Fertilization capacity evaluation of cryopreserved Delta Smelt sperm

Abstract Objective The Delta Smelt Hypomesus transpacificus, an endangered species endemic to the San Francisco Estuary, faces a high risk of extinction due to habitat loss, water diversions, and changing environmental conditions. This study aimed to develop and validate the first sperm cryopreservation protocol for Delta Smelt as a tool to support conservation aquaculture, genetic banking, and reintroduction efforts. Methods Using captive fish maintained at University of California–Davis and Livingston Stone National Fish Hatchery, we conducted preliminary cryopreservation trials between 2020 and 2023. The finalized protocol employed 10% methanol as a cryoprotectant, a 1:15 sperm-to-extender dilution ratio, and a two-step freezing method using 0.25-mL French straws. Fertilization capacity was assessed in 2024 through two replicated trials comparing cryopreserved and fresh sperm. Fertilization success, expressed as eye-up rate, was statistically analyzed using a linear mixed-effects model. Results Fertilization rates varied across treatments, with cryopreserved sperm achieving eye-up rates ranging from 12% to 68% and fresh sperm from 17% to 76%. Despite variability in egg quality, cryopreserved sperm maintained an average relative eye-up rate of 83 ± 7% compared with fresh controls, demonstrating consistent fertilization performance. Statistical analysis revealed no significant difference in fertilization success between treatments. Male and female fork lengths and their interaction also had no significant effect. Conclusions The cryopreservation protocol developed in this study effectively preserves Delta Smelt sperm fertilization capacity, demonstrating its reliability for conservation applications. This approach provides a foundational tool for genetic resource management and captive propagation of endangered estuarine fishes.