biology Article Red-Light Irradiation of Horse Spermatozoa Increases Mitochondrial Activity and Motility through Changes in the Motile Sperm Subpopulation Structure Jaime Catalán 1,2,3 , Marion Papas 1 , Sabrina Gacem 1 , Yentel Mateo-Otero 2,3 , Joan E. Rodríguez-Gil 1 , Jordi Miró 1, *,† and Marc Yeste 2,3, *,† 1 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, ES-08193 Bellaterra (Cerdanyola del Vallès), Spain; dr.jcatalan@gmail.com (J.C.); papas.marion@gmail.com (M.P.); swp.sabrina.gacem@gmail.com (S.G.); juanenrique.rodriguez@uab.cat (J.E.R.-G.) 2 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain; yentel.mateo@udg.edu 3 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain * Correspondence: jordi.miro@uab.cat (J.M.); marc.yeste@udg.edu (M.Y.); Tel.: +34-93-5814293 (J.M.); +34-972-419514 (M.Y.) † These authors contributed equally to this work.  Received: 19 August 2020; Accepted: 27 August 2020; Published: 29 August 2020  Abstract: Previous studies in other mammalian species have shown that stimulation of semen with red-light increases sperm motility, mitochondrial activity, and fertilizing capacity. This study sought to determine whether red-light stimulation using a light emitting diode (LED) at 620–630 nm affects sperm motility and structure of motile subpopulations, sperm viability, mitochondrial activity, intracellular ATP levels, rate of O2 consumption and DNA integrity of horse spermatozoa. For this purpose, nine ejaculates were collected from nine different adult stallions. Upon collection, semen was diluted in Kenney extender, analyzed, its concentration was adjusted, and finally it was stimulated with red-light. In all cases, semen was packaged in 0.5-mL transparent straws, which were randomly divided into controls and 19 light-stimulation treatments; 6 consisted of a single exposure to red-light, and the other 13 involved irradiation with intervals of irradiation and darkness (light-dark-light). After irradiation, sperm motility was assessed using a Computerized Semen Analysis System (CASA). Flow cytometry was used to evaluate sperm viability, mitochondrial membrane potential and DNA fragmentation. Intracellular levels of ATP and O2 consumption rate were also determined. Specific red-light patterns were found to modify kinetics parameters (patterns: 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5 min), the structure of motile sperm subpopulations (patterns: 2, 2-2-2, 3-3-3, and 4-1-4 min), mitochondrial membrane potential (patterns: 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 min), intracellular ATP levels and the rate of O2 consumption (pattern: 4 min), without affecting sperm viability or DNA integrity. Since the increase in some kinematic parameters was concomitant with that of mitochondrial activity, intracellular ATP levels and O2 consumption rate, we suggest that the positive effect of light-irradiation on sperm motility is related to its impact upon mitochondrial activity. In conclusion, this study shows that red LED light stimulates motility and mitochondrial activity of horse sperm. Additional research is needed to address the impact of red-light irradiation on fertilizing ability and the mechanisms through which light exerts its effects. Keywords: photobiology; red-light; horse; semen; motility; mitochondrial activity Biology 2020, 9, 254; doi:10.3390/biology9090254 www.mdpi.com/journal/biology Biology 2020, 9, 254 2 of 17 1. Introduction Artificial insemination (AI) is an effective technique to improve the use of stallions in breeding programs [1], and it is used worldwide not only for the reproduction of horses, but also for that of farm animals, companion animals, and wild species [2]. Its advantages are so vast that it is considered as a biotechnology with great impact on equine reproduction; in effect, when used correctly, a stallion can leave hundreds of descendants throughout its reproductive life [3]. While pregnancy rates following AI with any semen source have increased enough to provide commercially acceptable margins of reliability in the horse [4], equine veterinarians often find owners seeking methods that can improve the reproductive performance of their stallions. In this context, different strategies have been developed to improve and handle sperm, thus maximizing their survival and fertilizing ability [5,6]. Studies conducted with pig semen have reported that reproductive performance