Sperm morphology, functionality, and cryopreservation challenges in wild Ocellate River stingrays Potamotrygon motoro

This study characterized the sperm morphology and morphometry of wild Ocellate River stingrays (Potamotrygon motoro) and evaluated the effects of cryopreservation on their structural integrity and functional viability. Semen was collected from four reproductively mature males (five individuals were captured in situ). Scanning electron microscopy revealed a helical head structure and a total spermatozoon length of 131.37 ± 4.13 µm. Spermatozoa were frequently observed forming head-to-head aggregates characteristic of spermatozeugmata, which dissociated during handling, but could prevent uniform cryoprotectant penetration. Motility analysis showed predominantly circular (spinning) and low-progressive movement, consistent with the specific conditions required by this species to express progressive motility in high-viscosity media. Cryopreservation increased morphological defects in the midpiece and tail regions, potentially impairing sperm propulsion. Post-thaw membrane integrity, assessed via fluorescent staining, ranged from 1.5% to 7.9%. Although no significant differences were observed between cryoprotectants, 10% dimethyl sulfoxide (DMSO) and 10% dimethylformamide (DMF) yielded the highest motility retention. These results enhance our understanding of the reproductive biology P. motoro and highlight the challenges of optimizing cryopreservation protocols for freshwater stingrays due to their unique sperm ultrastructure and sensitivity to cryoinjury.