One testis was homogenized in ice-c

One testis was homogenized in ice-cold Tris–HCL buffer (150 mM,pH 7.4). The ratio of the tissue to the homogenization buffer was 1:10 w/v. Assessment of testicular antioxidant defense mechanisms was carried out in tissue homogenate. Determination of malondialdehyde (MDA), marker of lipid peroxidation, in testicular homogenate is based on its reaction with thiobarbituric acid to form a pink complex with an absorption maximum at 535 nm [31]. Testicular nitric oxide (NO) level was measured as total nitrite/nitrate, the stable degradation products of NO, by reduction of nitrate into nitrite using copperized cadmium, followed by color development with Griess reagent in acidic medium [27]. Evaluating reduced glutathione (GSH) concentration is done by using a spectrophotometric kit (Biodiagnostic, Egypt). In brief, the method is based on that the sulfhydryl component of GSH reacts with
5,5-dithio-bis-2-nitrobenzoic acid (Ellman's reagent) producing 5-
thio-2-nitrobenzoic acid having a yellow color, that was measured colorimetrically
at 405 nm. Superoxide dismutase (SOD) activity in testicular
tissues was determined according to the method described
previously [17]. One unit of SOD is described as the amount of enzyme