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.

中文翻译：

---

甲醇对斑马鱼（斑马鱼）精子冷冻保存的时间和浓度影响。


我们评估了冷冻保存过程中不同浓度、暴露时间和阶段的甲醇对斑马鱼精子的冷冻保护作用。通过压碎解剖的睾丸或剥离腹部来收集样品。暴露于 0%、2%、5%、8% 和 10% 甲醇 0–11 分钟后，新鲜精子 (1 × 10 ^{6 个}细胞/mL) 的质膜完整性未出现变化（通过流式细胞仪测量） ，但暴露于 8% 或 10% 后观察到细胞大小变化（光散射）。暴露 0-60 分钟后，新鲜精子 (1 × 10 ^{8 个}细胞/mL) 的存活率或膜完整性没有表现出显着变化。与 0 之间的其他浓度相比，在 5%、8% 和 10% 甲醇中冷冻保存的精子显示出较高的解冻后存活率，在 5% 和 8% 中显示出较高的解冻后运动性，在 5% 中显示出最高的解冻后膜完整性。 %和10%。解冻后 0-60 分钟内，任何浓度的细胞存活率和膜完整性均未发现显着差异 ( p ≥ 0.269)。比较 5% 和 8% 甲醇对解剖睾丸 ( n = 20) 的影响，结果显示解冻后活力、膜完整性、细胞存活、受精或孵化、胚胎活力没有差异；对于剥离的精子（ n = 10），在解冻后膜完整性、受精和胚胎活力方面没有观察到差异，但是，在 5% 甲醇中检测到比在 8% 甲醇中更高的运动性和存活率。因此，根据解冻后的存活率和活力，5% 的甲醇浓度似乎最适合冷冻保存斑马鱼精子。