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ARS Home » Southeast Area » Stoneville, Mississippi » Warmwater Aquaculture Research Unit » Research » Publications at this Location » Publication #427538
Research Project: Biophotonics - Emerging Imaging Technologies for Food Animal Research

Location: Warmwater Aquaculture Research Unit

Title: Production of live tiger salamander offspring using cryopreserved sperm

Author
item CHEN, DEVIN - Mississippi State University
item LAMPERT, SHAINA - Mississippi State University
item CHEN, LIDUNN - Mississippi State University
item BURGER, ISSABELLA - Mississippi State University
item VANCE, CARRIE - Mississippi State University
item ALLEN, PETER - Mississippi State University
item SONGSASEN, NUCHARIN - Smithsonian Institute
item ROTH, TERRI - Cincinnati Zoo & Botanical Garden
item KOUBA, ANDREW - Mississippi State University

Submitted to: Nature Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/16/2025
Publication Date: 5/5/2025
Citation: Chen, D., Lampert, S., Chen, L., Burger, I., Vance, C., Allen, P., Songsasen, N., Roth, C., Kouba, A. 2025. Production of live tiger salamander offspring using cryopreserved sperm. Nature Scientific Reports. 15 (15702). https://doi.org/10.1038/s41598-025-99052-2.
DOI: https://doi.org/10.1038/s41598-025-99052-2

Interpretive Summary: The findings from this study support the use of diluted HAM’s F-10 medium as an extender and DMFA as a cryoprotectant for freezing salamander sperm. Further work is needed into methodologies for washing tiger salamander sperm cryopreserved with DMSO to warrant its future use, as the chemical appears toxic during the fertilization process. The results from this study suggest that sperm motility post-cryopreservation may not be the optimal functional assay for sperm fertilization potential in tiger salamanders, and more studies should consider IVF to test the functionality of frozen sperm.

Technical Abstract: Amphibian conservation breeding programs often rely on assisted reproductive technologies (ART) to produce offspring due to the difficulty of replicating natural reproductive cues under human care. Reproductive management using in-vitro fertilization (IVF) with cryopreserved sperm has been successfully applied to a variety of anurans, yet the approach has seen limited use for internally-fertilizing caudates. This study aimed to test two cryoprotectants, dimethyl formamide (DMFA) and dimethyl sulfoxide (DMSO), in combination with three different sperm extenders, HAM’s F-10, diluted HAM’s F-10, and 10% Holtfreter’s, for the production of salamander larvae from cryopreserved sperm. Fresh sperm was collected from tiger salamanders (n'='12) and diluted with one of three extenders, then subsequently mixed 1:1 with cryoprotectant. Samples were frozen using liquid nitrogen and later thawed to be applied in IVF. The diluted HAM’s and DMFA treatment resulted in the highest (p'<'0.05) fertilization rate, with over 40% of eggs cleaving. The DMFA treatments resulted in 87 hatched offspring, while DMSO treatments resulted in only one offspring. This research highlights the potential of biobanking to produce internally-fertilizing salamander larvae, with DMFA being the likely preferred sperm cryoprotectant. These findings can inform conservation breeding programs by increasing caudate population sustainability through genetic management, facilitated by ART.