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In this study, certain enzymes in ram semen involved in reactive oxygen species elimination and their changes during the cryopreservation process were characterized in order to investigate the hypothesis that the antioxidant defense system is involved in the maintenance of frozen sperm quality. Glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities were quantified in ram sperm samples subjected to cooling and freezing/thawing processes. In addition, their distribution on the sperm surface and the changes due to cryoinjury were determined by indirect immunofluorescence. SOD showed the highest antioxidant activity, which was also twice as high in fresh and cooled samples as in frozen/thawed ones. Enzymatic activity of GPx and GR showed no significant change throughout the freezing process. Seminal plasma proteins (SPPs) added alone or with other compounds showed a protective effect and accounted for an increase in the sperm quality parameters and enzyme activity levels not only in the fresh sample but also after cooling and freezing/thawing. These antioxidant enzymes were distributed over several sperm regions, and we were able to define several subpopulations according to the obtained sperm immunofluorescence patterns. The sperm membrane distribution of SOD, GPx, and GR changed considerably during cryopreservation, and the type and percentage of the immunofluorescence patterns found in fresh samples were severely modified. This remodeling was strongly affected by the use of different cryoprotectants. The mixture of SPPs, oleic/linoleic acids, and vitamin E was able to partly maintain and recover the fresh enzyme distribution, particularly of SOD.

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The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this study, we investigate the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides the evaluation of viability and the capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the phosphatidylserine translocation and caspase-3 and -7 activities. This is the first report, to our knowledge, that evidences the presence of active caspases in ram sperm. The results confirm the better ability of the dextran/swimup procedure to select nonapoptotic spermatozoa, in addition to viable and noncapacitated sperm, compared with other sperm preparation methods. This should be considered to enhance results of artificial insemination techniques in ovine reproduction.