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) which 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 group is the addition of HT. Samples of control and treatment group repeated at least five times.
We selected 6 healthy Large White Pigs from the national core conservation farms in Xingping City, Shaanxi Province, China (34° 12’ N, 108° 17’ E). The pigs were in the same housing and management conditions, the environment was controlled at 15-25°C, individual fences in the building, the windows were exposed to natural daylight and supplemental light, a total of 16 hours of light per day (at the pig eye level ≥ 150lx light intensity). According to the breeding standards of adult AI pigs, they could get water freely and fed commercial forage. A total of 30 samples were collected (collect 6 samples from different pigs each time, every week and a total of five times), the first pre-sperm fraction was not collected and the gelatinous portion was discarded. The test was carried out by hand-collecting, and the sperm was collected and filtered twice with 0.22 μm filter membrane. The sperm motility was detected by computer-assisted sperm analysis CASA system (Hamilton Thorne Research, Beverly, MA, 87 USA). The test used only sperm samples with ejaculation volume ≥200 ml, milky white, and slightly smelly, with vitality >85%. We use BTS to dilute sperm to adjust the concentration to approximately 3×108 sperm/ml . Various concentrations of HT (0, 40, 80, 120, 160, 200 μmol/L) were added to the sperm dilution.
Sperm motility assay
The sperm motility of each preservation group was tested using the CASA system and measured at 0, 1, 2, 3, 4, 5 days. The method was as follows: 8 μL of each group sample was pre-warmed on a clean slide and then to be observed at 37°C. The CASA system automatically detected the sperm motility in the field of view (We selected five randomly fields for each sample and a minimum of 300 sperm cells were recorded for each view). Samples of control and treatment group repeated at least five times.
Analysis of ROS content
The ROS accumulation was measured at 0, 1, 3, 5 days during storage. 300 μL of specimen was taken from each sperm sample. 10 μL of the active oxygen fluorescent probe DCFH-DA was added and incubated 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)). Samples of control and treatment group repeated at least five times.
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 sperm . Sperm plasma: 0.1 μL SYBR-14 working solution, 0.5 μL PI (propidium iodide) working solution and 80 μL sample heated in a 37°C water bath for 10 minutes. 10 μL sample was placed on a clean glass slide and observed at 37°C. Sperm acrosome: 0.1 μL DAPI (4', 6-diamidino-2-phenylindole) working solution was pre-incubated with 80 μL sample for 30 minutes at 37°C and 25 μL sample smeared on the microslide. After fixation for 10 minutes in methanol, 5 μL of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) working solution was stained. After incubating for 20 minutes at 37°C, the slide was observed at 37°C. Each 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 (v6.0). Samples of control and treatment group repeated at least five times.
Pig sperm DNA damage test (Comet electrophoresis test)
When the pig sperm was preserved to day 3, the DNA damage of the control group and the HT 120 μmol/L group were observed by the comet assay. The test method was carried out according to the instruction of the DNA Damage Detection Kit (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: G010 -1-1)). Samples of control and the HT 120 μmol/L group repeated at least five times.
Analysis of sperm antioxidant ability
The Catalase (CAT) and Total antioxidant capacity (T-AOC) were determined using kits according to the manufacturer’s instruction (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A007-1-1, A015-1-2)). The preparation of sample followed the instructions of the operation. The CAT activity was measured at 405 nm and T-AOC was measured at 520 nm in the fluorescent microplate reader (Boster, Co., USA). Samples of control and treatment group repeated at least five times.
Superoxide Dismutase (SOD) and Glutathione Peroxidase (GSH-PX) analysis in this study were performed according to the manufacturer’s instruction (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A001-3-2, A005-1-2). Samples were measured at 550 nm for SOD and 412 nm for GSH-PX in the fluorescent microplate reader (Boster, Co., USA). Samples of control and treatment group repeated at least five times.
The Malondialdehyde (MDA) content was assessed using assay kit (thiobarbituric acid (TBA) method) (Nanjing Institute of Bioengineering, Co., Nanjing, China (Product No.: A003-1-2)) according to the manufacturer’s instruction. The result was measured at 532 nm in the fluorescent microplate reader (Boster, Co., USA). Samples of control and treatment group repeated at least five times.
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.
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 17 ℃ 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.
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 17 ℃ and pooled, desalted and dried by vacuum centrifugation.
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.
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.
The resulting MS/MS data were processed using the Maxquant search engine (v.22.214.171.124). 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.
We use three statistical analysis methods, namely, principal component analysis (PCA), relative standard deviation (RSD) and Pearson's Correlation Coefficient, so that we could evaluate the quantitative repeatability of protein (Supplement Figure1. a, b, c).
We perform statistics on the distribution of differentially expressed proteins in GO secondary annotations. GO annotations are divided into three major categories: Biological Process, Cellular Component and Molecular Function, which explain the biological role of proteins from different perspectives. Then, we used software to perform subcellular structure localization prediction and classification statistics for differentially expressed proteins (Figure4. d; Supplement Figure2. c. d).
Artificial insemination test
In order to study the reproductive effect of the control group and the HT 120 μmol/L group after 3 days of storage. We chose 198 healthy Large White multiparous sows and only a small difference in body weight (99 Large White sows in the control group and treatment group). The experimental animals were selected from the Huayang breeding farm in Luonan County, Shaanxi Province, China (33° 52’ N, 109° 44’ E). All sows are in the same management environment, and the sows were fed and managed according to commercial standards before and after artificial insemination.
Artificial insemination was performed by skilled technicians in accordance with standardized artificial insemination procedures and each sow insemination with 40 to 50 ml sperm (the sperm concentration of sperm doses are approximately 3.0×108 sperm/ml, ensure that at least 3 billion active sperm). The sows were fertilized in the morning and fertilized again after 12 hours (completing all works within 10 days).
After 35 days of artificial insemination, the hand-held ultrasonic detector was used to check the pregnancy status of all sows (Guangzhou sonostar V6, China). When the piglets were born, we recorded the following data: Laying births, Live number, Live rate, Healthy number, Health rate, Primary weight.
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.