Reagents

Unless stated otherwise, all reagents used in the present study were of analytical grade and purchased from Sigma (Merck, Darmstadt, Germany). Fluorochromes were acquired from ThermoFisher Scientific (Waltham, MA, USA).

Animals and samples

All semen samples used in the present study were provided by a local farm (Gepork S.L.; Masies de Roda, Spain), which follows the ISO certification (ISO-9001:2008). All the procedures that involved animals were performed by the AI centre in accordance with the EU Directive 2010/63/EU for animal experiments, the Animal Welfare Law issued by the Regional Government of Catalonia, and the current regulation on Health and Biosafety issued by the Department of Agriculture, Livestock, Food and Fisheries, Generalitat de Catalunya, Spain. As no animal was manipulated to conduct the present experiment, since ejaculates were commercially acquired from a local farm (AI-centre), no permission from an Ethics Committee was required.

Ejaculates from healthy and sexually mature Pietrain boars (1–3 years old) were collected using the gloved-hand method. Immediately after collection, semen samples were diluted to a final concentration of 33 × 106 sperm/mL using a commercial extender (Vitasem LD, Magapor S.L., Zaragoza, Spain) and stored at 17 °C for 24 h.

Experimental design

Twenty-four ejaculates from 24 boars (one ejaculate per boar) were used to conduct the analyses described below. Each ejaculate, considered as a biological replicate, was split into three aliquots: the first was used to assess sperm quality, in terms of sperm motility, morphology and viability; the second was intended to IVF; and the third aliquot was stored at – 80 °C until alkaline and neutral Comet assays were carried out.

Evaluation of sperm qualitySperm motility

Sperm motility was assessed through a computer-assisted sperm analysis (CASA) system (Integrates Sperm Analysis System, ISAS V1.0; Proiser S.L.; Valencia, Spain) and Olympus BX41 microscope (Olympus; Tokyo, Japan) with a negative phase contrast field (Olympus 10 × 0.30 PLAN objective, Olympus). Semen samples were incubated for 15 min at 38 °C, and 5 µL of each sample were analysed in a pre-warmed Leja20 counting chamber (Leja Products BV; Nieuw-Vennep, The Netherlands). Two technical replicates were examined, evaluating 1000 sperm per replicate.

Several sperm velocity parameters were recorded: VSL, VAP, curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), LIN, percentage of straightness (STR) and WOB. Total motility and progressive motility were also recorded, and sperm were considered motile when VAP was ≥ 10 µm/s, and progressively motile when STR was over 45%.

Sperm morphology

After diluting semen samples with 0.12% formaldehyde in saline solution (PanReac AppliChem; Darmstadt, Germany; 1:1, v:v), sperm morphology was analysed under a phase-contrast microscope at 1000 × magnification (Nikon Labophot; Nikon; Tokio, Japan). Two hundred sperm cells were counted and those without morphology alterations were considered as normal. Moreover, primary and secondary alterations were recorded [67].

Sperm viability assessment

The LIVE/DEAD sperm viability kit (Molecular Probes, Eugene, OR, USA) following the protocol of Garner and Johnson [68] was used to evaluate plasma membrane integrity. This kit includes SYBR-14, which stains the nuclei of all sperm, and propidium iodide (PI), which only stains those of sperm that have lost their plasma membrane integrity. In brief, semen samples were diluted to a final concentration of 4 × 106 sperm/mL in phosphate buffered saline 1 × (PBS). Next, sperm were stained with SYBR-14 (final concentration: 32 nM) and PI (final concentration: 7.5 µM) at 38 °C in the dark for 15 min. Next, stained samples were analysed using a CytoFLEX cytometer (Beckman Coulter; Fullerton, CA, USA). SYBR-14 fluorescence was detected by the fluorescein isothiocyanate (FITC) channel (525/40), and that of PI through the PC5.5 channel (690/50). Both fluorescent probes were excited with a 488-nm laser, and no spill compensation was applied. For each sample, three technical replicates containing at least 10,000 sperm were evaluated. Throughout the entire experiment, flow rate, laser voltage and sperm concentration remained unchanged. The percentages of viable (SYBR-14+/PI−) and non-viable sperm (SYBR-14−/PI+ and SYBR-14+/PI+) were recorded and used for the subsequent statistical analysis.

