Temporal and Concentration Effects of Methanol on Cryopreservation of Zebrafish (Danio rerio) Sperm

We evaluated the cryoprotective effects of methanol on zebrafish sperm at different concentrations, exposure times, and stages during cryopreservation. Samples were collected by crushing of dissected testes or abdominal stripping. After exposure to 0%, 2%, 5%, 8%, and 10% methanol for 0-11 min, fresh sperm (1 × 10⁶ cells/mL) did not show changes in plasma membrane integrity (measured by flow cytometer), but cell size changes (light scatter) were observed after exposure to 8% or 10%. After exposure for 0-60 min, fresh sperm (1 × 10⁸ cells/mL) did not show significant changes in survival or membrane integrity. Sperm cryopreserved in 5%, 8%, and 10% methanol showed high post-thaw survival, in 5% and 8% showed high post-thaw motility, and in 5% showed highest post-thaw membrane integrity compared to other concentrations between 0% and 10%. Within 0-60 min after thawing, no significant differences in cell survival and membrane integrity were found for any concentration (p ≥ 0.269). Comparison of 5% and 8% methanol for dissected testes (n = 20) revealed no difference in post-thaw motility, membrane integrity, cell survival, fertilization, or hatching, embryo viability; for stripped sperm (n = 10), no differences were observed in post-thaw membrane integrity, fertilization, and embryo viability, however, higher motility and survival were detected in 5% than in 8% methanol. Thus, a concentration of 5% methanol seems most suitable for cryopreserving zebrafish sperm based on post-thaw survival and motility.