The aim of the study was to characterize the enzymatic and non-enzymatic components comprising the antioxidant system in spermatozoa and individual fractions of dog ejaculate. Ejaculates were collected from six dogs of mixed-breeds. Total protein content, activity of antioxidant enzymes and the content of low-molecular antioxidants, such as L-glutathione (GSH), L-ergothioneine (ERG), L-ascorbic acid and total SH-group, were analyzed in the ejaculated spermatozoa and seminal plasma of the pre-spermatic, spermatic and post-spermatic fractions. The total antioxidant status (TAS) and antiperoxidant activity of the seminal plasma were also determined. The enzymatic antioxidant system of canine spermatozoa is mainly represented by superoxide dismutase (SOD) activity and, to a lesser extent, by glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) activity. Catalase activity was not detected either in the spermatozoa or in the different ejaculate fractions. GSH and ERG were detected in each fraction. Furthermore, a high level of L-ascorbic acid was observed in fractions of the ejaculate. Proteins and low-molecular weight antioxidants could influence the total antioxidant status and antiperoxidant activity of the seminal plasma. Increased suppressive activity against lipid peroxidation was shown only in the pre-spermatic and post-spermatic fractions. The different fractions of dog ejaculate, which are the main source of enzymatic antioxidants and low-molecular antioxidants, play an important role in protecting spermatozoa against reactive oxygen species.
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