Abstract An understanding of how seasonality impacts amphibian's sperm quality is crucial for the development and implementation of assisted reproductive technologies for threatened species. We analysed sperm quality both within and outside the breeding season for the common natterjack toad (Epidalea calamita) in a population close to Madrid (Spain). Fresh hormonally induced sperm samples were assessed for sperm concentration, total activated sperm (TAS), forward motility (FM), quality of motility (QM) and DNA integrity (DNAi). Motility and DNAi were re-evaluated after cold-storage at 5 °C for 24 h. After freezing and thawing DNAi was re-analysed and a fertilization trial performed to test post-thaw sperm functionality. More males presented sperm after hormone administration during the breeding season compared to out of season (78.3 vs. 52.2%), although the difference was not significant. Fresh sperm motility and DNAi were not affected by season (p > 0.05). However, sperm concentration was higher (p = 0.037) during the breeding season (5.2 vs. 2.9 × 106 sperm/ml). After cold-storage sperm obtained in the breeding season presented higher FM (36.1 ± 4.6 vs. 22.0 ± 3.5%; p = 0.013) and QM (1.28 ± 0.2 vs. 0.68 ± 0.2; p = 0.017) than samples from out of season. DNAi decreased after freeze-thawing in both seasons (p

中文翻译：

---

季节性对菖蒲（蟾科，两栖动物）激素诱导精子及其冷藏和冷冻保存的影响

摘要 了解季节性如何影响两栖动物的精子质量对于受威胁物种辅助生殖技术的开发和实施至关重要。我们分析了马德里（西班牙）附近种群中常见的蝾螈蟾蜍（Epidalea calamita）在繁殖季节内外的精子质量。评估新鲜的激素诱导精子样本的精子浓度、总活化精子 (TAS)、前向运动 (FM)、运动质量 (QM) 和 DNA 完整性 (DNAi)。在 5°C 下冷藏 24 小时后，重新评估运动性和 DNAi。冷冻和解冻后，重新分析 DNAi，并进行受精试验以测试解冻后精子的功能。与非季节相比，在繁殖季节服用激素后，更多的雄性出现了精子（78.3 对 52.2%），虽然差异并不显着。新鲜精子活力和 DNAi 不受季节影响（p > 0.05）。然而，在繁殖季节，精子浓度较高（p = 0.037）（5.2 对 2.9 × 106 精子/毫升）。在繁殖季节获得的精子在冷藏后表现出比非季节样品更高的 FM（36.1 ± 4.6 对 22.0 ± 3.5%；p = 0.013）和 QM（1.28 ± 0.2 对 0.68 ± 0.2；p = 0.017） . 两个季节冻融后 DNAi 均下降 (p p = 0.017）比反季节的样品。两个季节冻融后 DNAi 均下降 (p p = 0.017）比反季节的样品。两个季节冻融后 DNAi 均下降 (p