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small and threatened populations in amphibian conservation programs. However, there is little to no evidence demonstrating reproductive maturity and viability of offspring generated with cryopreserved sperm, potentially limiting widespread integration of these technologies. The purpose of this report is to demonstrate that amphibian sperm can be cryopreserved and thawed to successfully produce individuals of an F1 generation that can reach adulthood and reproductive maturity, to generating viable gametes and an F2 generation. Species-specific exogenous hormones were administered to both F0 and F1 adults to stimulate spermiation and oviposition in the eastern tiger salamander (Ambystoma tigrinum), dusky gopher frog (Lithobates sevosa), and Puerto Rican crested toad (Peltophryne lemur). Sperm cells collected non-lethally from F0 adults were cryopreserved, thawed, and used for in vitro fertilization (IVF) to produce F1 offspring. Individuals of the F1 generation are shown to reach adulthood, express viable gametes, and produce offspring through facilitated breeding, or IVF. The production of amphibian F2 generations shown here demonstrates that amphibian sperm collected non-lethally can be banked and used to generate reproductively viable animals of subsequent generations, thus maintaining valuable genetic linages and diversity in threatened amphibian species. The incredible value that cryopreservation of sperm has for long-term genetic management aids in the sustainability of both in situ and ex situ conservation efforts for this taxon.<\/jats:p>","DOI":"10.3390\/ani13010053","type":"journal-article","created":{"date-parts":[[2022,12,23]],"date-time":"2022-12-23T03:02:15Z","timestamp":1671764535000},"page":"53","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Sperm Cryopreservation as a Tool for Amphibian Conservation: Production of F2 Generation Offspring from Cryo-Produced F1 Progeny"],"prefix":"10.3390","volume":"13","author":[{"given":"Shaina S.","family":"Lampert","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Isabella J.","family":"Burger","sequence":"additional","affiliation":[{"name":"Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Allison R.","family":"Julien","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amanda B.","family":"Gillis","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew J.","family":"Kouba","sequence":"additional","affiliation":[{"name":"Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Diane","family":"Barber","sequence":"additional","affiliation":[{"name":"Department of Ectotherms, Fort Worth Zoo, Fort Worth, TX 76110, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8466-6077","authenticated-orcid":false,"given":"Carrie K.","family":"Kouba","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"coac032","DOI":"10.1093\/conphys\/coac032","article-title":"Development of an Amphibian Sperm Biobanking Protocol for Genetic Management and Population Sustainability","volume":"10","author":"Burger","year":"2022","journal-title":"Conserv. 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Online"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Peris-Frau, P., Soler, A.J., Iniesta-Cuerda, M., Mart\u00edn-Maestro, A., S\u00e1nchez-Ajofr\u00edn, I., Medina-Ch\u00e1vez, D.A., Fern\u00e1ndez-Santos, M.R., Garc\u00eda-\u00e1lvarez, O., Maroto-Morales, A., and Montoro, V. (2020). Sperm Cryodamage in Ruminants: Understanding the Molecular Changes Induced by the Cryopreservation Process to Optimize Sperm Quality. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21082781"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"172","DOI":"10.1002\/jez.a.10192","article-title":"Structural and Functional Aspects of Bufo Americanus Spermatozoa: Effects of Inactivation and Reactivation","volume":"295","author":"Kouba","year":"2003","journal-title":"J. Exp. Zool. A Comp. Exp. 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Mississippi State University. manuscript in preparation: To be submitted."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/1\/53\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:48:57Z","timestamp":1760147337000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/1\/53"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,23]]},"references-count":21,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2023,1]]}},"alternative-id":["ani13010053"],"URL":"https:\/\/doi.org\/10.3390\/ani13010053","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,23]]}}}