Lipid peroxidation assay

The measurement of the tissue lipid peroxidation (LPx) level was taken with the thiobarbituric acid (TBA)-reactive substance assay, as described by Ohkawa et al.¹⁷ A 1.15% KCl solution was used to homogenize 250 mg of testis tissue and 0.2 ml of 1.8% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid solution (pH 3.5), 0.8% TBA and 0.6 ml distilled water were added to 0.2 ml of tissue homogenate. The mixtures were heated at 95°C for one hour. After cooling with tap water, 5 ml of the mixture of n-butanol and pyridine (15:1, v/v) was added and then centrifuged at 4000 rpm for 20 min. This organic layer was measured at 532 nm absorbance. 1,1,3,3-tetramethoxypropane (TMP) was used as an external standard, and the level of lipid peroxides was expressed as nmol/mg wet tissue.