Oocyte maturation, in vitro fertilisation, and embryo culture

First, ovaries from pre-pubertal gilts were collected at a local abattoir (Frigorífics Costa Brava; Riudellots de la Selva, Girona) and transported to the laboratory in 0.9% NaCl supplemented with 70 µg/mL kanamycin at 38 °C. Cumulus-oocyte complexes (COC) were retrieved from follicles and only those with complete and compact cumulus mass were selected and washed in Dulbecco’s PBS (Gibco, ThermoFisher) supplemented with 4 mg/mL of BSA.

For oocyte maturation, TCM-199 (Gibco) supplemented with 0.57 mM cysteine, 0.1% (w:v) polyvinyl alcohol, 10 ng/mL human epidermal growth factor, 75 µg/mL of penicillin-G potassium, and 50 µg/mL of streptomycin sulphate was used. Groups of 40–50 COCs were transferred to a four-well multi-dish (Nunc, ThermoFisher; Waltham, MS, USA) containing 500 µL of pre-equilibrated maturation media supplemented with 10 IU/mL equine chorionic gonadotropin (eCG; Folligon; Intervet International B.V.; Boxmeer, The Netherlands) and 10 IU/mL human chorionic gonadotropin (hCG; Veterin Corion; Divasa Farmavic S.A.; Gurb, Barcelona, Spain). After 20–22 h, oocytes were transferred to 500 µL of pre-equilibrated maturation media without hormones.

For the fertilisation protocol, denuded mature oocytes were placed in 50-µL drops of pre-equilibrated IVF medium containing 1 mM caffeine. The basic medium used for IVF was a modified Tris-buffered medium [69]. After adjusting semen samples to a final concentration of 1000 sperm per oocyte in IVF medium, oocytes and sperm were co-incubated for 5 h.

The presumptive zygotes were washed and transferred (40 zygotes/well) into a four-well multi-dish containing 500 μL of NCSU23 medium [70] supplemented with 0.4% BSA, 0.3 mM pyruvate and 4.5 mM lactate. After 2 days, cleaved embryos were counted to calculate the fertilisation rate; embryos were changed to NCSU23 medium supplemented with 0.4% BSA and 5.5 mM glucose, and cultured for 5 days. Embryos were classified following Balaban and Gardner [71] criteria and the percentages of morulae, early blastocysts/blastocyst, hatching/hatched blastocysts and total embryos (sum of morulae, early blastocysts/blastocyst and hatching/hatched blastocysts) were calculated on day 6 post-fertilisation. Moreover, two different ratios were determined: (i) the developmental potential of morulae on day 6, calculated as the percentage of early blastocysts/blastocysts plus hatched/hatching blastocysts divided by the percentage of morulae; and (ii) the developmental competency of fertilised embryos, calculated as the ratio between the number of embryos on day 2 and on day 6.

All procedures (oocyte maturation, IVF, and embryo culture) were carried out at 38.5 °C under a humidified atmosphere of 5% CO2 in air. Each of the 24 ejaculates was used as a biological replicate, obtaining at least 40 zygotes per semen sample.

Neutral and alkaline Comet assays

The neutral Comet assay was used to quantify the amount of DSB, and the alkaline Comet assay was conducted to determine the whole amount of DNA breaks, including both SSB and DSB. In order to infer the amount of SSB, the neutral Comet OTM was subtracted from the alkaline Comet outcome. The protocols used for both Comet assays were previously adapted to pig sperm by Ribas-Maynou et al. [42].

Sperm fixation and lysis

First, samples were diluted to 5 × 105 sperm/mL, and mixed with low melting point agarose (37 °C) at a final concentration of 0.66%. Quickly, two drops of the mixture (6.5 µL each) were poured onto two agarose pre-treated slides, one designated for neutral Comet and the other for alkaline Comet, and covered with an 8-mm round coverslip. Thereafter, agarose was allowed to jellify at 4 °C for 5 min and coverslips were gently removed. Both slides were incubated in three lysis solutions: (1) 0.8 M Tris–HCl, 0.8 M DTT and 1% SDS for 30 min; (2) 0.8 M Tris–HCl, 0.8 M DTT and 1% SDS 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 for 180 min.

