Advanced sperm preservation techniques in yellow spotted mountain newts Neurergus derjugini enhance genetic management and conservation efforts

Advances in cold storage and cryopreservation of amphibian sperm are critical for the genetic management and conservation of threatened species. This study represents the first investigation into the sperm of the yellow spotted mountain newt (Neurergus derjugini), focusing on both short-term and long-term storage for future reproductive efforts. We examined the effects of seven extenders on sperm motility over time at three storage temperatures (4 ± 1 °C, 9 ± 1 °C, and 20 ± 1 °C). Additionally, we assessed the impact of 16 cryoprotectants on sperm motility and morphology post-thawing. Following the identification of the most effective freezing medium, we evaluated sperm DNA fragmentation to ensure viability. Our results indicate that 10% Holtfreter's solution is the optimal extender for short-term storage at all three temperatures, maintaining sperm motility for up to 15 days at 4 °C. For long-term storage, a combination of 10% Holtfreter's solution and 10% DMSO was found to best preserve sperm motility, morphology, and minimize DNA fragmentation after thawing. These findings underscore the importance of specific extenders and temperature treatments in enhancing sperm functionality, thereby supporting successful assisted reproductive technologies (ART) for endangered species.