Testicular Oxidative Stress Assessment

Malondialdehyde (MDA) levels in testis tissues were assessed using reagents provided by Northwest Life Science Specialties LLC (Vancouver, WA, USA). Briefly, MDA reaction of a colored complex was measured at an absorbance of 532 nm using a kinetic spectrophotometer (Spectra Max 180, Molecular Devices, Sunnyvale, CA, USA). MDA levels are expressed as μmole of MDA/mg of wet testis tissue. Testicular ROS/RNS levels were quantified using an OxiSelect in Vitro ROS/RNS Assay Kit (STA-347, Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Fluorescence of dichlorofluorescein (DFC) was measured with a SpectraMax Gemini XS fluorimeter (Molecular Devices, Sunnyvale, CA, USA) at excitation/emission wavelengths of 480/530 nm as described previously [1 (link)]. Activities of testicular superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase were detected with commercially available kits (item no. 706002, 703102, and 707002, respectively, Cayman Chemical, Ann Arbor, MI, USA) as per the manufacturer’s instructions. Testicular SOD, GPx, and catalase values are expressed per milligram of protein.