Péricard Louise, Le Mével Sébastien, Marquis Olivier, Locatelli Yann, Coen Laurent
Physiologie Moléculaire et Adaptation, UMR 7221 Centre National de la Recherche Scientifique, Muséum National d'Histoire Naturelle, Cedex 05, 75231 Paris, France.
Parc Zoologique de Paris, Muséum National d'Histoire Naturelle, 75012 Paris, France.
Animals (Basel). 2025 Jul 1;15(13):1941. doi: 10.3390/ani15131941.
The significant decline in amphibians worldwide is demanding the development of reliable techniques to save species and their genetic diversity. Considerable efforts are currently in progress to develop assisted reproductive technologies (ARTs), focusing mainly on sperm cryopreservation and in vitro fertilization (IVF). In , a simple and efficient transgenesis method based on the intracytoplasmic injection (ICSI) of cryoconserved sperm was developed several decades ago, allowing for quick generation of large numbers of transgenic animals, for biological research. Such a methodology could be critical for the recovery of species and their genetic diversity, contributing to amphibian conservation. However, this approach raised the question of whether the sperm preservation method used with ICSI is compatible with long-term storage. To address this question, animals were generated by ICSI using a twenty-year-old cryopreserved sperm preparation. Their development, behavior, and reproduction ability were compared with those of animals obtained using a recently frozen sperm preparation and those of animals obtained via IVF using fresh semen. Although lower than with IVF, we showed that fertilization rates using ICSI after 20 years of cryopreservation are similar to those of a recent preparation, with viable offspring leading to normal F2 generation. This pioneering achievement is proof of concept for long-term sperm cryopreservation using simple and readily available technologies for the conservation of endangered amphibians.
全球两栖动物数量的显著下降促使人们开发可靠的技术来拯救物种及其遗传多样性。目前正在大力开展工作以开发辅助生殖技术（ARTs），主要集中在精子冷冻保存和体外受精（IVF）方面。几十年前，人们开发了一种基于胞质内注射（ICSI）冷冻保存精子的简单高效的转基因方法，可快速产生大量用于生物学研究的转基因动物。这种方法对于物种及其遗传多样性的恢复可能至关重要，有助于两栖动物的保护。然而，这种方法引发了一个问题，即与ICSI一起使用的精子保存方法是否与长期储存兼容。为了解决这个问题，使用一份保存了20年的冷冻精子制剂通过ICSI生成了动物。将它们的发育、行为和繁殖能力与使用最近冷冻的精子制剂获得的动物以及使用新鲜精液通过体外受精获得的动物进行了比较。尽管低于体外受精，但我们表明，冷冻保存20年后使用ICSI的受精率与最近制剂的受精率相似，有活力的后代可产生正常的F2代。这一开创性的成果证明了使用简单且易于获得的技术对濒危两栖动物进行长期精子冷冻保存的概念验证。
