Fish sperm subpopulations detection, separation, and application: current state and trends

Sperm subpopulations represent a fundamental manifestation of intra-ejaculate heterogeneity in fish, arising from differences in structural features, motility patterns, and physiological states of sperm cells. This variability is increasingly recognised as biologically significant, influencing fertilisation success, sperm competition, and egg selection. Advances in computer-assisted sperm analysis, flow cytometry, and fluorescent staining have enabled the identification of distinct subgroups based on kinematic, morphological, and biochemical traits. However, techniques for their physical separation and functional assessment remain in early development for fish, limiting their routine application in aquaculture. Understanding sperm heterogeneity offers innovative perspectives for reproductive biology, providing insights into species with complex mating strategies and guiding the refinement of aquaculture practices, particularly in situations where sperm quality is reduced or highly variable. Applications include optimising artificial fertilisation, improving cryopreservation protocols, and enhancing genetic stability by isolating high-quality subpopulations, especially when optimal volumes of high-quality sperm are not available. Importantly, sperm subpopulation analysis also holds promise for conservation programs, where selecting and preserving viable gametes is critical for maintaining genetic diversity and supporting the recovery of endangered and threatened fish species. By reducing abnormal larval development and improving fertilisation efficiency, this approach strengthens hatchery success and contributes to long-term sustainability. Overall, framing sperm heterogeneity as both a biological phenomenon and a biotechnological resource highlights its importance and innovation. This review synthesises current knowledge, explores possible origins of sperm subpopulations, and outlines future directions for their application in aquaculture and conservation, with particular relevance in contexts where reproductive performance is compromised or variable.