## 2 Materials and Methods
### Methods
#### 2.2.9 Determination of ROS Production  
ROS production in sperm was evaluated by nitroblue tetrazolium (NBT) assay. NBT is a pale-yellow colored substance and interacted with superoxide and generated tetrazoliumyl radical. It was then dismutated to form formazan crystals [17]. The sperm concentration was 1 \(\times\) 10\({}^{6}\) cells/mL and 0.3 mL of NBT solution (0.1 mg/mL NBT, 5% FCS, 3% DMSO in 10 mL RPMI) was added and incubated at 37 \({}^{\circ}\)C for 60 min. After centrifugation (500\(\times\)_g_, 10 min), the supernatant was removed and washed with PBS. After, 200 \(\upmu\)L of dimethyl sulfoxide (DMSO) was added, and the absorbance was measured at 570 nm [15].  
2.10 Determination of Tumor Necrosis Factor-\(\alpha\) (TNF-\(\alpha\)) and Interleukin (IL)-6 Levels  
TNF-\(\alpha\) and IL-6 ELISA kits were used to determine the IL-6 and TNF-\(\alpha\) levels. These cytokines were produced during the inflammatory phase and promote cell death [18; 19]. The capture antibody was diluted 200\(\times\) with a 1\(\times\) coating buffer in a 96-well plate (100 \(\upmu\)L/mL) at 4\({}^{\circ}\)C overnight. Later, the supernatant was removed and washed 4 times with a washing buffer (400 \(\upmu\)L/mL). Then, 1\(\times\) assay buffer (400 \(\upmu\)L/well) was added and incubated for 1 h at room temperature. After washing (4 times), standard solutions and test samples (100 \(\upmu\)L/well) were added and incubated for 2 h. Later, 100 \(\upmu\)L/well of the diluted detection antibody (200\(\times\) with 1\(\times\) assay buffer) was added after washing and incubated for 1 h at room temperature. The plates were washed with washing buffer 4 times and then diluted. Avidin-HRP enzyme (100 \(\upmu\)L/mL) was added and incubated for 30 min. Later,100 \(\mu\)L/mL substrate solution was added after washing (5 times) and incubated at room temperature for 20 min. Finally, stop solution (100 \(\mu\)L/mL) was added and the absorbance was measured at 450 nm [15].  
2.11 Determination of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Urea, and Creatinine Levels  
ALT and AST kits were used to analyze the ALT and AST contents, respectively. They helped identify the liver problem. For ALT determination, 10 \(\mu\)L of plasma was mixed with 100 \(\mu\)L of reagent (15 mmol/mL \(\alpha\)-ketoglutarate, 1.2 U/mL LD, 0.18 mmol/mL NADH). After mixing, the absorbance was measured at 340 nm at 1, 2, and 3 min. The AST content was analyzed by the AST kit. In total, 10 \(\mu\)L of plasma was mixed with 100 \(\mu\)L of reagent (12 mmol/mL \(\alpha\)-ketoglutarate, 420 U/mL MDH, 600 U/mL LD, 0.18 mmol/mL NADH) and the absorbance was measured at 340 nm for 3 min. High levels of BUN and creatinine were observed under renal disease [20]. The urea kit was used to analyze the BUN content. A total of 1 \(\mu\)L of plasma was mixed with 100 \(\mu\)L enzyme reagent (10 U/mL urease, 2 U/mL GLDH, 0.26 mmol/mL NADH, 3 mmol/mL adenosine-5-diphosphate, 14 mmol/mL \(\alpha\)-oxoglutarate) and the absorbance was measured at 340 nm for 1, 2, and 3 min. A creatinine kit was used to analyze the content of creatinine. Then, 20 \(\mu\)L of plasma was mixed with 100 \(\mu\)L picric acid (35 mmol/mL) and 100 \(\mu\)L sodium hydroxide (0.32 mol/mL). After mixing, the absorbance was determined at 492 nm for 1 and 2 min.  
\[\text{ALT or AST acidity }(\text{U/mL})=1746\times\frac{A\text{ }sample-A\text{ } blank}{min} \tag{3}\]  
Urea or Creatinine concentration\((\mu\text{mol/mL})=\left(\frac{\Delta\text{ A sample}}{\Delta\text{ A standard}}\right)\times\text{standard concentration}\) (4)