Animals and samples

All ejaculates were provided by a local pig farm (Grup Gepork S.L., Masies de Roda, Spain), which follows the ISO certification (ISO-9001:2008) in the production of doses. The Centre follows all the regulations concerning the production and selling of seminal doses (Directive 2010/63/EU; Animal Welfare Law issued by the Regional Government of Catalonia, Spain; and the regulation on Health and Biosafety issued by the Department of Agriculture, Livestock, Food and Fisheries, Regional Government of Catalonia, Spain). As authors did not manipulate any animal but samples were rather provided by the farm, no permission from the local ethics committee was needed.

All seminal samples intended to the experiments defined below came from healthy and sexually mature Pietrain boars (1–3 years old). Ejaculates were collected with the standard hand-gloved method for this species. Immediately after collection, samples were diluted to a final concentration of 33 × 106 sperm/mL in a commercial extender (Vitasem LD, Magapor S.L.; Zaragoza, Spain) and transported at 17 °C to the laboratory.

Experimental design

Different experiments were designed to test whether the SCF mechanism operates in ejaculated sperm, and to elucidate if it may be triggered in vitro upon incubation with different ions in a dose-dependent manner. In the first and second experiments, whether the activation of the SCF mechanism increases the incidence of DSBs and if these DSBs are located in the TLRs was investigated. The third experiment determined if evoking SCF with Mn2+/Ca2+ and Mg2+/Ca2+ has any repercussion on other sperm functional variables, such as motility, viability, agglutination and intracellular levels of reactive oxygen species.

Experiment 1: Dose-dependent effect of Mn2+/Ca2+ and Mg2+/Ca2+ on the generation of DNA breaks

To assess the generation of DNA breaks after exposure to Mn2+ and Mg2+ ions, the first experiment incubated ejaculated sperm with these ions, at different concentrations and for different times. In addition, these experiments were conducted in permeabilized and non-permeabilized samples with the aim to address if the SCF mechanism is triggered outside the cell or involves the intracellular machinery. Briefly, three semen doses, each from a separate boar (sperm concentration: 33 × 106 sperm/mL), were pooled and centrifuged at 600 g for 10 min at room temperature to remove preservation medium. Sperm were resuspended in phosphate buffered saline buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4, pH = 7.5). In the case of permeabilized samples, they were further incubated with 0.25% Triton X-100 for 10 min on ice. Following this, every sample was split into two aliquots (one for Mn2+/Ca2+ and the other for Mg2+/Ca2+), and each of these aliquots was in turn divided into 10 tubes of equal volume (five to assess the effects of the dose, and five to determine those of the incubation time). To evaluate the dose–response, samples were incubated with Mn2+/Ca2+ or Mg2+/Ca2+ at 0 mM [Control], 0.1 mM, 1 mM, 5 mM or 50 mM (prepared with the appropriate volumes of MnCl2, MgCl2 and CaCl2, all at 0.5 M), for 10 min at 37 °C. To evaluate the time-response, samples were incubated with 10 mM Mn2+/Ca2+ or Mg2+/Ca2+ at 37 °C for 0 min (Control), 2 min, 10 min, 30 min or 60 min. After incubation, the incidence of DNA breaks was determined through the Comet assay.

Experiment 2: Assessment of the size of the DNA fragments generated by Mn2+/Ca2+ and Mg2+/Ca2+ incubations

Pulsed-field Gel Electrophoresis (PFGE) was run to identify the size of the DNA fragments generated after inducing SCF through incubation with Mn2+/Ca2+ or Mg2+/Ca2+. Based on the results of experiment 1, 7 mL of non-permeabilized ejaculated sperm from three boars (concentration: 33 × 106 sperm/mL) were centrifuged at 600 g for 10 min at room temperature to remove preservation medium. Then, samples were resuspended in PBS buffer and incubated with 10 mM Mn2+/Ca2+ or 10 mM Mg2+/Ca2+ (prepared with the appropriate volumes of MnCl2, MgCl2 and CaCl2, all at 0.5 M) at 37 °C for 30 min. Samples were subsequently subjected to PFGE, as described below, in order to assess the size of the resulting DNA fragments. Negative controls consisted of non-treated sperm in PBS without Mn2+/Ca2+ or Mg2+/Ca2+ incubated at 37 °C for 30 min.

