Unless stated otherwise, the reagents used in the present study were purchased from Sigma (Merck, Darmstadt, Germany).

All semen samples were acquired from an artificial insemination centre (Gepork S.L.; Masies de Roda, Spain). Management of animals by this centre is in accordance with the ISO certification (ISO-9001:2008), the EU Directive 2010/63/EU for animal experimentation, the Catalan Animal Welfare Law, and the current regulation on Health and Biosafety issued by the Department of Agriculture, Livestock, Food and Fisheries, Regional Government of Catalonia, Spain. No Ethics Committee permission was required to conduct this work because ejaculates were purchased from the artificial insemination centre and, therefore, no animal was manipulated for the purpose of the present study.

Semen samples were collected from healthy, sexually mature Pietrain boars (1–3 years old) using the gloved-hand method. Next, ejaculates 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 17ºC for 24 h. Before being included, basic sperm quality parameters including sperm viability and motility were assessed to check semen samples met the standard minimum requirements (> 80% of viable sperm and > 70% of motile sperm).

Moreover, ovaries were retrieved from pre-pubertal gilts sacrificed for food purposes at a local abattoir (Frigorífics Costa Brava; Riudellots de la Selva, Girona, Spain).

The present study was divided into three experiments in order: (i) to explore the relationship between mitochondrial activity, ROS production and IVF outcomes; (ii) to validate the effect of cyanide on sperm function, mitochondria and intracellular ROS levels; and (iii) to evaluate whether mitochondrial activity influences sperm DNA integrity. For the first experiment, thirty-three ejaculates (each from a separate boar; i.e., 33 boars) were split into two aliquots. The first one was intended to assess sperm function, which included MMP, intracellular levels of total ROS and O2●−, and oxygen consumption rate (OCR). The second aliquot was used to perform IVF. For the second experiment, a total of twelve pools (each one composed of three ejaculates from three separate boars; i.e. three boars per pool) were split into three aliquots containing either 5 mM or 10 mM of cyanide, or H2O as a vehicle control. After 1 h of incubation, sperm function (sperm viability, sperm motility, MMP, intracellular levels of total ROS and O2●−, and OCR) were assessed. The third experiment was also carried out using six ejaculate pools (each one composed of three ejaculates from separate boars; i.e. three boars per pool). Again, each pool was divided into three aliquots containing 0, 5, or 10 mM of cyanide. Sperm function (sperm viability, sperm motility, MMP and intracellular ROS levels) were assessed at three time points (0 h, 24 h, and 72 h). In addition, 30 µL of each treatment per time point were snap-frozen at -80ºC in order to determine sperm DNA integrity by the Comet assay. All incubations with cyanide were performed at 17ºC in order to evaluate basal metabolic activity in sperm.

After pre-warming semen samples at 38ºC for 15 min, 3 µL was placed into a Leja20 counting chamber (Leja Products BV; Nieuw-Vennep, The Netherlands). Each sample was evaluated individually using a Olympus BX41 microscope (Olympus; Tokyo, Japan) with a negative phase contrast objective (Olympus 10 × 0.30 PLAN objective, Olympus), through a computer-assisted sperm analysis system (Integrated Sperm Analysis System, ISAS V1.0; Proiser S.L.; Valencia, Spain). At least 1,000 sperm were examined per sample. The percentage of motile sperm, defined as those with an average path velocity ≥ 10 μm/s, was recorded.

Sperm viability, MMP, and intracellular levels of total ROS and O2●− were determined using a CytoFLEX cytometer (Beckman Coulter; Brea, CA, USA). Subcellular debris and cell aggregates were excluded using forward and side scatter, and gain and flow rate remained constant throughout the experiment. A minimum of 10,000 sperm events per sample were examined. All fluorochromes were purchased from ThermoFisher Scientific (MA, USA).

Samples were adjusted to a final concentration of 4 × 106 sperm/mL in 1× PBS before fluorochrome staining. Sperm viability was assessed following the protocol of Garner and Johnson [19], which uses SYBR-14 to stain sperm nuclei, and propidium iodide (PI) to label sperm with compromised plasma membrane integrity. Briefly, samples were incubated with SYBR-14 (final concentration: 32 nM) and PI (final concentration: 7.5 µM) at 38ºC in the dark for 15 min. Fluorescence of SYBR-14 was detected through the fluorescein isothiocyanate channel (FITC; 525/40), and that of PI using the PC5.5 channel (690/50). Both fluorochromes were excited with the 488-nm laser and no compensation was applied. The percentage of viable sperm corresponded to the SYBR-14+/PI− population, after subtracting the percentage of debris particles (SYBR-14−/PI−) in the analysis.