can be increased through light-stimulation of semen prior to AI [7,8]. In effect, previous works using different light sources, such as lasers and light-emitting diodes (LED), have suggested that light-stimulation has a positive effect on the motility and ability of sperm to fertilize the oocyte in pigs [7–9], mice [10], and sheep [11]. Remarkably, no study has reported any detrimental effect of visible light on DNA integrity [12–14]. Light-stimulation of semen consists of irradiating sperm samples at a fixed (laser) or variable (LED) wavelength for a short period of time [15]. Data from previous studies indicate that the effects of light-stimulation depend on the state of the sample, the irradiation of the light beam used [13], the time or pattern of exposure [7], and the species [11]. While the mechanisms through which light exerts its effects on sperm are largely unknown, mounting evidence gives endogenous cellular photosensitizers, especially those present in mitochondria, a crucial role. Consequently, when these molecules are stimulated with light, both the production of ATP and the entry of Ca2+ into the sperm cell raise [14]. This raise in energy supply underlies the subsequent increase of sperm motility [14,16–18]. In addition to this, opsins, which are present in sperm, and the potential influence of light on the conformation of other proteins like those belonging to the Transient Receptor Potential (TRP) family may also be involved in the sperm response to light [14,19]. The evidence accumulated so far in studies conducted with both low-level laser therapy devices and LED agree with these hypotheses, since they indicate that light irradiation accelerates mitochondrial respiration and ATP production in sperm [14,20,21]. Furthermore, it has become apparent that the sperm response to light-stimulation is biphasic, as low doses produce stimulating effects, moderate doses have no effect, and high doses exert cytotoxic effects [14]. As far as light devices are concerned, the positive results of low-level lasers and LED suggest that any method can be used safely [13]; however, practically speaking, not only are LED-based systems much cheaper and easier to maintain than lasers, but they also show high photonic efficiency [7]. Among all visible light spectra tested, red-light has been shown to be the one that improves sperm motility the most [15]. In addition to this, it has been reported that red-light stimulation (620–630 nm) by LED increases the fertilizing ability of pig sperm [7–9], without affecting their viability [7,9]. In spite of all the aforementioned, the effects of red-light stimulation on freshly ejaculated horse sperm are yet to be investigated. Therefore, the present work aimed at evaluating, for the first time, whether stimulation with red LED light (620–630 nm) affects the viability, motility, mitochondrial activity, intracellular levels of ATP, O2 consumption rate, DNA integrity, and motile sperm subpopulations of fresh horse spermatozoa. 2. Materials and Methods 2.1. Suppliers All reagents used were of analytical grade and were purchased from Boehringer-Mannheim (Mannheim, Germany), Merck (Darmstadt, Germany), and Sigma-Aldrich (Saint Louis, MO, USA). As far as fluorochromes are concerned, and unless otherwise stated, all were purchased from Molecular Biology 2020, 9, 254 3 of 17 Probes (Thermo Fisher Scientific; Waltham, MA, USA) and were previously resuspended with dimethyl sulfoxide (Sigma-Aldrich). Plastic materials were provided by Nunc (Roskilde, Denmark). 2.2. Animals and Ejaculates This study included nine ejaculates from nine different adult stallions, with proven fertility, housed at the Equine Reproduction Service, Autonomous University of Barcelona (Bellaterra, Cerdanyola del Vallès, Spain). This is an EU-approved semen collection center (Authorization code: ES09RS01E) that operates under strict protocols of animal welfare and health control. All animals were semen donors and were collected under the health conditions set by the EU (free of equine arteritis, infectious anemia, and contagious metritis). Since this service already runs under the approval of the Regional Government of Catalonia (Spain) and because no manipulation to animals other than semen collection was carried out, the ethics committee of our institution indicated that no further ethical approval was required. Ejaculates were collected through a Hannover artificial vagina (Minitüb GmbH, Tiefenbach, Germany) and an in-line nylon mesh filter was used to separate the gel fraction from the semen. Upon collection, gel-free semen was diluted 1:5 (v:v) with Kenney extender [22], previously prewarmed to 38 ◦ C. Sperm concentration was determined in all samples through a hemocytometer (Neubauer Chamber, Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). 