Table 1.

Direct and indirect tests used to measure oxidation. A. Direct tests measure ROS production and sperm cell oxidation. B. Indirect tests evaluate oxidation by measuring the downstream markers of injury from ROS.

Test	Function	Method of measurement
A. Direct tests
Chemiluminescence assay	Detection of oxidation or reduction through generation of light (by-product of reaction)	Uncharged and charged probes undergo either oxidation or reduction to generate light as a by-product
Flow cytometry	Measurement of ROS	Incubation with dye emits fluorescence when excitated by a light source at specific wavelengths
Electron spin resonance (ESR)	Direct detection of free radicals (but not at low concentrations or short half-lives)	ESR obtains absorption spectra of the spin energy of unpaired electrons in an applied magnetic field
Cytochrome c reduction	Measurement of ROS on the cell membrane	Superoxide radicals being reduced to ferricytochrome c are identified
Nitroblue tetrazolium tests	Localisation of reactions between superoxide ions and sperm cells or leucocytes	Nitroblue tetrazolium reacts with superoxide ions and turns from yellow to purple/blue when ROS are present
B. Indirect tests
Myeloperoxidase test	Detection of granulocytes in semen	Benzidine is used to buffer and assess samples for peroxidase positivity through staining
Lipid peroxidation level	Detection of lipid peroxidation through identification of by-products	Levels of mutagenic MDA and toxic 4-HNE by-products of lipid peroxidation are measured using thiobarbituric acid assays and colorimetric reactions
MiOxsys	Measurement of oxidation–reduction potential	A galvanostat-based system is used to measure the transfer of electrons from reductant to an oxidant in millivolts
Total antioxidant capacity	Evaluation of antioxidant status of a biological sample	The addition of an enhanced chemo-luminescent assay to seminal plasma measures suppression of chemiluminescence and time to recovery