The MMP was evaluated following the protocol set by Ortega-Ferrusola et al. [20], which uses 5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethyl-benzimidazolylcarbocyanine iodide (JC-1). Briefly, sperm were incubated with JC-1 (final concentration: 750 nM) at 38ºC in the dark for 30 min. In sperm with high MMP, JC-1 aggregates and emits orange fluorescence, which was here collected through the PE (585/42) channel. On the contrary, JC-1 is found in its monomeric form in sperm with low MMP and emits green fluorescence, which was collected through the FITC channel (525/40). Both JC-1 aggregates and monomers were excited using the 488-nm laser. The MMP of each sample was evaluated by the ratio between the mean fluorescence intensities of JC-1 aggregates and JC-1 monomers.

Total ROS levels were determined using the CellROX™ Deep Red Reagent. CellROX™ Deep Red is a cell-permeant dye that emits red fluorescence when oxidised by ROS. Sperm were incubated with CellROX™ Deep Red (final concentration: 2 µM) at 38ºC in the dark for 30 min. Then, PI (final concentration: 7.5 µM) was added and further incubated for 5 min at the same conditions. CellROX™ Deep Red was excited with the 638-nm laser and detected through the APC channel (660/20), whereas PI was excited with the 488-nm laser and detected through the PC5.5 channel (690/50). The CellROX™ Deep Red fluorescence intensity of viable sperm with high levels of intracellular total ROS (CellROX™ Deep Red+/PI−) was recorded to assess total ROS levels.

Intracellular O2●− levels were assessed following the protocol of Guthrie and Welch [21], which uses hydroethidine (HE), a molecule that is oxidized into ethidium (E+) in the presence of O2●−. Briefly, samples were incubated with HE (final concentration: 5 µM) and YO-PRO-1 (final concentration: 31.25 nM) at 38ºC in the dark for 20 min. Samples were excited with the 488-nm laser, and fluorescence emitted by E+ and YO-PRO-1 was collected through PE (585/42) and FITC (525/40) channels, respectively. The fluorescence intensity of E+ in viable sperm with high levels of intracellular O2●− (E+/ YO-PRO-1−) was recorded to assess O2●− levels in semen samples.

The SensorDish® Reader system (PreSens Gmbh; Regensburg, Germany) was used to evaluate OCR in sperm samples. One mL from each sample was transferred onto an Oxodish® OD24 plate, and the dish was then sealed with Parafilm®. Negative controls were performed by transferring the same volume of the cell-free medium in order to measure the background concentration of O2. Samples were then incubated at 38ºC for 1 h, and the O2 concentration in each well was measured every 30 s. The background of O2 concentration in cell-free wells was subtracted from every sample. Data were normalised against the percentage of viable cells, and the OCR was subsequently calculated as µM O2/h×106 viable sperm.

Ovaries were transported to the laboratory in 0.9% NaCl supplemented with 70 µg/mL kanamycin at 38ºC. Cumulus-oocyte complexes (COC) were extracted from follicles and only those exhibiting a complete, compact cumulus mass were included in the experiment. Selection of COCs was carried out using Dulbecco’s PBS (Gibco, ThermoFisher) supplemented with 4 mg/mL BSA.

Oocytes were in vitro matured using 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 penicillin-G potassium, and 50 µg/mL streptomycin sulphate. First, groups of 50–60 COCs were matured in four-well multi-dishes (Nunc, ThermoFisher; Waltham, MS, USA) containing 500 µL of pre-equilibrated maturation medium 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 22 h, oocytes were moved to 500 µL fresh pre-equilibrated maturation medium without hormones.

After mechanically removing cumulus cells, the matured oocytes were transferred into 50-µL drops of pre-equilibrated IVF medium (Tris-buffered medium [22]) containing 1 mM caffeine in groups of 20–30 oocytes. In parallel, semen samples were adjusted to 1,000 sperm per oocyte in IVF medium. Next, gametes were co-incubated at 38.5ºC and 5% CO2 for 5 h. A total of 100 oocytes per semen sample were inseminated. The putative zygotes were then moved onto 500 µL NCSU23 medium [23] supplemented with 0.4% BSA, 0.3 mM pyruvate, and 4.5 mM lactate for in vitro embryo culture. After two days, fertilisation rate was assessed by calculating the percentage of cleaved embryos, which were then transferred into NCSU23 medium supplemented with 0.4% BSA ad 5.5 mM glucose. Six days after fertilisation, the resulting embryos were classified following Balaban & Gardner [24] criteria. Specifically, percentages of morulae, early blastocysts/blastocysts, hatching/hatched blastocysts and total embryos (sum of morulae, early blastocysts/blastocysts and hatching/hatched blastocysts) were evaluated. In addition, two different ratios were calculated: (i) the developmental potential at day 6, which resulted from dividing the percentage of morulae, early blastocysts/blastocysts and hatched/hatching blastocysts by the percentage of 2–8 cell embryos; and (ii) the developmental competency of fertilised embryos, which was the number of embryos at day 6 divided by the number of embryos at day 2.

