*4.3. OPs Characterization by LC-ESI/MS/MS*

The molecular weight and amino acid sequences of the OPs were identified by liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). The sample (1 μL) was injected with an autosampler and subsequently separated by a C18 column and eluted with conditions of mobile phase A (H2O, 0.1% formic acid) and mobile phase B (95% acetonitrile, 0.1% formic acid). Peptides were first eluted with a linear gradient from 2% to 35% of mobile phase B for 40 min, and then from 35% to 80% of mobile phase B for 10 min, running temperature of 25 ◦C at a flow rate of 0.3 μL/min. After chromatography, ESI-MS/MS was carried out using a Q-EXACTIVE mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with the electrospray ionization (ESI) source. The spectrometer worked in the positive ion mode, and the parameters of the ESI ionization interface were set as follows: capillary voltage 1.6 kV, Resolution 70,000, AGC target:1e5, NCE/stepped NCE: 27. Samples were analyzed with a full-scan MS mode in the range of 350–2000 m/z to obtain the total ion chromatogram. Then, the Mascot search engine was used to analyze the chromatographic peaks corresponding to the mass spectrometry of the custom oyster protein database. Only peptides identified with *p*< 0.05 significance were recorded.

#### *4.4. Determination of Amino Acid Composition*

The amino acid composition and content of OPs were determined by an amino acid autoanalyzer (L-8900, Hitachi, Tokyo, Japan). The sample and 6 mol/L HCl were added into a tube containing phenol for hydrolysis. After the tube was vacuumed, the mixture was washed with nitrogen and hydrolyzed at 110 ◦C for 22 h. After the filtrate had cooled, it was heated to 40–50 ◦C with a test tube concentrator and dried under reduced pressure until evaporated. Sodium citrate buffer solution was added to the dried test tube and dissolved. The solution was transferred to the injection flask of the instrument for determination by the amino acid analyzer [47]. According to the peak area in comparison with the standard, amino acid contents were calculated.

#### *4.5. Animals and Treatment*

The Laboratory Animal Committee of Guangdong Ocean University, China (no. GDOU-LAE-2020-014) approved all the animal protocols and experimental procedures for this study. Sixty adult ICR male mice (specific pathogen-free, approval no.11032201101487454) were purchased from the Huafukang Biotech Co., Ltd. (Beijing, China) and acclimatized for 7 days before the start of the experiments. All mice were housed at 4–5 mice per cage (23 cm × 32 cm × 15 cm) and randomly fed a normal diet in an environmentally controlled room (temperature was 25 ± 2 ◦C and relative humidity was 50–65%, with a 12 h light/dark cycle). Animals were randomly divided into six groups (n = 10). Control group: mice were given 0.9% NaCl orally and intraperitoneally, once a day; TP (model group): mice were pretreated with 0.9% NaCl and intraperitoneally injected with TP at a

dose of 120 μg/kg; TP + VE (positive control group): mice were orally administrated with VE at a dose of 7.5 mg/kg, and intraperitoneally injected with TP (120 μg/kg) after 1 h every day; TP + OPs: Mice were orally administrated with OPs at the dose of 100 mg/kg (TP + OPs-L), 200 mg/kg (TP + OPs-M), 400 mg/kg (TP + OPs-H) and intraperitoneally injected with TP (120 μg/kg) after 1 h every day. Administration methods and doses of TP-induced testicular injury were conducted according to Wang et al., Ma et al., and Zhang et al. [8,13,32]. VE was determined as the positive control drug according to the study results of Li et al. and Hamza et al. [22,48]. After 28 days of continuous treatment, mice were sacrificed by enucleation of the eye and bled for the collection of blood, and all testes were removed and weighted to measure the testis index (testis weight/body weight). The right epididymis of mice was collected for sperm analysis, and the right testis of mice was stored at −80 ◦C for protein expression and other parameter detection. The left testis was fixed in 4% paraformaldehyde for further histological analysis.

### *4.6. Sperm Analysis*

The entire right epididymis was placed in 1 mL of prewarmed saline and minced into small sections, incubating for 10 min (37 ◦C, 5% CO2) to allow the spermatozoa to swim out from the epididymal tubules. The sperm suspension was dropped into a Neobar's hemocytometer, and the sperm count was estimated under a cover glass. The sperm motility was observed under a 400× light microscope (Olympus Corporation, Tokyo, Japan) and calculated and expressed as a percentage of motile sperm according to the World Health Organization manual criteria. The sperms were stained using the Quick sperm stain kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. Additionally, images of sperm after staining were observed under a light microscope and acquired by photographing using a Leica DMI4000B microscope with Leica DFd4500 imaging system (Leica Corporation, German). The rate of abnormal morphology of sperm was evaluated and calculated by observing sperm staining images. Sperm parameters of mice (count, motility, and morphology) were analyzed in each group according to the methods adopted by Qiu et al. and Oghbaei et al. [49,50].

#### *4.7. Histopathological and Ultrastructural Assessment*

The left testes of mice dehydrated in gradient concentrations of alcohol, and cleared with xylene, then embedded in paraffin at 65 ◦C. After cooling at −20 ◦C, the sections were cut at 4 μm and stained with hematoxylin and eosin (H&E). The sections of testis tissue were observed under a light microscope and photographed at magnifications of 200× and 400× using a Nikon Eclipse E100 microscope with Nikon DS-U3 imaging system (Nikon Corporation, Japan). The testicular structure of each animal was assessed, and Johnsen's score was assessed based on spermatogenic function and germ cell count from 1 (no spermatogenic epithelium) to 10 (normal spermatogenesis). All analyses were performed by an evaluator who was unaware of the treatment group.

#### *4.8. Measurements of Enzyme and Hormone*

The homogenates of right testis tissue were prepared to determine the levels of testicular superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA), and testicular marker enzymes containing lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and acid phosphatase (ACP) according to the specific steps in the kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The protein concentrations of testicular tissue homogenates were determined by the bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology, Shanghai, China). Sex hormones of mice including testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in serum were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits (Mmbio, Jiangsu, China) following the instructions.
