Al-Kass Ziyad, Morrell Jane M, Ntallaris Theodoros
Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Mosul, Mosul 41002, Iraq.
Department of Clinical Sciences, Swedish University of Agricultural Sciences, P.O. Box 7054, SE-75007 Uppsala, Sweden.
Animals (Basel). 2025 Jun 25;15(13):1881. doi: 10.3390/ani15131881.
Sperm quality is adversely affected by cryopreservation due to the increased production of reactive oxygen species, which affects the integrity of sperm membranes, motility, and DNA fragmentation. Three methods for removing seminal plasma, washing (centrifuging extended semen at 800× for 10 min) and Single Layer Centrifugation with high or low density Equicoll, were used to prepare 29 ejaculates from ten stallions for freezing. Sperm quality parameters (kinematics, plasma membrane integrity, superoxide and hydrogen peroxide production, mitochondrial membrane potential, and DNA fragmentation) were evaluated before and after freezing using kinematic and flow cytometric analysis. The parameters for fresh samples were within the normal range for stallion semen but were lower after thawing. There were few differences between the three preparation methods. Interestingly, DNA fragmentation was affected most by the sperm preparation method, being lowest for SLC through high density Equicoll, although SLC through low density Equicoll was effective for some stallions. Some differences were observed in the proportions of live or dead spermatozoa positive for hydrogen peroxide. In conclusion, all of these methods would be suitable for the preparation of semen prior to cryopreservation, but Single Layer Centrifugation through high density Equicoll was the most effective in removing spermatozoa with damaged DNA.
由于活性氧的产生增加，冷冻保存会对精子质量产生不利影响，活性氧会影响精子膜的完整性、活力和DNA碎片化。使用三种去除精浆的方法，即洗涤（将稀释后的精液以800×离心10分钟）以及使用高密度或低密度Equicoll进行单层离心，从10匹种马中制备了29份射精样本用于冷冻。在冷冻前后，使用运动学和流式细胞术分析评估精子质量参数（运动学、质膜完整性、超氧化物和过氧化氢产生、线粒体膜电位以及DNA碎片化）。新鲜样本的参数在种马精液的正常范围内，但解冻后较低。三种制备方法之间差异不大。有趣的是，DNA碎片化受精子制备方法影响最大，通过高密度Equicoll进行单层离心时最低，不过通过低密度Equicoll进行单层离心对某些种马也有效。在过氧化氢呈阳性的活精子或死精子比例上观察到了一些差异。总之，所有这些方法都适用于冷冻保存前精液的制备，但通过高密度Equicoll进行单层离心在去除DNA受损的精子方面最为有效。
