{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T19:25:09Z","timestamp":1790709909935,"version":"4.1.0"},"reference-count":44,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T00:00:00Z","timestamp":1751328000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"CRYO-GPA"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>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 Xenopus, 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.<\/jats:p>","DOI":"10.3390\/ani15131941","type":"journal-article","created":{"date-parts":[[2025,7,1]],"date-time":"2025-07-01T06:56:54Z","timestamp":1751353014000},"page":"1941","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Intracytoplasmic Sperm Injection Using 20-Year-Old Cryopreserved Sperm Results in Normal, Viable, and Reproductive Offspring in Xenopus laevis: A Major Pioneering Achievement for Amphibian Conservation"],"prefix":"10.3390","volume":"15","author":[{"given":"Louise","family":"P\u00e9ricard","sequence":"first","affiliation":[{"name":"Physiologie Mol\u00e9culaire et Adaptation, UMR 7221 Centre National de la Recherche Scientifique, Mus\u00e9um National d\u2019Histoire Naturelle, Cedex 05, 75231 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4357-7945","authenticated-orcid":false,"given":"S\u00e9bastien","family":"Le M\u00e9vel","sequence":"additional","affiliation":[{"name":"Physiologie Mol\u00e9culaire et Adaptation, UMR 7221 Centre National de la Recherche Scientifique, Mus\u00e9um National d\u2019Histoire Naturelle, Cedex 05, 75231 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8215-1221","authenticated-orcid":false,"given":"Olivier","family":"Marquis","sequence":"additional","affiliation":[{"name":"Parc Zoologique de Paris, Mus\u00e9um National d\u2019Histoire Naturelle, 75012 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2657-9327","authenticated-orcid":false,"given":"Yann","family":"Locatelli","sequence":"additional","affiliation":[{"name":"Physiologie Mol\u00e9culaire et Adaptation, UMR 7221 Centre National de la Recherche Scientifique, Mus\u00e9um National d\u2019Histoire Naturelle, Cedex 05, 75231 Paris, France"},{"name":"R\u00e9serve Zoologique de la Haute Touche, Mus\u00e9um National d\u2019Histoire Naturelle, 36290 Obterre, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2643-7668","authenticated-orcid":false,"given":"Laurent","family":"Coen","sequence":"additional","affiliation":[{"name":"Physiologie Mol\u00e9culaire et Adaptation, UMR 7221 Centre National de la Recherche Scientifique, Mus\u00e9um National d\u2019Histoire Naturelle, Cedex 05, 75231 Paris, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,7,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Browne, R.K., Luo, Q., Wang, P., Mansour, N., Kaurova, S.A., Gakhova, E.N., Shishova, N.V., Uteshev, V.K., Kramarova, L.I., and Venu, G. 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