Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me2SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me2SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me2SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me2SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.

中文翻译：

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脱脂奶粉用作非渗透性冷冻保护剂可减少冷冻保存的斑马鱼精子中的氧化和 DNA 损伤

冷冻保护剂在冷冻保存过程中起着至关重要的作用，保护生物样品免受冷冻损伤。在这里，我们评估了不同增量剂与可渗透和不可渗透冷冻保护剂的组合和相互作用对斑马鱼精子冷冻保存的影响，并通过广泛的变量方法分析冷冻保存的影响。使用了两种稀释剂，Hank 平衡盐溶液 (HBSS) 和 Ginsburg 溶液。使用了八种冷冻保护溶液 (CS)：CS1 (HBSS + Me2SO 8%)、CS2 (HBSS + 甲醇 8%)、CS3 (HBSS + Me2SO 8% + 脱脂奶粉 15%)、CS4 (HBSS + 甲醇 8% +脱脂奶粉 15%)、CS5 (Ginsburg + Me2SO 8%)、CS6 (Ginsburg + 甲醇 8%)、CS7 (Ginsburg + Me2SO 8% + 脱脂奶粉 15%) 和 CS8 (Ginsburg + 甲醇 8% + 脱脂奶粉末 15%）。将样品在冷冻管中在干冰上冷冻保存 20 分钟，储存在液氮中，在 38°C 下解冻 10 秒，然后进行分析。除了提高活力外，我们还发现奶粉还可以更好地保存细胞膜和正常细胞形态，并保护精子细胞免受冷冻保存引起的 DNA 损伤和氧化应激。