natural reproduction and growth. Cryopreservation technology overcomes spatiotemporal constraints by enabling the long-term storage of high-quality sperm for future use. This study optimized cryopreservation protocols for silver pomfret sperm, evaluation key parameters including extenders, cryoprotectants, dilution ratios, cooling heights, and thawing temperatures. Sperm quality was assessed post thaw via enzyme activity assays and electron microscopy. Results demonstrated that modified plaice Ringer solution (MPRS) extender yielded the highest post-thaw motility (95.98 ± 1.59)%. The optimal cryopreservation conditions for silver pomfret sperm were established as follows: MPRS diluent, 20% EG, a 1:6 dilution ratio, a 7 cm cooling height, and a 28 °C thawing temperature. This protocol yielded post-thaw sperm with motility and motion parameters most closely resembling those of fresh sperm. Ultrastructural observations and enzyme activity assays, however, confirmed that cryopreservation induced sublethal damage, including significant reduction in ATPase activity, as well as structural anomalies such as head deformation, membrane damage, and organelle disarray. This work establishes a foundational cryopreservation protocol, providing critical tools for conserving the genetic resources of this declining species and supporting sustainable aquaculture and wild population restoration efforts.