Chemicals and sources
Unless otherwise stated, all chemicals used were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The extender used in this study was Beltsville thawing solution (BTS), the diluent was composed of 37.5g fructose, 1.25 g of ethylenediaminetetraacetic acid (EDTA)-Na2, 6.0g sodium citrate, 6.0 g of citric acid two sodium, 0.75g of potassium chloride, 1.25g of NaHCO3 and 0.2 mg of gentamicin in 1000mL of deionized water. The only difference between control group and treatment groups is the addition of hydroxytyrosol. Samples of control and treatment groups repeated at least five times.
Semen collection
The experimental animals were selected 6 healthy Large White boars from the national core conservation farms in Xingping City, Shaanxi Province (34° 12’ N, 108° 17’ E). The boars are in the same housing and management conditions, the environment is controlled at 15-25°C, individual fences in the building, the windows are exposed to natural daylight and supplemental light, a total of 16 hours of light per day (at the boar eye level ≥ 150lx light intensity). According to the nutritional needs of adult AI boars, they can get water freely and feed commercial forage. The test was carried out by hand-collecting, and the semen was collected and filtered twice with 0.22 μm filter membrane. Besides, the first pre-sperm fraction was not collected and the gelatinous portion was discarded. The sperm motility was detected by computer-assisted sperm analysis CASA system (Hamilton Thorne Research, Beverly, MA, 87 USA). The test used only semen samples with ejaculation volume ≥200 ml, milky white, and slightly smelly, with vitality >85%. Use BTS (Beltsville Thawing Solution) to dilute semen to adjust the concentration to approximately 1.5×107 sperm/ml. Various concentrations of hydroxytyrosol (0, 40, 80, 120, 160, 200 μM) were added to the sperm dilution.
Sperm motility assay
The sperm motility of each preservation group was tested using the CASA system and was measured at 0, 1, 2, 3, 4, 5 days. The method is as follows: 8 μL of each group is pre-warmed on a clean slide to be observed at 37°C. The CASA system automatically detects the sperm motility and related motion parameters in the field of view (A minimum of 300 sperm cells were recorded for each sample from five randomly selected fields per replicate).
Analysis of ROS content
The ROS accumulation was measured at 0, 1, 3, 5 days during liquid preservation of semen. 300 μL of semen was taken from each semen sample, 10 μL of the active oxygen fluorescent probe DCFH-DA was added, and incubation was carried out at 37°C for 30 minutes. Flow cytometry was used to detect the fluorescence intensity of the probe (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: E004-1-1)).
Analysis of plasma membrane and acrosome integrity
The sperm membrane and acrosome integrity were measured at 0, 1, 3, 5 days during liquid preservation of semen [8]. Sperm membrane: 0.1 μL SYBR-14 working solution, 0.5 μL PI (propidium iodide) working solution and 80 μL pig sperm used water bath heating 10 minutes at 37°C. 10 μL was taken on a clean glass slide and the slide was observed at 37°C . Acrosome staining: 0.1 μL DAPI (4', 6-diamidino-2-phenylindole) working solution was pre-incubated with 80 μL sperm for 30 minutes at 37°C, then 25 μL of uniform smear was taken. After fixation for 10 minutes in methanol, 5 μL of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) working droplets were stained, after incubating for 20 minutes at 37°C, the slides were taken and observed. Each field of view was not less than 300 sperm. The sperm plasma membrane and the acrosome integrity were detected using an inverted fluorescence microscope (Leica DMI8). The regression equation was obtained by least squares analysis. Image Pro-Plus software was used to quantify fluorescence intensity.
Pig sperm DNA damage test (comet electrophoresis test)
When the pig semen was preserved to day 3, the DNA damage of the control group and the hydroxytyrosol 120 μmol/L group were observed by the comet assay. The test method was carried out in strict accordance with the instructions of the DNA Damage Detection Kit (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: G010 -1-1)).
Analysis of semen antioxidant ability
The MDA competencies were assessed using a malondialdehyde test kit (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A007-1-1)) according to the manufacturer’s instructions. Briefly, the samples and test reagent were mixed by vortexing. The tubes were then sealed with plastic wrap and punctured with a small needle to produce a small hole. The samples were used water bath heating 40 minutes at 95°C. When the water cooling, centrifuged 10 minutes at 1500g. The supernatants were collected and the absorbance was measured at 532 nm in the fluorescent microplate reader (Boster, Co., USA).
The CAT and T-AOC were determined using kits according to the manufacturer’s protocols (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A007-1-1, A015-1-2)). The preparation of the sample followed the instructions of the operation. Finally, the CAT activity was measured at 405 nm and T-AOC was measured at 520 nm in the fluorescent microplate reader.
SOD and GSH-PX in this study were performed by SOD kit and GSH-PX kit according to the manufacturer’s protocols (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A001-3-2, A005-1-2). The absorbance of samples was determined at 550 nm for SOD and 412 nm for GSH-PX at the end of reaction in the fluorescent microplate reader.
Sequencing analysis of pig sperm protein group
Protein sample was sonicated three times on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail). The remaining debris was removed by centrifugation at 12,000 g at 4°C for 10 minutes. Ultimately, the supernatant was collected and the protein concentration was determined with BCA kit according to the manufacturer’s instructions.
Trypsin Digestion:
For digestion, the protein solution was reduced with 5 mM dithiothreitol for 30 min at 56°C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM TEAB to urea concentration less than 2 M. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratios for a second 4 h-digestion.
TMT Labeling (optional):
After trypsin digestion, peptide was desalted by Strata XC18SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Briefly, one unit of TMT/iTRAQ reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 hours at room temperature and pooled, desalted and dried by vacuum centrifugation.
HPLC Fractionation (optional):
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, and 250 mm length). Briefly, peptides were first separated with a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 18 fractions and dried by vacuum centrifuging.
LC-MS/MS Analysis:
The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto a home-made reversed-phase analytical column (15-cm length, 75 μm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min and climbing to 80% in 3 min then holding at 80% for the last 3 min, all at a constant flow rate of 400 nL/min on an EASY-nLC 1000 UPLC system.
The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTMPlus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
Database Search:
The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against databases concatenated with the reverse decoy database. Trypsin/P was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the First search and 5 ppm in the Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and oxidation on Met was specified as variable modifications. FDR was adjusted to < 1% and the minimum score for peptides were set > 40.
Artificial insemination test
The experimental animals were selected from the Huayang breeding farm in Luonan County, Shaanxi Province (33° 52’ N, 109° 44’ E), with 198 healthy Large White sows and a small difference in body weight.
Pre-treatment of each concentration of pig semen by artificial insemination, batching the test sows in batches (33 batches per batch, preferentially selecting sows with good estrus conditions and recent uninfected diseases, completing all breeding within 10 days), Maintain the same nutritional physiology and management level during breeding. The sow was fertilized in the morning and fertilized again the next morning. Artificial insemination is operated by skilled technicians in accordance with standard artificial insemination procedures. Each sow is inseminated with 40 to 50 ml of semen.
After 35 days of artificial insemination, the hand-held ultrasonic detector was used to check the pregnancy status of the sow. When the sows were born, the size of litters were checked and recorded, and the newborn weight of the piglets were also weighed.
Statistical analysis
All results were expressed as the mean ± SD. Sperm activity, plasma membrane integrity, acrosome integrity, T-AOC activity, MDA content, CAT activity, ROS accumulation, SOD content and GSH-PX activity were compared using Duncan’s multiple-range test. Statistical analyses were performed using Statistical Product and Service Solutions (SPSS 21; SPSS, Chicago, IL, USA). Statistically significant differences between variable were determined at P < 0.05.