2.3. Experimental Design Prior to light-stimulation, sperm concentration of samples was adjusted to 25 × 106 spermatozoa/mL. Following this, samples were packed into 0.5-mL transparent straws (Minitüb GmbH; Tiefenbach, Germany). After semen packaging, straws were placed in a programmable photo-activation system (Maxicow, IUL, S.A.; Barcelona, Spain). In this system, each straw is in contact with a triple LED configuration system that emits red-light (wavelength window: 620–630 nm). The system is equipped with a supporting software (IUL, S.A.) that allows the regulation of intensity and exposure time. In all cases, intensity was set at 100%. A total of 19 irradiation protocols (red light) were evaluated. This aimed at determining which light exposure protocols had greater effects, which ones had no effect and which ones caused deleterious effects on sperm motility, viability, mitochondrial activity, intracellular levels of ATP, O2 consumption rate, or DNA integrity. Six of these protocols consisted of single periods of light emission (1, 2, 3, 4, 5, and 10 min), whereas the other 13 treatments consisted of light-dark-light intervals (1-1-1, 2-1-2, 2-2-2, 3-1-3, 3-3-3, 4-1-4, 4-4-4, 5-1-5, 5-5-5, 10-5-10, 10-10-10, 15-5-15, and 15-15-15 min). Control samples were not irradiated. In order to determine the impact of each irradiation pattern, sperm viability, motility and motile subpopulations, mitochondrial activity, intracellular levels of ATP, O2 consumption rate, and DNA fragmentation were assessed. Prior to evaluation, irradiated and control samples, previously transferred into 1.5-mL tubes, were incubated in a water bath at 38 ◦ C for 5 min. 2.4. Analysis of Sperm Motility Sperm motility was evaluated using a computer-assisted sperm-analysis (CASA) system (Integrated Sperm Analysis System V1.0; Proiser S.L.; Valencia, Spain) following incubation at 38 ◦ C for 5 min. Five µL of each sperm sample was placed onto a Makler chamber (Sefi Medical Instruments; Haifa, Israel) previously warmed at 38 ◦ C. Samples were analyzed with a 10 × negative phase-contrast objective and an Olympus BX41 microscope (Olympus), and at least 1000 sperm cells per analysis were counted. In each evaluation, percentages of total (%TMOT) and progressively motile spermatozoa (%PMOT) were recorded together with the following kinetic parameters: curvilinear velocity (VCL, µm/s), which is the mean path velocity of the sperm head along its actual trajectory; straight-line velocity (VSL, µm/s), which is the mean path velocity of the sperm head along a straight line from its first to its last position; average path velocity (VAP, µm/s), which is the mean velocity of the sperm head along its average trajectory; percentage of linearity (LIN, %), which is the quotient between VSL and VCL multiplied by 100; percentage of straightness (STR, %), which is the quotient between VSL and VAP multiplied by 100; percentage of oscillation (WOB, %), which is the quotient between VAP and VCL multiplied by Biology 2020, 9, 254 4 of 17 100; mean amplitude of lateral head displacement (ALH, µm), which is the mean value of the extreme side-to-side movement of the sperm head in each beat cycle; and frequency of head displacement (BCF, Hz), which is the frequency at which the actual sperm trajectory crosses the average path trajectory (Hz). In addition, individual kinetic parameters for each spermatozoon were also recorded and used to investigate the effects of light-stimulation upon sperm motile subpopulations. Settings of the CASA system were those recommended by the manufacturer, i.e., frames/s: 25 images captured per second; particle area >4 and <75 µm2 ; connectivity: 6; minimum number of images to calculate the ALH: 10. Cut-off values were VAP ≥ 10 µm/s for a sperm cell to be considered as motile, and STR ≥ 75% for being considered as progressively motile. 