BACKGROUND Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians. METHODS The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined. RESULTS Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials. CONCLUSIONS Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.

中文翻译：

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欧亚蟾蜍（Epidalea calamita）精子的激素诱导。

背景技术由于栖息地丧失、入侵物种、气候变化和疾病，两栖动物多样性正在以惊人的速度下降。已经为一些处于危险中的物种建立了圈养保证群；然而，许多物种在圈养条件下繁殖不佳，辅助生殖技术 (ART) 的发展对于帮助维持遗传多样性至关重要。迄今为止，大部分工作已经在美洲大陆和澳大利亚的物种中完成，并且需要解决欧亚和非洲两栖动物物种的类似育种挑战。方法本研究的目的是开发一种激素方案，用于刺激 Epidalea calamita 中的精子生成，作为欧亚蟾蜍的模型。因此，评估了三种剂量的绒毛膜促性腺激素（5、10和15 IU hCG/g）随时间（1至24小时）对精子产生和质量的影响。此外，还检查了精子的冷藏（5°C）24和48小时以及三种频率的激素治疗（每周、每两周和每月）。结果 激素浓度为 10 或 15 IU 的 hCG 可以诱导 100% 的男性精子生成，并产生质量相当的精子，而 5 IU hCG 只能刺激 40% 的男性精子生成。总动力在 10 IU hCG 治疗后 1 至 4 小时内达到峰值，而 15 IU hCG 治疗后 2 至 6 小时内达到峰值。冷藏 24 小时后，10 和 15 IU 处理的总动力下降了 20%，前向动力下降了 10%。每周施用激素导致试验之间所有运动参数的差异较大，并且总体总运动和向前运动较低。此外，外源激素治疗的效果在最后两项试验中的每周频率上有重叠。在所有频率的第一个试验中，精子浓度较高，但其他试验之间没有差异。结论 总的来说，这些结果表明，在为欧亚两栖动物制定激素刺激方案时，应牢记激素浓度、治疗后时间、激素治疗频率和冷藏。