7. Biochemical analyses

Testicular tissues were separated into small pieces after weighing. These tissues were diluted 10-fold with 50 mM phosphate-buffered saline solution and homogenized using a homogenizer (TissueLyser LT; Qiagen, Dublin, Ireland). The homogenate was centrifuged at 10,000 rpm for 15 minutes at 4℃. The total antioxidant status (TAS) in testicular tissues was measured using an auto-analyzer (AU5800; Beckman Coulter Inc., Brea, CA, USA) and the Rel Assay kit (Rel Assay Diagnostics, Gaziantep, Turkey) following Erel [14]. This kit works according to the principle of blue-green 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ATBS) or the ABTS radical being reduced to its colorless form by antioxidant molecules. The change in absorbance at 660 nm is related to the total antioxidant level of the sample. Trolox, the water-soluble analogue of vitamin E, was used as the calibrator. The results are expressed as equivalent/g.

Total oxidant status (TOS) in testicular tissue was measured using the AU5800 auto-analyzer and the Rel Assay kit following Erel [15]. This kit works by the principle of oxidant molecules reducing the ferrous form of iron ions to the ferric form. The iron ions form a colorful complex with chromogen in an acidic environment. The color intensity measured with a spectrophotometer is related to the number of total oxidant molecules in the sample. Hydrogen peroxide was used as the calibrator, and the results are expressed as µmol H₂O₂ equivalent/g.

The oxidative stress index (OSI) is the ratio of TOS to TAS [15]. The results are expressed as an arbitrary unit (AU).