2.5. Flow Cytometry Analyses 2.5.1. General Information about Flow Cytometry Analyses Information about flow cytometry analyses is given according to the recommendations of the International Society for Advancement of Cytometry [23]. Prior to staining, sperm concentration (except for SCSA test) was adjusted to 1 × 106 total spermatozoa per mL in a final volume of 0.5 mL with HEPES buffered saline solution (10 mM HEPES, 150 mM NaCl, 10% BSA; pH = 7.4). In addition, a correction procedure that consisted of differentiating into DNA-containing and non-DNA-containing particles was made for JC1 test, since the presence of alien particles could overestimate the percentages of intact spermatozoa [24,25]. All flow cytometry assessments were conducted using a Cell Laboratory QuantaSC cytometer (Beckman Coulter; Fullerton, CA, USA), and samples were excited with an argon ion laser (488 nm) set at a power of 22 mW. For each event, the cytometer provided the electronic volume (EV, equivalent to forward scatter, FS, in other equipment) and the side scatter (SS). Calibration of this device was made periodically through 10-µm Flow-Check fluorospheres (beads; Beckman Coulter); the bead size was positioned at channel 200 on the volume scale. A total of three optical filters (FL1, FL2 and FL3), with the following particular characteristics, were used: FL1 (green fluorescence): Dichroic/Splitter, DRLP: 550 nm, BP filter: 525 nm; FL2 (orange fluorescence): DRLP: 600 nm, BP filter: 575 nm; and FL3 (red fluorescence): LP filter: 670/730 nm. Signals were logarithmically amplified and photomultiplier settings were adjusted to particular staining methods. FL1 was used to detect green fluorescence (SYBR14, JC1 monomers and SCSA), FL2 was used to detect orange fluorescence (JC1 aggregates, JC1agg ), and FL3 was used to detect red fluorescence (PI and SCSA). When required, and as stated below, compensation was used to minimize spill over between channels. Sheath flow rate was set at 4.17 µL/min in all analyses, and EV and SS were recorded in a log-linear mode (in EV vs. SS dot plots) for a minimum of 10,000 events per assessment. The analyzer threshold was adjusted on the EV channel to exclude subcellular debris and cell aggregates, and the sperm-specific events were positively gated on the basis of EV/SS distributions. Information on the events was collected in list-mode data files (*.LMD), and files were subsequently analyzed through the Cell Lab QuantaSC MPL Analysis Software (version 1.0; Beckman Coulter). Three replicates using independent tubes were evaluated, and the corresponding mean ± standard error of the mean (SEM) was subsequently calculated. 2.5.2. Plasma Membrane Integrity Sperm membrane integrity was assessed using the LIVE/DEAD Sperm Viability Kit (SYBR14/PI; Molecular Probes, ThermoFisher Scientific, Waltham, Massachusetts, MA, USA), according to the protocol described by Garner and Johnson [26] adapted to horse spermatozoa. Briefly, sperm samples were incubated at 37.5 ◦ C for 10 min with SYBR14 at a final concentration of 100 nM, and then with PI at a final concentration of 12 µM for 5 min at the same temperature. All incubations were performed in the dark. FL1 was used to measure the green fluorescence from SYBR14, and FL3 was used to detect the red fluorescence from PI. Three sperm populations were identified: (i) spermatozoa with an intact plasma Biology 2020, 9, 254 5 of 17 membrane, stained in green (SYBR14+ /PI− ); (ii) spermatozoa with a damaged plasma membrane, stained in red (SYBR14− /PI+ ); and (iii) spermatozoa with a damaged plasma membrane, stained in orange (SYBR14+ /PI+ ). Non-sperm particles (debris) were found in the SYBR14− /PI− quadrant, and were used to correct JC1-data. Single-stained samples were used for setting the EV-gain, FL1 and FL3 photomultiplier (PMT)-voltages, and for compensation of SYBR14 spill over into the PI channel (2.45%). 2.5.3. Evaluation of Mitochondrial Membrane Potential (∆Ψm, JC1) Mitochondrial membrane potential of horse spermatozoa was determined after modification of the protocol described in [27]. Sperm samples were incubated with JC1 (5,50 ,6,60 -tetrachloro- 1,10 ,3,30 tetraethyl-benzimidazolylcarbocyanine iodide) at a final concentration of 0.5 µM at 37.5 ◦ C for 30 min in the dark. Green fluorescence from JC1-monomers was collected through FL1, and orange fluorescence from JC1 aggregates (JC1agg ) was collected through FL2. Two populations were distinguished: (i) spermatozoa with low MMP, in which all mitochondria were stained in green (FL1+ /FL2− ); and (ii) spermatozoa with high MMP (JC1agg ), which contained spermatozoa with heterogeneous mitochondria stained both in green and orange in the same cell (FL1+ /FL2+ ) and spermatozoa that had all their mitochondria stained in orange (FL1− /FL2+ ). Spillover of FL1 into FL2-channel was compensated (68.5%). Following the protocol of Petrunkina et al. [24], the percentages of non-sperm, debris particles found in the SYBR14/PI test (SYBR14− /PI− ) were used to correct the percentages of non-stained events in the sperm population with low MMP; the percentages of the sperm population with high MMP were recalculated. 