Md 2529

Pancreatic homogenate samples were used for the spectrophotometric assessment of the malondialdehyde (MDA) (a lipid peroxidation end product) and reduced glutathione (GSH) levels by commercially available kits (MD 25 29 and GR 25 11, respectively Bio-diagnostic co., Giza, Egypt) following the instructions of the manufacturer^{27 (link)}.

CAT (CA 2517, BioDiagnostic, Egypt), GPX (1.11.1.9, BioAssay Systems, United States), and SOD (SD 2521, BioDiagnostic, Egypt) were determined colorimetrically by a commercial kit as described formerly (37 (link)–39 (link)) at 520, 340, 560, and 534 nm, respectively. Lipid peroxidation (marked by MDA level) was evaluated in the sperm cell homogenate using commercial kits (MD 2529, BioDiagnostic, Egypt). Lipid peroxidation in spermatozoa was measured by the reaction of thiobarbituric acid (TBA) with MDA. The level of MDA was measured colorimetrically at 534 nm according to Ohkawa et al. (39 (link)).

Determination of MDA and SOD content in sciatic nerve homogenates was performed using specific colorimetric kits obtained from Bio-diagnostic (Catalog No: MD2529 and SD2521, respectively, Cairo, Egypt) as per the kits’ instruction manual.

Homogenization of heart tissues was carried out in a 10-fold volume of ice-cooled phosphate-buffered saline (PBS) (ice-cooled, pH 7.4). Following centrifugation at 10,000× g for 20 min and 4 °C, the supernatant was then collected for analysis of oxidative and inflammatory markers. ELISA kits were used in the assessment of the hearts’ content of malondialdehyde (MDA), reduced glutathione (GSH), enzymatic activities of superoxide dismutase (SOD), catalase (CAT) (Cat. No. MD 2529, GR 2511, SD 2521, and CA 2517, Biodiagnostic, Giza, Egypt, respectively), and protein carbonyl, interlukin-6 (IL-6), and tumor necrosis factor α (TNF-α) (Cat. No. ab238536, ab234570, and ab100785, Abcam, Cambridge, UK, respectively).
Nuclear fractions of tissue homogenates were obtained using NE-PER nuclear and cytoplasmic extraction kit (Cat. No. 78833, Thermo Fisher Scientific, Waltham, MA, USA). Protein content of the nuclear extracts was determined, and a volume equivalent to 80 μg was employed in the assessment of the DNA-binding activity of NF-kB p65 using NF-κB p65 ELISA Kit (Cat. No. ab133112, Abcam, Cambridge, UK). Results are expressed as fold change of control.

Total antioxidant capacity (TAC) in serum was determined by colorimetric method using a trade available kit (Cat. no# TA 2513) obtained from Bio-Diagnostics Company, Egypt, following previously outlined procedures (Koracevic et al., 2001 (link)). The anti-oxidative capacity in rat serum is measured by the reaction of antioxidants in the sample with a known amount of exogenously supplied hydrogen peroxide (H₂O₂), resulting in removing a specific amount of H₂O₂. The remaining H₂O₂ is estimated colorimetrically by an enzymatic reaction, which involves the conversion of 3, 5, dichloro–2–hydroxyl benzensulphonate to a colored product. The level of serum malondialdehyde (MDA), a major lipid peroxidation product, is measured colorimetrically using a commercially available kit (Cat. no# MD2529) obtained from Bio-Diagnostics Company, Egypt, following previously outlined instructions (Ohkawa et al., 1979 (link)). This assay is based on the reaction of thiobarbituric acid (TBA) with MDA in an acidic medium at a temperature of 95°C for 30 min to form a thiobarbituric acid reactive product with a pink color, with its absorbance measured at 534 nm. Serum TAC is measured in mM/L, while serum MDA is measured in nmol/ml.

Semen samples at 24, 48, and 72 postchilling were centrifuged at 1,000 × g for 20 min, and the supernatant, composed of semen extender and seminal fluid, was used to assess oxidative stress biomarkers, using MDA contents and assessing the concentration of glutathione peroxidase (GPx), SOD, and CAT. Pellets of spermatozoa were washed by suspending them in cold fresh phosphate buffer saline, centrifuged at 3,000 × g for 10 min, and then frozen in liquid nitrogen for RNA extraction. Lipid peroxidation was evaluated by quantifying the MDA content (nmol/mL) using a commercial kit (MD 2529, Bio-Diagnostic, Egypt, with a sensitivity of 0.1 nmol/ml and detection range up to 50 nmol/ml) according to manufacturer instructions. MDA was measured with 2-thiobarbituric acid, monitoring the absorbance change at 532 nm with the spectrophotometer (Shanghai Spectrophotometer Co. Ltd., China). Antioxidant concentrations of CAT, SOD, and GPx were determined with commercial kits: CAT (CA 2517, Bio-Diagnostic, Egypt, with a sensitivity of 0.22 U/ml and detection range up to 200 U/ml), SOD (SOD 2521, Bio-Diagnostic, Egypt, with a sensitivity of 2.0 mU/ml and detection range up to 500 U/ml), and GPx (1.11.1.9, Bio-Assay Systems, United States, with a sensitivity of 1 mU/ml and detection range up to 500 mU/ml), according to manufacturer's instruction.