C2037

Cytochrome c oxidase (COX) activity was measured according to [75 (link)] using a microplate combining 5 μL of supernatant, 192 μL of K phosphate buffer (50 mM, 1 mM EDTA, pH 7.2), and 3 μL of 2 mM reduced cytochrome c (c2037; Sigma-Aldrich; USA; reduced with ascorbic acid 20:1) to initiate the reaction. The COX activity was measured in duplicate at 37 °C, with an absorbance of 550 nm using a microplate reader (Varioskan Flash-Spectral Scanning Multimode Microplate Reader 183, Thermo Scientific, MA, USA). The results were expressed as nmol/min/mg of protein. Protein concentration was measured with the Bradford method.

To evaluate the activity of mitochondrial respiratory complex activity, sperm cells were resuspended in 20 mmol/L potassium phosphate buffer (pH 7.0) to obtain a final concentration of 2 × 10⁸ spermatozoa/mL and then disrupted by freeze-thawing before analysis.
The catalytic activities of mitochondrial complexes I, II, III, and IV were evaluated spectrophotometrically [37 (link),39 (link)]. Complex I activity was determined following the decrease in absorbance at 340 nm, using decylubiquinone (D7911, Sigma Aldrich) as electron acceptor and NADH (N8129, Sigma Aldrich) as donor. Complex II activity assay was evaluated following the decrease in absorbance at 600 nm, using succinate (S3674, Sigma Aldrich) as donor and DCPIP (119814, Sigma Aldrich) as electron acceptor. Complex III and complex IV activities were determined by measuring the variation of absorbance at 550 nm, resulting from reduction and oxidation of cytochrome c (C2037, Sigma Aldrich), respectively. Catalytic activities expressed as nmoles/min.