2.5.4. DNA Integrity (SCSA Test) DNA fragmentation of horse spermatozoa in control and irradiated samples was evaluated through SCSA test [28,29], as modified by Morrell et al. [30]. Briefly, sperm samples were diluted in a buffer solution (TNE; 0.15 M NaCL, 0.01 M Tris-HCl, 1 mM EDTA, pH = 7.4) to a final concentration of 2 × 106 spermatozoa/mL. Next, 200 µL of this solution containing 2 × 106 spermatozoa/mL were mixed with 400 µL of an acid-detergent solution (80 mM HCl, 150 mM NaCl, and 0.1% Triton X-100; pH = 1.2) in ice. After 30 s, 1.2 mL of an acridine orange (AO) solution (6 µg/mL in 37 mM citric acid, 126 mM Na2 HPO4 , 1.1 mM EDTA, 150 mM NaCl, pH = 6.0) was added, and samples were kept in ice for further 3 min. Immediately after this time, samples were evaluated and green and red fluorescence were collected through FL1 and FL3 filters, respectively. Percentages of DNA fragmentation (DNA fragmentation index, %DFI), which was the ratio between red (ssDNA) fluorescence and red (ssDNA) + green (dsDNA) fluorescence, mean fluorescence intensity of single stranded DNA (ssDNA, mean DFI) and percentages of high DNA stainability (HDS) were determined. 2.6. Determination of Intracellular ATP Levels Intracellular ATP levels were determined following the protocol set by Chida et al. [31]. Briefly, after irradiated, 1-mL semen aliquots were centrifuged at 17 ◦ C for 30 s and pellets were immediately plunged into liquid N2 . Frozen pellets were subsequently stored at −80 ◦ C for three weeks. Thereafter, pellets were resuspended in 300 µL ice-cold 10 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES) buffer containing 250 mM sucrose (pH was adjusted to 7.4). Resuspended pellets were sonicated (10 kHz, 20 pulses; Bandelin Sonopuls HD 2070; Bandelin Electronic GmbH and Co., Berlin, Germany), while being kept on ice to avoid specimen heating. Samples were subsequently centrifuged at 1000× g and 4 ◦ C for 10 min and supernatants were collected. Twenty µL was used to determine total protein content, and the remaining volume was mixed with 300 µL ice-cold 10% (v:v) trichloroacetic acid and kept at 4 ◦ C for 20 s. Samples were subsequently centrifuged at 1000× g and 4 ◦ C for 30 s, and supernatants were carefully separated from the pellet and again centrifuged at 1000× g and 4 ◦ C for 10 min. Resulting supernatants were mixed with two volumes of 1 M Tris-acetate buffer (pH = 7.75), and ATP content was determined in these final suspensions using the Invitrogen® ATP Determination Kit (ThermoFisher Bioscientific; Waltham, MA, USA; catalogue number: A22066) Biology 2020, 9, 254 6 of 17 following the manufacturer’s instructions. Determinations of ATP content were carried out through an Infinite F200 fluorimeter (TECAN® ), using 96-wells microplates for fluorescence-based assays (Invitrogen® ). To normalize data, total protein of samples was determined through the Bradford method [32] using a commercial kit (Bio-Rad laboratories; Hercules, CA, USA). 2.7. Determination of O2 Consumption Rate For the determination of the O2 consumption rate, the unirradiated control sample and five light stimulation patterns of short and long exposure to light were used, which had obtained significant differences in some kinetic parameters of sperm motility and mitochondrial activity, these parameters were; 2, 4, 3-3-3, 5-5-5, and 15-5-15 min. Determination of O2 consumption rate was performed using the SensorDish® Reader (SDR) system (PreSens Gmbh; Regensburg, Germany). One-mL semen aliquots, previously exposed to red-light, were transferred onto Oxodish® OD24 plates (24 wells/plate) specifically designed for this device. Plates were sealed with Parafilm® , introduced in the SDR system, and incubated at 37 ◦ C (controlled atmosphere) for 2 h. During that time, O2 concentration was recorded in each well at a rate of one reading per min. Data were exported to an Excel file and final O2 consumption rate was normalized against the total number of viable spermatozoa per sample, which was determined through flow cytometry (SYBR14+ /PI− spermatozoa) using another aliquot. In addition, O2 consumption rates in irradiated samples were standardized against the control (O2 consumption rate irradiation pattern /O2 consumption rate control × 100). 