Taheri-Khas Zeynab, Gharzi Ahmad, Vaissi Somaye, Heshmatzad Pouria, Kalhori Zahra
Department of Biology, Faculty of Science, Razi University, Kermanshah, Iran.
Sci Rep. 2025 Mar 18;15(1):9334. doi: 10.1038/s41598-025-93284-y.
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.
两栖动物精子冷藏和冷冻保存技术的进步对于濒危物种的遗传管理和保护至关重要。本研究首次对黄斑山螈（Neurergus derjugini）的精子进行了调查，重点关注短期和长期保存，以用于未来的繁殖工作。我们研究了七种稀释液在三种储存温度（4 ± 1°C、9 ± 1°C和20 ± 1°C）下随时间对精子活力的影响。此外，我们评估了16种冷冻保护剂对解冻后精子活力和形态的影响。在确定最有效的冷冻培养基后，我们评估了精子DNA片段化情况以确保精子的活力。我们的结果表明，10%的霍尔特弗雷特氏溶液是在所有三种温度下进行短期保存的最佳稀释液，在4°C下可使精子活力维持长达15天。对于长期保存，发现10%的霍尔特弗雷特氏溶液和10%的二甲基亚砜组合能最好地保持精子活力、形态，并使解冻后的DNA片段化最小化。这些发现强调了特定稀释液和温度处理在增强精子功能方面的重要性，从而为濒危物种的成功辅助生殖技术（ART）提供支持。
