A003 1

Right lung specimens from 10 mice in each group were taken. The microplate reader
was used to measure MDA and SOD values in lung tissues. The lung tissue samples
were crushed and ground in a mortar and weighed. Phosphate buffer solution (PBS)
(4°C) was added according to a mass-to-volume ratio of 1:9 and the mixture was
homogenized on ice. The homogenate was centrifuged to collect the supernatant
and used as a sample for subsequent experiments. According to the manufacturer's
instructions (Nanjing Jiancheng, A003-1), the prepared samples were mixed with
vortex mixer and soaked in water at 95°C for 40 min. After cooled with running
water, it was centrifuged for 10 min (2000 rpm/min). The absorbance of 300
µL of supernatant was measured at 532 nm for malondialdehyde (MDA)
values. According to the manufacturer's instructions (Nanjing Jiancheng,
A003-1), the prepared samples were evenly mixed, incubated at 37°C for 20 min,
and the absorbance values of each sample were measured at 450 nm for SOD
values.

For each straw, 200 µL of thawed semen were centrifuged at 1600 × g for 5 min, and the precipitate was collected. The sperm precipitate was mixed with dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, China) medium without fetal bovine serum (FBS) to make the 10 µM DCFH-DA working solution for ROS detection. Samples were protected from light and incubated at 37 °C for 20 min. Spermatozoa were washed gently three times with PBS and the OD was measured at 525 nm with the microplate reader (TECAN, Switzerland).
The spermatozoa were mixed with 400 µL of distilled water and then centrifuged repeatedly to destroy the sperm structure. Finally, they were centrifuged at 4000 × g for 30 min and the supernatants were collected which was regarded as the enzyme crude extract. Lipid peroxidation, oxidative DNA damage and total antioxidant capacity (T-AOC) in the enzyme crude extracts were estimated by using the MDA assay kit (A003-1, Jiancheng, China), 8-OHdG (8-hydroxydeoxyguanosine) ELISA kit (E-EL-0028c, Elabscience, China) and the T-AOC assay kit (A015, Jiancheng, China), respectively. The processes were performed according to the manufacturers’ instructions. The MDA, 8-OHdG and T-AOC levels were assessed by using thiobarbituric acid (TBA), Fe³⁺ reduction and horseradish peroxidase-labeled avidin methods, and measured at 532, 520 and 450 nm on a microplate reader, respectively.