2.8. Statistical Analyses Statistical analyses were conducted using a statistical package (IBM® SPSS® 25.0 for Windows; IBM corp., Armonk, NY, USA). Data were first tested for normality and homogeneity of variances with √ Shapiro–Wilk and Levene tests, respectively. When required, data were transformed through arcsin x. The effects of red-light stimulation patterns on total and progressive motility, kinematic parameters, percentages of viable spermatozoa, percentage of spermatozoa with high MMP, intracellular ATP levels, O2 consumption rates, and DNA fragmentation were evaluated through one-way analysis of variance (ANOVA) followed by post-hoc Sidak’s test. Sperm motile subpopulations were set according to the procedure described in [33]. Briefly, individual kinematic parameters obtained for each spermatozoon (VSL, VCL, VAP, LIN, STR, WOB, ALH, and BCF) were used as independent variables in a principal component analysis (PCA). These kinematic parameters were sorted into PCA components and the obtained matrix was subsequently rotated using the Varimax procedure with the Kaiser normalization. For each spermatozoon, regression scores of the resulting PCA components were worked out. Based on the regression scores of each individual spermatozoon, a two-step cluster analysis based on the log-likelihood distance and the Schwarz’s Bayesian Criterion was run. Following identification of four motile sperm subpopulations, percentages of spermatozoa belonging to each subpopulation (SP1, SP2, SP3, or SP4) were calculated in each treatment and replicate. These percentages were subsequently used to evaluate the effects of red-light stimulation on sperm subpopulations through one-way ANOVA and Sidak’s post-hoc test. In all analyses, the level of s for statistical significance were set at p ≤ 0.05. Data are shown as mean ± standard error of the mean (SEM). 3. Results 3.1. Effects of Red-Light Irradiation on Sperm Viability Figure 1 shows representative histograms and dot-plots for the control (Figure 1a,b) and one light-stimulation pattern (15-15-15; Figure 1c,d). Figure 2a shows, as mean ± SEM, the percentages of viable spermatozoa following light-stimulation. No significant differences between control and irradiation patterns were observed (p > 0.05). Biology Biology 2020, 2020, 9, x 9, x 7 of 718of 18 viable viable Biology spermatozoa spermatozoa 2020, 9, 254 following following light-stimulation. light-stimulation. NoNo significant significant differences differences between between control control andand 7 of 17 irradiation irradiation patterns patterns were were observed observed (p >(p0.05). > 0.05). (a) (a) (b) (b) (c) (c) (d) (d) Figure Figure Figure 1. 1.1.SpermSperm Sperm viability viability viability (SYBR14/PI). (SYBR14/PI). (SYBR14/PI). Representative Representative Representative SYBR14 SYBR14 SYBR14 (FL1)(FL1) (FL1) histograms histograms histograms for forfor the thethe control control control(a)(a)(a)and and and 15-15-15 15-15-15 light-stimulation light-stimulation pattern pattern (c). (c). Representative Representative FL1/FL3 FL1/FL3 15-15-15 light-stimulation pattern (c). Representative FL1/FL3 dot-plots for dot-plots dot-plots for for the the control control (b) (b) and and 15- control (b) and 15-15-15 15- 15-15 15-15 light-stimulation light-stimulation pattern pattern (d). (d). Viable Viable spermatozoa spermatozoa + (SYBR14 (SYBR14 +/PI+-)/PI-) appear in the lower right light-stimulation pattern (d). Viable spermatozoa (SYBR14 /PI ) appear in the lower right quadrant. − appear in the lower right Noquadrant. quadrant. No No significant significant significant differences differences differences between thebetween between the the control andcontrol control andand treatments treatments treatments were were observed were observed observed (p >(p (p > 0.05). > 0.05). 0.05). (a) (a) (b) (b) Figure 2. (a) Percentages of spermatozoa with an intact plasma membrane (SYBR14+ /PI− ; viable spermatozoa) in the control and the different light-stimulation patterns. No significant differences Biology 2020, 9, x 8 of 18 BiologyFigure 2020, 9, 2. 