For the evaluation of DNA integrity (single- and double-strand breaks), the Comet assay protocol set by Ribas-Maynou et al. [25] was followed. First, samples were diluted to 5 × 105 sperm/mL, and mixed with 0.66% low melting point agarose (37ºC). Then, two 6.5-µL drops of the mixture were poured onto agarose pre-treated slides. Next, drops were covered with an 8-mm round coverslip and agarose was jellified at 4ºC for 5 min. After gently removing the coverslips, 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. Then, a denaturalisation step was performed in cold (4ºC) alkaline solution (0.03 M NaOH, 1 M NaCl) for 5 min before slides were electrophoresed in an alkaline buffer (0.03 M NaOH, pH = 13) at 1 V/cm for 4 min. Finally, the electrophoresed slides were incubated in neutralisation solution (0.4 M Tris-HCl, pH = 7.5) for 5 min, and dehydrated in an ethanol series (70%, 90% and 100%) for 2 min each. Sperm DNA was stained with 5 µL of 1× Safeview DNA stain (NBS biological, Huntingdon, UK), and covered with a coverslip.

Comets were observed and captured under an epifluorescence microscope (Zeiss Imager Z1; Carl Zeiss AG, Oberkochen, Germany). At least 100 sperm cells per sample were captured at 100× magnification, with a resolution of 1388 × 1040 pixels and using Axiovision 4.6 software (Carl Zeiss AG, Oberkochen, Germany). Capture time was constant for all samples.

Fluorescence intensities of Comet heads and tails were analysed through open-access CometScore v2.0 software (Rexhoover, www.rexhoover.com). Captures not corresponding to cells, overlapping comets, or those that showed impurities in the head or tail signal were manually removed. For the quantification of the amount of sperm DNA breaks, olive tail moment (OTM), calculated as (Tail mean intensity – Head mean intensity) × Tail DNA / 100, was chosen as a reference parameter.

Statistical analyses were conducted with a statistical package (IBM SPSS for Windows Ver. 27.0; IBM Corp., Armonk, NY, USA). Plots were elaborated using GraphPad Prism 8.0 Software (GraphPad, San Diego, USA). Data was first tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene test). The level of significance was set at P ≤ 0.05.

For the first experiment, Pearson correlations between mitochondria-related parameters, total ROS and O2●− levels, and IVF outcomes were calculated. In addition, sperm samples were classified by their in vitro fertility potential. A principal component analysis was run using the percentage of cleaved embryos at day 2, morulae, early blastocysts/blastocysts, hatching/hatched blastocysts, and total embryos (sum of morulae, early blastocysts/blastocysts and hatching/hatched blastocysts) at day 6. Varimax with Kaiser normalization was utilised as the rotation method. The analysis yielded two components explaining 77.1% of the total variance. Thereafter, a two-step cluster analysis was performed using regression factors of these two components to classify sperm samples on the basis of their in vitro fertility potential (measure of distance: log-likelihood; clustering criterion: Schwarz’s Bayesian Criterion, BIC), with the number of groups being determined automatically. Then, mitochondria-related parameters were compared between the two groups through a t-test for independent samples. In addition to this, sperm samples in the first experiment were also clustered considering OCR, total ROS and developmental potential through a two-step cluster analysis (measure of distance: log-likelihood; clustering criterion: BIC) to compare the OCR, ROS and embryo development potential between sperm samples.

For experiments 2 and 3, data of each biological replicate were first normalised to their control within each time point. Then, one-way ANOVA for each timepoint was performed, followed by Dunnett’s post-hoc test. For Comet analysis, OTM values of each spermatozoon were used to run a two-step cluster analysis (measure of distance: log-likelihood; clustering criterion; BIC), with the number of groups being determined automatically. This resulted in two subpopulations of sperm (high and low sperm DNA fragmentation), the cut-off value being 27.80. Following this, the proportions of each sperm subpopulation in every sample were calculated. Data were standardised by calculating the ratio between the proportion of each sperm subpopulation at a given time point (0 h, 24 h, 72 h) and treatment (control, 5 mM, 10 mM cyanide) with respect to the control at that time point. The resulting ratios were then used to run a linear mixed model (repeated measures) where the inter-subject factor was cyanide concentration, and the intra-subject factor was the time of incubation. Pair-wise comparisons were made with Bonferroni’s test.