The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane‐derived oxygen scavenger (Oxyrase) on post‐thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris‐egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post‐thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo‐3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine‐phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F‐pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose‐dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase‐fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose‐dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post‐thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

中文翻译：

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大肠杆菌膜衍生的氧还原酶系统 (Oxyrase) 在冷冻保存过程中保护 bubaline 精子。

本研究的目的是确定使用大肠杆菌膜衍生的氧清除剂 (Oxyrase) 对精液补充剂脱氧对水牛 ( Bubalus bubalis ) 精子解冻后质量的有效性。16 份精液，4 只来自四头公牛，每份 4 份，每份被分成 5 个等份，用 Tris-蛋黄补充剂稀释，添加不同浓度的氧化酶（0、0.3、0.6、0.9 和 1.2 U/ml），指定为处理分别为 T1、T2、T3、T4 和 T5，并冷冻保存。解冻后，Oxyrase 并没有立即改善精子的动力学和活力；然而，它提高了 T3 的保存质量（在孵育 120 分钟后显着降低了解冻后精子活力的恶化）。此外，T3 减少（p < .05) 胆固醇流出并保护精子质膜的完整性。Fluo-3 AM/碘化丙啶 (PI) 双染色流式细胞术显示最高 ( p < .05) T3 中细胞内钙含量低的活精子的比例。在金霉素试验中，酪氨酸磷酸化蛋白（32、75 和 80 kDa）的低表达和相对较低的 F 型（无获能的精子）百分比证实了氧化酶补充剂防止精子过早获能。重要的是，氧化酶强化以剂量依赖性方式降低了超氧阴离子，表明精子线粒体水平的氧气可用性降低。同样，在氧化酶强化的精子中，脂质过氧化指标丙二醛浓度也以剂量依赖性方式降低。综上所述，