*4.10. Western Blotting*

Testicular tissues were homogenized over ice using a homogenizer with 1% protease inhibitor cocktail (Service bio). Then, after repeated mixing for 30 min in ice, the supernatant was collected after centrifugation at 12,000 g and 4 ◦C for 10 min. Total protein concentrations were determined by a BCA protein assay kit (Service bio) and adjusted to the same level. A total of 30 μg of protein extracts from testicular issues were separated by 5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and followed by blocking with 5% skimmed milk at room temperature for 1 h. The primary antibodies were added into the membrane and then incubated overnight at 4 ◦C. Primary antibodies to the following proteins were used: Keap1 (sc-514914; 1:500), Heme Oxygenase 1 (sc-390991; 1:1000), NQO1 (sc-376023; 1:1000), Bax (sc-20067; 1:1000), JNK (sc-7345; 1:500), p-JNK (sc-6254; 1:500), cleaved PARP (sc-56196; 1:1000) (Santa Cruz, CA) and Nrf2(16396-1-AP; 1:1000), Bcl2 (26593-1-AP; 1:1000), Caspase-3 (19577-1-AP; 1:1000) (Proteintech, USA). After washing, the membranes were incubated with secondary antibodies at 1:2000 at room temperature for 2 h, and the marked proteins were illuminated using ECL Reagent Kit (Service bio). The bands were visualized by the scanner EPSON V300 (Seiko Epson Corporation, Japan) as well as its intensity was analyzed by ImageJ software and normalized to GAPDH levels.

#### *4.11. Statistical Analysis*

Results were expressed as the mean ± standard deviation (SD) and analyzed by SPSS version 17. All the experimental data were analyzed using One-way ANOVA. Differences at *p* < 0.05 was considered statistically significant.

**Author Contributions:** Conceptualization, X.Q. and X.Z.; methodology, X.Z. and Z.P.; formal analysis, X.Z.; investigation, X.Z.; resources, C.Z.; data curation, X.Z.; writing—original draft preparation, X.Z.; writing—review and editing, X.Z., Z.P., H.Z. and H.L.; supervision, X.Q.; project administration, X.Q.; funding acquisition, X.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was financially supported by the China Agricultural Research System of MOF and MARA, High Value Aquatic Products Processing and Utilization Innovation Team of Higher Education Institutions of Guangdong Province (GDOU2016030503).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Laboratory Animal Committee of Guangdong Ocean University, China (no. GDOU-LAE-2020-014).

**Data Availability Statement:** Data are available upon request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

