Understanding sperm quality and its impact on reproductive success is essential for the aquaculture, conservation, and management of protected fish species. At the Fish Conservation and Culture Laboratory at the University of California, Davis, where all species were maintained under controlled environment, we studied the sperm in three California smelt species: the endangered delta smelt (Hypomesus transpacificus), the threatened longfin smelt (Spirinchus thaleichthys), and the introduced wakasagi (H. nipponensis), and applied the knowledge to inform the aquaculture practices and conservation management. Using advanced imaging and analysis tools, including the OpenCASA plugin and Scanning and Transmission Electron Microscopy (SEM and TEM), we examined sperm motility and ultrastructure (Figure 1). Key findings revealed that sperm quality, characterized by motility traits such as velocity and motility, was significantly affected by preservation time, salinity, recovery phases, and body size. Notably, sperm motility in delta smelt peaked within five seconds post-activation, highlighting the importance of precise collection timing. Delta smelt, longfin smelt, and wakasagi exhibited unique responses to sperm activation salinity, underscoring species-specific adaptive strategies. We also looked into the morphology of their sperm, revealing structural differences that may influence motility and fertilization outcomes. In summary, a comprehensive assessment of sperm quality across these smelt species under aquaculture conditions provides valuable knowledge for enhancing hatchery efficiency and supporting broader conservation efforts. By integrating these findings, aquaculture operations can better align with conservation objectives, ensuring the sustainable propagation of protected fish populations.