精子冷冻损伤发生在快速冷冻阶段之后：流式细胞术方法及冷冻保存不同阶段的抗氧化酶活性
Sperm cryodamage occurs after rapid freezing phase: flow cytometry approach and antioxidant enzymes activity at different stages of cryopreservation.
作者信息
Castro L S, Hamilton T R S, Mendes C M, Nichi M, Barnabe V H, Visintin J A, Assumpção M E O A
机构信息
Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
Laboratory of Spermatozoa Biology, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil ; Laboratory of In Vitro Fertilization, Cloning and Animal Transgenesis, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of Sao Paulo, Sao Paulo, Brazil.
出版信息
J Anim Sci Biotechnol. 2016 Mar 5;7:17. doi: 10.1186/s40104-016-0076-x. eCollection 2016.
BACKGROUND
In order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry.
RESULTS
There was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters.
CONCLUSION
Based on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.
背景
为提高牛精子冷冻保存过程的效率，了解精子如何应对温度差异以及恢复自身代谢的能力非常重要。流式细胞术方法与抗氧化酶活性相结合，能够在冷冻保存过程中更灵敏地评估精子细胞。本研究的目的是评估冷冻保存过程不同阶段的精子属性和抗氧化酶活性。从荷斯坦公牛采集的精液样本（n = 4）分为3种处理：新鲜（37°C）；冷却（5°C）；解冻。在孵育后0小时和2小时进行评估。通过流式细胞术评估膜完整性、线粒体膜电位（MMP）和DNA损伤；通过分光光度法测量过氧化氢酶、超氧化物歧化酶和谷胱甘肽过氧化物酶等抗氧化酶的活性。
结果
与新鲜和冷却组相比，解冻组中DNA损伤精子的百分比增加，并且与0小时相比，孵育2小时时也增加。考虑MMP时，解冻组中中等电位细胞的百分比与新鲜和冷却组相比增加。相反，解冻组中高线粒体电位的细胞百分比下降。同样在解冻组中，与新鲜和冷却组相比，顶体和膜受损的细胞增加。抗氧化酶活性与膜或线粒体参数之间存在显著相关性。
结论
根据我们的结果，我们得出结论，冷冻保存会影响细胞和DNA完整性，关键时期是精子细胞暴露于冷冻温度时。此外，我们的研究表明细胞内抗氧化机制（超氧化物歧化酶和谷胱甘肽过氧化物酶）不足以控制冷冻损伤。
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