**2.5 Test of metabolic activity**

The mitochondrial toxicity test (MTT test) is a colorimetric test often used for the determination of cell metabolic activity. The main principle of the test is the application of yellow tetrazolium bromide (Sigma-Aldrich, St. Louis, USA) and its subsequent reaction with insoluble mitochondrial succinate dehydrogenase produced by cell mitochondria. This reaction ends with the formation of a blue-violet formazan [15]. The amount of formed formazan is directly proportional to the level of metabolic activity of the cells. We evaluated this test spectrophotometrically at 570 against 620 nm (Multiscan reader, Thermo Fisher, Vantaa, Finland).

## **2.6 Production of superoxide radicals**

For the detection of the superoxide radical concentration, we used the nitroblue tetrazolium or NBT test. The principle of this method is the application of nitroblue tetrazolium (Sigma-Aldrich, St. Louis, USA) to the semen sample. This substance reacts with cellular superoxide to form derivates of formazan. Following washing with PBS and centrifugation (1250 rpm, 5 min), the concentration of formasan derivates was evaluated spectrophotometrically [16] at wavelengths of 620 against 570 nm (Multiscan reader, Thermo Fisher, Vantaa, Finland).

#### **2.7 DNA fragmentation**

This type of DNA damage is characterized by both single and double DNA strand breaks. Several types of DNA damage may be observed in mammalian germ cells and are often associated with male infertility. Nowadays various tests are available to detect sperm DNA damage. DNA fragmentation arises from various reasons such as deficiencies in recombination during spermatogenesis, abnormal sperm maturation, abortive apoptosis, or oxidative stress [17, 18]. In our research we used the chromatin-dispersion test by using the Halomax diagnostic kit (Halotech, Madrid, Spain). This kit can analyze the integrity of the DNA molecule and is based on a controlled DNA denaturation process to facilitate the subsequent removal of the proteins contained in each spermatozoon. The main principle of this method is that damaged spermatozoa create halos formed by loops of fragmented DNA at the head of the sperm which are not present in normal spermatozoa. For the evaluation we used a fluorescent microscope (Leica, Holzheim, Germany) and a magnification of 40×. At least 300 cells were evaluated for DNA fragmentation in each slide containing agarose with processed spermatozoa.

#### **2.8 Lipid peroxidation**

Lipid peroxidation is a process of membrane lipid degeneration caused by free radicals [19]. The extent of lipid peroxidation in our samples was expressed as the amount of malondialdehyde (MDA) production following the addition of thiobarbituric acid (Sigma-Aldrich, St. Louis, USA) and exposure to heat (100°C, 1 h). The MDA concentration in the samples was evaluated spectrophotomerically at a wavelength of 540 nm (Multiscan reader, Thermo Fisher, Vantaa, Finland).
