A number of studies have explored the use of membrane permeable (usually metabolizable) and membrane impermeable saccharides to protect cells in general, and sperm in particular during cryopreservation. Critical concentrations for protective levels of sugars frequently range between 50 mmol/L and 500 mmol/L, where efficacy is attributed to the sugar's membrane stabilizing and glass forming attributes and colligative effects that reduce intra- and extracellular salt concentrations during freezing. Many studies on bull sperm have demonstrated that both permeating and non-permeating sugars have negligible positive effects on post-thaw viability. Recently, however, a non-metabolizable sugar, 3-O-Methylglucose (3-OMG), was shown to protect hepatocytes during liver cryopreservation at 0.1-0.3 mol/L. Because glucose is readily transported into sperm, we hypothesized that 3-OMG could be a new class of cryoprotectant to explore in bull sperm. Here we present positive results demonstrating that 3-OMG improves post thaw viability in bull sperm, and that this effect is not likely due to improved glass forming capabilities. In particular, in experiment 1, 3-OMG was added to the Tris-egg yolk-glycerol base media at levels from 0 mmol/L to 200 mmol/L. Semen from four bulls was collected and diluted with one of the cryopreservation media, cooled, and frozen following industry standard practices. Motility and mitochondrial activity were negatively impacted when concentration of 3-OMG was more than 25 mmol/L. Therefore, we explored lower concentrations in experiment 2, where semen from eight bulls was used to evaluate concentrations 5 mmol/L, 15 mmol/L and 25 mmol/L of 3-OMG compared with control. Motility and progressive motility in 5 mmol/L 3-OMG and in the control were significantly higher than 15 mmol/L and 25 mmol/L 3-OMG, whereas mitochondrial activity and acrosome integrity in 5 mmol/L 3-OMG were significantly better than the control freezing medium. In experiment 3, to evaluate whether the improved effects of 3-OMG are due to its non-metabolizing property, or due to colligative effects, we compared post-thaw viability in semen from four bulls cryopreserved with 5 mmol/L glucose, sucrose, or 3-OMG. Motility and progressive motility was significantly improved in 3-OMG compared to glucose or sucrose groups which were comparable to the EY control. In conclusion, 3-OMG at a concentration of 5 mmol/L in Tris-egg yolk-glycerol medium improves the post thaw motility, progressive motility and viability of bull sperm. The mechanism of action is not understood but because the efficacy is maximal at low concentrations, it is not likely due to improved intra- or extracellular glass forming abilities and may demonstrate a different protective mechanism than was shown in hepatocytes.

中文翻译：

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低浓度的 3-O-甲基葡萄糖可改善冷冻保存的牛精子的解冻后恢复

许多研究探索了使用膜渗透性（通常可代谢）和膜非渗透性糖类来保护一般细胞，特别是在冷冻保存过程中的精子。糖的保护水平的临界浓度通常在 50 mmol/L 和 500 mmol/L 之间，其功效归因于糖的膜稳定和玻璃形成属性以及在冷冻过程中降低细胞内和细胞外盐浓度的依数效应。许多关于公牛精子的研究表明，渗透性和非渗透性糖对解冻后活力的积极影响都可以忽略不计。然而，最近，一种不可代谢的糖，3-O-甲基葡萄糖 (3-OMG)，显示在 0.1-0.3 mol/L 的肝脏冷冻保存过程中保护肝细胞。因为葡萄糖很容易被输送到精子中，我们假设 3-OMG 可能是在公牛精子中探索的一类新的冷冻保护剂。在这里，我们展示了积极的结果，证明 3-OMG 提高了公牛精子的解冻后活力，并且这种效果不太可能是由于提高了玻璃形成能力。具体而言，在实验 1 中，将 3-OMG 以 0 mmol/L 至 200 mmol/L 的水平添加到 Tris-蛋黄-甘油基础培养基中。收集四头公牛的精液并用其中一种冷冻保存介质稀释、冷却并按照行业标准做法冷冻。当 3-OMG 浓度超过 25 mmol/L 时，运动性和线粒体活性受到负面影响。因此，我们在实验 2 中探索了较低的浓度，其中使用八头公牛的精液来评估与对照相比 5 mmol/L、15 mmol/L 和 25 mmol/L 的 3-OMG 浓度。5 mmol/L 3-OMG 和对照组的运动性和渐进性运动性显着高于 15 mmol/L 和 25 mmol/L 3-OMG，而 5 mmol/L 3-OMG 的线粒体活性和顶体完整性明显更好比对照冷冻介质。在实验 3 中，为了评估 3-OMG 的改善效果是由于其非代谢特性，还是由于依数效应，我们比较了用 5 mmol/L 葡萄糖、蔗糖、或 3-OMG。与与 EY 对照相当的葡萄糖或蔗糖组相比，3-OMG 的运动性和渐进性运动性显着改善。总之，在 Tris-卵黄-甘油培养基中浓度为 5 mmol/L 的 3-OMG 可提高公牛精子的解冻后运动性、渐进运动性和活力。