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Published-Ahead-of-Print February 22, 2008, DOI:10.2164/jandrol.107.003459

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Effect of the Cryopreservation Process on the Activity and Immunolocalization of Antioxidant Enzymes in Ram Spermatozoa

Elena Marti , Jose I. Marti , Teresa Muiño-Blanco , and Jose A. Cebrian-Perez *

* To whom correspondence should be addressed. E-mail: pcebrian{at}unizar.es.

In this study, certain enzymes in ram semen involved in ROS elimination and their changes during the cryopreservation process were characterized in order to investigate the hypothesis that the antioxidant defence 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 that 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 (SPP) 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 remodelling was strongly affected by the use of different cryoprotectants. The mixture of SPP, oleic-linoleic acids and Vitamin E were able to partly maintain and recover the fresh enzyme distribution, particularly of SOD.


Key words: Cryopreservation • Reactive Oxygen • Sperm • ROS • antioxidant enzymes • immunolocalization






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