Fernández-Santos María R, Martínez-Pastor Felipe, García-Macías Vanesa, Esteso Milagros C, Soler Ana J, Paz P, Anel L, Garde José J
Biology of Reproduction Group, Department of Game Resources (IDR), Castilla-La Mancha University (UCLM), Albacete, Spain.
J Androl. 2007 Mar-Apr;28(2):294-305. doi: 10.2164/jandrol.106.000935. Epub 2006 Nov 1.
The main goal of this study was to investigate the potential protective effects of enzymatic and nonenzymatic antioxidants on cryopreservation injuries to red deer epididymal spermatozoa. In Experiment 1, the effects on sperm freezability of the enzymatic antioxidants catalase, superoxide dismutase, and a combination thereof were studied. In Experiment 2, sperm cryoresistance was evaluated when different nonenzymatic antioxidants, such as vitamin E, vitamin C, and butylated hydroxytoluene (BHT), were added to the freezing extender. Sperm quality was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and acrosome (ie, spermatozoa with normal apical ridges; % NAR) and membrane (by means of the HOS test) integrity. To address fully these topics, we incorporated a new set of functional sperm tests for mitochondrial function, membrane phospholipid disorder, and sperm chromatin stability. Samples were evaluated after freezing and thawing, and after a 2-hour period of incubation at 37 degrees C. The present study demonstrates that the addition of enzymatic antioxidants to freezing extenders improves sperm viability after cooling, and improves sperm motility, acrosome integrity, and mitochondrial status (P<.05) after thawing. After a 2-hour incubation period at 37 degrees C in the presence of enzymatic antioxidants, an improvement in membrane integrity (P<.05) was observed. However, when nonenzymatic antioxidants were present in the freezing diluents, no positive effects on thawed sperm parameters were noted. The chromatin stability test did not show significant differences between the treatments. We conclude that enzymatic antioxidants should be present in the early steps of cryopreservation of epididymal spermatozoa from red deer, so as to improve motility and acrosome integrity.
本研究的主要目的是探讨酶促抗氧化剂和非酶促抗氧化剂对马鹿附睾精子冷冻损伤的潜在保护作用。在实验1中，研究了酶促抗氧化剂过氧化氢酶、超氧化物歧化酶及其组合对精子冷冻能力的影响。在实验2中，当向冷冻稀释液中添加不同的非酶促抗氧化剂，如维生素E、维生素C和丁基羟基甲苯（BHT）时，评估了精子的抗冻性。通过显微镜评估单个精子活力（SMI）、存活率、顶体（即具有正常顶嵴的精子；%NAR）和膜（通过HOS试验）完整性来体外判断精子质量。为了全面解决这些问题，我们纳入了一组新的功能性精子测试，用于评估线粒体功能、膜磷脂紊乱和精子染色质稳定性。在冷冻和解冻后以及在37℃孵育2小时后对样本进行评估。本研究表明，向冷冻稀释液中添加酶促抗氧化剂可提高冷却后精子的存活率，并改善解冻后精子的活力、顶体完整性和线粒体状态（P<0.05）。在酶促抗氧化剂存在的情况下，在37℃孵育2小时后，观察到膜完整性有所改善（P<0.05）。然而，当冷冻稀释液中存在非酶促抗氧化剂时，解冻后精子参数未观察到积极影响。染色质稳定性测试在各处理之间未显示出显著差异。我们得出结论，在马鹿附睾精子冷冻保存的早期步骤中应存在酶促抗氧化剂，以提高精子活力和顶体完整性。