Experiment 3: Impact of Mn2+/Ca2+ and Mg2+/Ca2+ dose and/or incubation time treatments on sperm function and survival

How incubation with different doses of Mn2+/Ca2+ and Mg2+/Ca2+ and for different periods affects sperm function and survival was evaluated on the basis of sperm motility with Computer Assisted Sperm Analysis (CASA); sperm agglutination under a phase-contrast microscope; and sperm viability, and levels of total ROS and superoxides through flow cytometry. For all treatments, 50 mL of each sample (N = 3, from three separate boars) were centrifuged at 600 g and room temperature for 10 min to remove the preservation medium. After centrifugation, the supernatant was discarded and the pellet was resuspended in 50 mL of PBS previously tempered at 37 °C. Then, sperm diluted in PBS were distributed in 1-mL aliquots according to the number of treatments and incubation times tested. Following this, the corresponding Mn2+/Ca2+ or Mg2+/Ca2+ treatments were added to sperm samples. To evaluate the dose- and incubation time-response, sperm samples were incubated with different concentrations of Mn2+/Ca2+ or Mg2+/Ca2+ (Control, 0.1 mM, 1 mM, 5 mM, 10 mM and 50 mM, prepared with the appropriate volumes of MnCl2, MgCl2 and CaCl2, all at 0.5 M) for different incubation times (2 min, 10 min, 30 min and 60 min) at 37 °C. Once done, flow cytometry and CASA analysis were conducted to assess the aforementioned variables.

Comet assay

The global incidence of sperm DNA breaks was determined with the Comet assay. The protocol used included a previous step for complete chromatin decondensation, which is known to be essential to accurately evaluate DNA fragmentation in pig sperm [58]. First, samples were diluted to 5 × 105 sperm/mL in PBS and mixed 1:2 (v:v) with prewarmed (37 °C) low melting point (LMP) agarose, achieving a final agarose concentration of 0.66% (w:v). Then, 6.5 µL of the mixture was placed onto an agarose-treated slide, which contained a layer of 1% LMP agarose (Thermo Fisher Scientific; Waltham, MA, USA); the drop was covered with an 8-mm diameter round coverslip. After allowing the agarose-sample mixture to solidify for 5 min on the top of a metal cold plate at 4 °C, the coverslip was gently removed. Subsequently, the slide was incubated in three lysis solutions: (1) 0.8 M Tris–HCl, 0.8 M DTT and 1% SDS (pH = 7.5) for 30 min; (2) 0.8 M Tris–HCl, 0.8 M DTT and 1% SDS (pH = 7.5) for 30 min; and (3) 0.4 M Tris–HCl, 0.4 M DTT, 50 mM EDTA, 2 M NaCl, 1% Tween20 and 100 µg/mL Proteinase K (pH = 7.5) for 180 min. Then, DNA was denatured through incubation in an alkaline solution (0.03 M NaOH, 1 M NaCl, pH = 13) at 4 °C for 5 min. Slides were electrophoresed at 1 V/cm for 4 min in an alkaline buffer (0.03 M NaOH, pH = 13). After electrophoresis, samples were incubated in a neutralization solution (0.4 M Tris–HCl, pH = 7.5) for 5 min, and slides were subsequently dehydrated in an increasing ethanol series (70%, 90% and 100% ethanol; 2 min per step). Samples were dried in horizontal position and stored until staining and analysis. For staining, slides were submerged into a solution containing 5 µL of 1 × SYTOX Orange (Invitrogen; Waltham, MA, USA) in 50 mL of distilled water for 15 min.