Electrophoresis

Electrophoresis was differently conducted depending on the Comet variant. For neutral Comet, slides were electrophoresed in TBE buffer (0.445 M Tris–HCl, 0.445 M boric acid and 0.01 M EDTA; pH = 8) at 1 V/cm for 4 min, and then washed in 0.9% NaCl for 2 min. For alkaline Comet, slides were denatured in cold (4 °C) alkaline solution (0.03 M NaOH, 1 M NaCl) for 5 min, and electrophoresed in an alkaline buffer (0.03 M NaOH, pH = 13) at 1 V/cm for 4 min.

Neutralization, dehydration, and staining

Both electrophoresed slides were incubated in neutralization solution (0.4 M Tris–HCl, pH = 7.5) for 5 min, dehydrated in ethanol series (70%, 90% and 100%) for 2 min each, and allowed to dry in horizontal position. Staining was conducted using 5 µL of 1 × Safeview DNA stain (NBS biological, Huntingdon, UK), and covered with a 20 × 20 coverslip.

Imaging and analysis

An epifluorescence microscope (Zeiss Imager Z1, Carl Zeiss AG, Oberkochen, Germany) was used to observe Comets. Captures of at least 100 sperm cells per sample were conducted at 100 × magnification and resolution of 1388 × 1040 pixels, through Axiovision 4.6 software (Carl Zeiss AG, Oberkochen, Germany). Exposure time was adjusted in each capture to avoid overexposure of staining.

The quantitative analysis of the fluorescence intensity of Comet heads and tails was conducted through the open-access CometScore v2.0 software (Rexhoover, www.rexhoover.com). After automatic analysis, a manual review of each analysed Comet was conducted to remove captures not corresponding to cells, overlapping comets, or those that showed impurities that affected head or tail signal. Also, this review served to correct any inaccurate interpretation of Comet heads by the software. At this point, if the final Comet number was less than 100, more captures were performed until this figure was reached.

For the quantification of the amount of DNA breaks, OTM calculated as (Tail mean intensity − Head mean intensity) × Tail DNA/100, was chosen as a reference parameter [72].

A representative composition of images for the alkaline and neutral Comet assays, including the analysis of DNA damage conducted by the CometScore v2.0 software is shown in Fig. 3.

Fig. 3

Representative images for alkaline and neutral Comet assay, and their respective analysis using the Cometscore v2 software. Purple lines indicate the intensity of the comet core, blue lines indicate the intensity of the comet tail, and yellow lines indicate the superposition between core and tail. OTM olive tail moment

Statistical analysis

Data were analysed through GraphPad Prism 8.0 Software (GraphPad, San Diego, USA), and Statistics Package for Social Sciences (SPSS) ver. 25.0 (IBM Corp.; Armonk, NY, USA). For all tests, the level of significance was set as P ≤ 0.05. First, normal distribution and homogeneity of variances were determined with Shapiro–Wilk and Levene tests, respectively. Thereafter, Spearman correlations between sperm DNA damage and sperm quality and IVF outcomes were run, and associations were assessed through multiple linear regression tests.

Subsequently, to determine the discriminant relevance of each DNA damage and sperm quality parameter for fertilisation on day 2 and total blastocyst percentage on day 6, these two IVF outcomes were divided into two groups below and above the median. A ROC analysis was used to determine the AUC of each variable, and the discriminant relevance was graded as: 0.0–0.5 no discriminant value, 0.5–0.6 failed discriminant value, 0.6–0.7 poor discriminant value, 0.7–0.8 fair discriminant value, 0.8–0.9 good discriminant value, and 0.9–1 excellent discriminant value. For all DNA damage and sperm quality parameters, sensitivity, specificity, and odds ratio were recorded.

Finally, in order to address if the addition of sperm DNA damage to the conventional semen analysis could have a higher discriminant value, a Principal Component Analysis (PCA) was generated including neutral OTM, alkaline OTM—neutral OTM, progressive motility, total motility, kinematic parameters, morphology and viability. These parameters were sorted into one PCA component, and the obtained data matrix was rotated through the Varimax procedure with Kaiser normalisation. Variables with a loading factor higher than 0.6 and lower than 0.3 in the rotated matrix were selected. The resulting coefficients were used to calculate regression scores that were assigned to each spermatozoon, and the variable was used to calculate a ROC curve for the prediction of fertilisation and blastocyst rates.