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

Abstract

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.

Cryopreservation of canine semen: the effect of two extender variants on the quality and antioxidant properties of spermatozoa

“…Frozen-thawed samples of bull semen showed more than a 50% drop in SOD activity (Bilodeau et al 2000). In cryopreserved human and ram spermatozoa a decrease in SOD activity was accompanied by stable levels of GPx activity (Alvarez and Storey 1992, Lasso et al 1994, Marti et al 2008. In our study an insignificant (ca.…”

Influence of bovine sperm DNA fragmentation and oxidative stress on early embryo in vitro development outcome

“…ROS are physiologically produced during sperm metabolism (Marti et al 2008). These free radicals are necessary for sperm physiological processes such as the maintenance of membrane fluidity, sperm capacitation, acrosome reaction, sperm-oocyte interactions, and the signaling pathways involved in the fertilization, implantation, and early embryo development process (de Lamirande et al 1997, Sakkas et al 1998, Sanocka & Kurpisz 2004, Bansal & Bilaspuri 2010).…”

Preservability of bull spermatozoa in Tris-egg yolk extender enriched with different concentrations of butylated hydroxytoluene

“…The generation of ROS resulting from cryopreservation [14] and the reduced activity of the antioxidant enzymes in semen postfreezing [11,35,36] bay be the causative factor of sperm cryoinjury, therefore, the addition of BHT as an antioxidant in buffalo semen extenders [28] in ram [37], in goat [7] and in canine [1] can improve postthawed semen quality.…”