The prepared Comet samples were visualized under an epifluorescence microscope (Zeiss Imager Z1, Carl Zeiss AG; Oberkochen, Germany) and captured using the Axiovision 4.6 software (Carl Zeiss AG) at a resolution of 1388 × 1040 pixels. At least 100 sperm cells per sample were captured at 100 × magnification, adjusting the exposure time in each capture to avoid overexposure of Comet heads or Comet tails. The quantitative analysis of fluorescence intensity was performed using the automatic function of the CometScore v2.0 software (Rexhoover, www.rexhoover.com). After reviewing the analysis, overlapping comets or signals that did not correspond to comets were eliminated. A minimum of 50 analyzable comets was set as the lowest limit to establish a mean DNA damage intensity, and more pictures were captured and analyzed when this figure was not reached. Data files including Comet head intensity (arbitrary units (AU)), tail intensity (AU), tail length (pixels) and percentage of DNA in the tail (Tail DNA) were exported from CometScore as.csv. To quantify the amount of DNA breaks, the Olive Tail Moment (OTM) was used as a standard parameter and calculated as OTM = (Tail mean intensity − Head mean intensity) × Tail DNA/100.

Pulsed-field gel electrophoresis (PFGE)

After each treatment, sperm concentration was adjusted to 400 × 106 sperm/mL through centrifugation at 600 g for 5 min and resuspension in PBS. Following this, samples were mixed 1:1 (v:v) with 2% LMP agarose (Thermo Fisher Scientific) previously melted at 38 °C, then poured onto BioRad plug molds (Bio-Rad; Hercules, CA, USA) and finally cooled at 4 °C for 15 min. Thereafter, plugs were unmolded, placed in 2 mL of lysis buffer (10 mM Tris–HCl, 10 mM EDTA, 100 mM NaCl, 20 mM DTT, 2% SDS and 20 mg/mL proteinase K; pH = 8.0), and incubated at 53 °C for 60 min. After incubation, plugs were washed three times in TE buffer (10 mM Tris–HCl, 0.1 mM EDTA; pH = 8).

A half of each plug was cut-off and loaded onto a well of 1% PFGE agarose gel (Pulsed-Field Certified Agarose BioRad; Hercules, CA, USA). A slice of the Low Range PFG DNA Marker (New England Biolabs; Ipswich, MA, USA), which was commercially embedded in agarose, was also loaded. The agarose gel with the standard casting platform frame was placed into a contour-clamped homogeneous electric field apparatus (Bio-Rad CHEF DRIII system) in 0.5 × TBE buffer (Tris–borate 50 mM, EDTA 0.1 mM) at 14 °C. Samples were run at 4 V/cm for 27.1 h with a rotation (angle) of 120° and a pulse change ramp from 6.7 to 33.7 s. Finally, the gel was stained with ethidium bromide and visualized and photographed under ultraviolet light using the GelDoc System (BioRad; Hercules, CA, USA).

For each sample, the intensity of the DNA smear in the gel was quantified employing the Image Studio Lite (LI-COR Biosciences, Lincoln, NE, USA). The DNA ladder was utilized to distinguish the size of DNA fragments as follows: (i) < 33 Kb, corresponding to DNA fragments shorter than a toroid; (ii) between 33 and 194 Kb, corresponding to DNA fragments with sizes compatible with one or multiple toroids; (iii) and > 194 Kb, corresponding to mostly intact DNA (i.e., where SCF did not induce DSBs).

Sperm motility

Sperm motility was assessed through a CASA system (Integrates Sperm Analysis System, ISAS V1.0; Proiser S.L.; Valencia, Spain) coupled to an Olympus BX41 microscope (Olympus; Tokyo, Japan) with a negative phase contrast field at 100 × (Olympus 10 × 0.30 PLAN objective, Olympus). Semen samples were incubated at 38 °C for 15 min, and 3 µL of each sample was placed into a pre-warmed 20-μm Leja chamber slide (Leja Products BV; Nieuw-Vennep, The Netherlands). Two technical replicates were examined, evaluating 1000 sperm per replicate. Total motility was recorded assuming that a sperm cell was motile when its average path velocity (VAP) was ≥ 10 μm/s.

