A Comparison of Two Different Cryoprotectants on Post-Thaw Sperm Parameters and Fertilization Success in Tiger Salamanders (Ambystoma tigrinum)

ORCID

CKouba: https://orcid.org/0000-0001-8466-6077

MSU Affiliation

College of Agriculture and Life Sciences; Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology; College of Forest Resources; Department of Wildlife, Fisheries, and Aquaculture

Creation Date

2026-10-30

Abstract

Different cryoprotectants have been found to have species-specific effects on post-thaw sperm parameters such as motility and egg fertilization. This study aimed to compare the effect of dimethyl sulfoxide (Me2SO) versus dimethylformamide (DMFA) on tiger salamander (Ambystoma tigrinum) post-thaw sperm parameters and fertilization capability. Milt samples from two male A. tigrinum were standardized to 1x106 sperm/mL and then mixed 1:1 with stock cryoprotectant for a final concentration of either 10% Me2SO + 0.5% bovine serum albumin (BSA) or 10% DMFA + 0.5% BSA; samples were frozen in straws (4-5 straws/treatment/animal) for 10 minutes at 10 cm height above liquid nitrogen. Straws were then thawed in a 40°C water bath for five seconds and applied to fresh eggs from three A. tigrinum females for in vitro fertilization. Post-thaw sperm motility, morphology, and viability as well as fertilization and hatch rates were compared between the two treatments using generalized linear mixed modeling. When comparing the post-thaw parameters, sperm treated with Me2SO had significantly higher (p< 0.001) relative post-thaw total motility (40.1 ± 14.3% vs. 20.8 ± 5.0%) compared to DMFA. Additionally, there was no significant difference (p>0.10) between Me2SO and DMFA for relative normal sperm morphology (92.2 ± 9.5% vs. 94.1 ± 8.1%) or viability (12.7 ± 7.6% vs. 9.8 ± 6.0%). Sperm treated with DMFA resulted in 11.6 ± 14.9% fertilization and 7.9 ± 13.8% hatch rates while sperm treated with Me2SO resulted in 0.01 ± 0.02% fertilization and 0.0% hatch rates. A total of 22 A. tigrinum larvae were produced using sperm frozen with DMFA. It is possible that Me2SO has a negative effect on salamander gametes compared to DMFA thus affecting fertilization potential. Overall, the use of DMFA in combination with BSA is a promising cryoprotectant treatment for salamander sperm to generate new offspring for conservation programs.

Publication Date

12-15-2022

Publication Title

Cryobiology: International Journal of Low Temperature Biology and Medicine

Publisher

Elsevier

First Page

49

Rights

© 2022 Elsevier

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Digital Object Identifier (DOI)

https://doi.org/10.1016/j.cryobiol.2022.11.158