Sample collection and preparation of seminal plasma
The boars (n=15) were chosen based on production records during 2-years. All boars were Landrace raised under the same management conditions and received the same nutrition, and they were stalled in commercial herds. Semen was collected using the gloved hand method. The semen collection rhythm was twice a week and one single ejaculate per boar was used in this study.
After collection, the spermatozoa-rich fraction of each ejaculate (80–100 mL) was filtered through gauze and subsequently divided into two aliquots of equal volume. The first one was used for seminal plasma separation from spermatozoa through centrifugation at 500×g and 4 °C for 30 min. Seminal plasma preparations were then examined using phase microscopy to ensure no spermatozoa remained. Clean seminal plasma samples were then stored in liquid nitrogen. The other spermatozoa-rich fraction aliquot was diluted in Androhep Plus (Minitube, Germany) at 2×108, and then used to cryopreserve.
Cryopreservation and thawing of sperm samples
Fifteen ejaculates (> 2×108 spermatozoa/mL; > 75% motility) were chosen to cryopreserve. Firstly, the semen samples were stored at 17 °C to cool, then the semen was centrifuged at 500×g for 10 min. Soft sperm pellets were subsequently diluted to 2×109 spermatozoa/mL in Androstar® Cryo Plus (Minitube, Germany) containing 20% egg yolk. Then the spermatozoa were cooled slowly to 5ºC for 5 h and subsequently diluted to 1 × 109 spermatozoa/mL with freezing medium containing 6% glycerol (Sigma–Aldrich, MO) at 5ºC. Afterward, sperm samples were packed in 0.5 mL labeled plastic straws (Minitube, Germany). The straws were then transferred to a programmable freezer CryoMed 7457 (Thermo Fisher, MA). The cooling ramp was as follows: Wait at 4°C→2°C /min to 2°C→Hold for 1 min at 2°C→35°C /min to -30°C→Hold for 1 min at -30°C→35°C /min to -150°C→ Hold for 4 min at -150°C. The straws were finally plunged into liquid nitrogen and stored before use.
Assessment of sperm quality
Sperm motility parameters obtained were those described by Yeste et al[22]. Sperm motility assessment was carried out utilizing a commercial computer assisted sperm analysis (CASA) system (CASAS-QH-III, Tsinghua Tongfang Co., Ltd.). After evaluating three replicates per sample (a minimum of 1000 spermatozoa was counted per replicate), the corresponding mean standard error of the mean (SEM) was calculated.
Sample classification into GFEs and PFEs
To classify seminal plasma samples into two groups (GFEs vs. PFEs), spermatozoa were cryopreserved and thawed and sperm quality assessments were carried out at three different points: pre-freeze, refrigerated semen at 17 °C and frozen–thawed spermatozoa at 30 min post thawing. To distinguish seminal plasma samples between two groups of good (GFE) and poor (PFE) freezability, Boar sperm were characterized by the reduced of sperm motility.
Ultra-high Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-qTOF-MS) data acquisition
Analysis data was acquired using a UHPLC-high definition quadrupole time-of-flight MS instrument (UHPLC-qTOF SYNAPT G1 HD-MS system, Waters) equipped with TripleTOF 6600 (Q-TOF, AB Sciex). A binary solvent method consisting of eluent A (25mM NH4Ac and 25mM NH4OH in water pH=9.75) and acetonitrile (B) was carried with elution gradient as follows: 0 min, 95% B; 0.5 min, 95% B; 7 min, 65% B; 8 min, 40% B; 9 min, 40% B; 9.1 min, 95% B; 12 min, 95% B, delivered at 0.5mL min-1. The Triple TOF mass spectrometer was used for its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Analyst TF 1.7, AB Sciex) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. In each cycle, 12 precursor ions whose intensity greater than 100 were chosen for fragmentation at collision energy (CE) of 30 V (15 MS/MS events with product ion accumulation time of 50 msec each). ESI source conditions were set as following: Ion source gas 1 as 60 Psi, Ion source gas 2 as 60 Psi, Curtain gas as 35 Psi, source temperature 600°C, Ion Spray Voltage Floating (ISVF) 5000 V or -4000 V in positive or negative modes, respectively.
Multivariate data (MVD) analysis
UHPLC-qTOF-MS data was analyzed using SIMCA 13 software (Umetrics, Umea, Sweden) and interactive XCMS (version 3.2). Before exporting the data to SIMCA for visualization and biomarker selection, the LC-MS raw data was firstly processed (noise elimination, peak picking, alignment and retention time correction) with MarkerLynxTM software (version 4.1, Waters Corporation, Milford, MA, USA). The following parameters were used for data processing: retention time (Rt) range of 2.5-11 min, mass range of 100-1000 Da, mass tolerance of 0.02 Da, Rt window of 0.2 min. The data matrix obtained from MarkerLynxTM processing was then exported into SIMCA 13 for PCA and OPLS-DA analyses. The data were Pareto-scaled and no transformation was used. For the XCMS analyses, the MassLynxTM raw data (.raw) were converted to NetCDF format using the DataBridge application in MassLynxTM (Waters, MA, USA). The converted data (NetCDF format) were then use XCMS for processing, statistical analysis, visualization and biomarker identification as described by [23]. The parameters were as follows: feature detection set as centWave method, minimum peak width = 5, maximum peak width = 20, retention time correction set as Obiwarp method, Profstep = 1, alignment set as m/z width = 0.015, min fraction = 0.5, bw = 5, and statistics set as statistical test = unpaired parametric t-test (Welch t-test), paired t-test and posthoc analysis with the threshold p-value = 0.01 and fold-change = 1.5.
Relative distribution and statistical analysis
Total intensity values (integrated area under the peak) from MarkerLynxTM XS software (Waters Corporation, Manchester, UK) pre-processed data matrixes were used for univariate statistical analyses. SPSS software (IBM SPSS Statistics for Windows, Version 22. Armonk, NY: IBM Corp.) was used for such descriptive statistics. Here, Univariate Analysis of Variance (ANOVA) was performed as 2-tailed complete randomized blocks, and used to compare the nontreated with the different time points of treated cells. ANOVA was followed by the Bonferroni post hoc test where differences between the means were considered significant at p < 0.05, and indicated in the Box-and-Whiskers plots.
Targeted metabolomics analysis
Targeted metabolomics analysis was performed using QTRAP 5500 (AB SCIEX), The target metabolomics metabolite extraction method is as same as UHPLC-qTOF-MS data acquisition. We perform absolute quantification of candidate differential metabolites based on standard products, and the standard products are purchased from Yuanye Biological Technology Co., Ltd.