Flow cytometry

Flow cytometry analysis were performed using a CytoFLEX flow cytometer (Beckman Coulter, Fullerton, CA, USA), equipped with red, blue and violet lasers (637, 488 and 405 nm). First, sperm concentration was adjusted to 1 × 106 sperm/mL in PBS. Two replicates per sample were examined in each test and three sperm parameters were evaluated: sperm viability, total ROS and superoxides. For this purpose, SYBR-14, 2′7′-dichlorodihydrofuorescein (H2DCFDA) and Hydroethidine (HE) fluorochromes were combined with Propidium Iodide (PI) or YO-PRO-1. All fluorochromes were excited with the 488 nm laser. The fluorescence emitted by SYBR-14, YO-PRO-1 and H2DCFDA was detected with the FITC channel (525/40), that emitted by HE was collected through the PE channel (585/42), and the fluorescence emitted by PI was detected through the PC5.5 channel (690/50). All fluorochromes were purchased from ThermoFisher (Waltham, MA, USA). Analysis of flow cytometry dot-plots was conducted through the CytExpert Software (Beckman Coulter; Fullerton, CA, USA), and the device was calibrated daily as recommended by the manufacturer.

Sperm viability

Sperm viability was determined by staining samples with SYBR-14 (final concentration of 32 nmol/L) and PI (final concentration of 7.5 μmol/L) at 38 °C in the dark for 15 min. The percentages of viable (SYBR-14+/PI−) and non-viable sperm (SYBR-14−/PI+ and SYBR-14+/PI+) were recorded and used for subsequent statistical analyses.

Total ROS levels (H 2 DCFDA)

Total ROS levels in sperm were detected through staining with H2DCFDA (final concentration of 100 μmol/L), a cell-permeant compound that is oxidized into DCF+ in the presence of ROS, and PI (final concentration of 5.6 μmol/L) at 38 °C in the dark for 20 min. After analysis, four subpopulations were identified and recorded: viable sperm with low levels of ROS (DCF−/PI−); viable sperm with high levels of ROS (DCF+/PI−); non-viable sperm with low levels of ROS (DCF−/PI+); and non-viable sperm with high levels of ROS (DCF+/PI+).

Superoxide levels (HE)

Intracellular superoxide levels (O2•−) were determined after incubation of samples with hydroethidine (HE) (final concentration of 5 µmol/L), an element that is oxidized into E+ in the presence of O2•−, and YO-PRO-1 (final concentration of 31.2 nmol/L) for 20 min at 38 °C in the dark. The fluorescence emitted by E+ was detected with the FITC channel and the one emitted by YO-PRO-1 was collected through the PE channel. Spill-over into these two channels was compensated (2.24% and 7.5%, respectively). Four separate subpopulations were identified and recorded: viable sperm with low levels of superoxides (E−/YO-PRO-1−); viable sperm with high levels of superoxides (E+/YO-PRO-1−); non-viable sperm with low levels of superoxides (E−/YO-PRO-1+); and non-viable sperm with high levels of superoxides (E+/YO-PRO-1+).

Sperm agglutination

Because sperm became agglutinated after incubation with some treatments, the degree of agglutination was also determined in this study. For this purpose, and following the protocol described by Harayama et al. [59], 250 sperm cells per sample were counted under a phase-contrast microscope at 100 × magnification. Each sperm cell was classified as either agglutinated or non-agglutinated. Two technical replicates per sample were examined.

Statistical analysis

Statistical analyses were conducted using IBM SPSS for Windows version 27.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 8 (La Jolla, CA, USA). First, normal distribution and homogeneity of variances were examined using Shapiro–Wilk and Levene tests. As parametric assumptions could not be assumed for the data obtained and because samples were paired (repeated measures), a non-parametric two-way ANOVA (Scheirer-Ray-Hare Test) was run. Factors were concentration of Mn2+/Ca2+ or Mg2+/Ca2+, and incubation time. Pair-wise comparisons were conducted using the Wilcoxon test. A P-value ≤ 0.05 was taken as the limit to consider values statistically significant.