Quantitative Assessment of Oxidative Stress in Testis

Malondialdehyde (MDA) levels in testis tissue homogenates were determined using a commercially available MDA assay kit (NWLSSTM malondialdehyde assay kit; Northwest Life Science Specialties LLC., Vancouver, WA, USA) according to the manufacturer’s instructions. Absorbance of colored complex was measured at a wavelength of 532 nm by kinetic spectrophotometric analysis using a Spectra Max 180 (Molecular Devices, Sunnyvale, CA, USA). MDA concentration in the sample was analyzed by comparing the measured absorbance value to the standard curve of MDA. The level of MDA was expressed as μmole per mg tissue. ROS/RNS levels in testis tissue homogenates were determined using a fluorescence kit (STA-347, OxiSelect^TM in vitro ROS/RNS assay kit, Cell Biolabs, Inc., San Diego, CA, USA). Absorbance values were measured at excitation and emission wavelengths of 480 and 530 nm, respectively, with a SpectraMax Gemini XS Fluorimeter, as described previously [26 (link)].