254(a) Percentages of spermatozoa with an intact plasma membrane (SYBR14+/PI-; viable 8 of 17 spermatozoa) in the control and the different light-stimulation patterns. No significant differences between the control and light-stimulation patterns were observed (p > 0.05). (b) Percentages of between the control and light-stimulation patterns were observed (p > 0.05). (b) Percentages of spermatozoa with high mitochondrial membrane potential (JC1agg) in the control and different spermatozoa with high mitochondrial membrane potential (JC1agg ) in the control and different irradiation patterns. The superscript (*) means significant differences (p ≤ 0.05) between the control irradiation patterns. The superscript (*) means significant differences (p ≤ 0.05) between the control and and the different light-stimulation patterns. Data are shown as mean ± SEM of nine independent the different light-stimulation patterns. Data are shown as mean ± SEM of nine independent experiments. experiments. 3.2. Effects of Red-Light Irradiation on Mitochondrial Membrane Potential (∆Ψm) 3.2. Effects of Red-Light Irradiation on Mitochondrial Membrane Potential (ΔΨm) As As shown shown inin Figure 2b, percentages Figure 2b, percentages of of spermatozoa spermatozoawith withhigh highJC1 JC1agg agg were were significantly significantly (p (p < 0.05) < 0.05) higher, higher,compared compared withwith the control samples the control (74.4%±±4.8%), samples(74.4% 4.8%),inin the the following following light-stimulation light-stimulation patterns: patterns: 44(82.7% ± 2.5%), 3-3-3 (82.3% ± 1.9%), 4-4-4 (82.9% ± 4.1%), 5-1-5 (82.7% ± 2.7%), 5-5-5 (83.4% (82.7% ± 2.5%), 3-3-3 (82.3% ± 1.9%), 4-4-4 (82.9% ± 4.1%), 5-1-5 (82.7% ± 2.7%), 5-5-5 (83.4% ± 2.8%), ± 2.8%), 15-5-15 (85.1% ± 15-5-15 (85.1% ± 1.7%), and 15-15-15 1.7%), and (85.5%±±1.8%). 15-15-15 (85.5% 1.8%).Figure Figure3 3shows showsrepresentative representative histograms histograms andand dot-plots for the control (Figure 3a,b) and 15-15-15 pattern (Figure dot-plots for the control (Figure 3a,b) and 15-15-15 pattern (Figure 3c,d). 3c,d). (a) (b) (c) (d) Figure 3. Mitochondrial membrane potential (∆Ψm, JC1). Representative JC1agg (FL2 channel) Figure 3. Mitochondrial histograms for the controlmembrane potential (a) and 15-15-15 (ΔΨm, JC1). Representative light-stimulation JC1agg (FL2 channel) pattern (c). Representative FL1/FL2 histograms for the control (a) and 15-15-15 light-stimulation pattern (c). Representative dot-plots for the control (b) and 15-15-15 light-stimulation pattern (d). Spermatozoa FL1/FL2 withdot- high plots for the membrane mitochondrial control (b) potential and 15-15-15 (JC1agglight-stimulation patternhalf ) appear in the upper (d). of Spermatozoa with high of the Figure. Percentages mitochondrial spermatozoa membrane with potential (JC1 high mitochondrial agg) appear in the upper half of the Figure. Percentages of membrane potential (JC1agg ) were significantly (p < 0.05) higher in the 15-15-15 light-stimulation pattern than in the control. Biology 2020, 9, x 9 of 18 spermatozoa with high mitochondrial membrane potential (JC1agg) were significantly (p < 0.05) higher Biology 2020, 9, 254 9 of 17 in the 15-15-15 light-stimulation pattern than in the control. 3.3. 3.3. Effects Effects of of Red-Light Red-Light Irradiation Irradiation on on Sperm Sperm Motility Motility Total Total and and progressive progressivespermspermmotility motilitydiddidnot notdiffer differbetween between control control andandirradiation irradiationpatterns patterns(p >(p0.05; Figure 4). However, red-light stimulation affected several sperm kinematic > 0.05; Figure 4). However, red-light stimulation affected several sperm kinematic parameters parameters when compared when comparedto the to control in a fashion the control that depended in a fashion that dependedon theonspecific lightlight the specific regime applied regime to cells. applied In to cells. effect, VSL increased after the application of irradiation patterns 3-3-3 (65.7 μm/s ± In effect, VSL increased after the application of irradiation patterns 3-3-3 (65.7 µm/s ± 4.8 µm/s) and 4.8 μm/s) and 5- 5-5 (66.7 μm/s ± 2.0 μm/s) when compared to control samples (56.9 μm/s ± 2.5 5-5-5 (66.7 µm/s ± 2.0 µm/s) when compared to control samples (56.9 µm/s ± 2.5 µm/s; see Table 1). μm/s; see Table 1). Furthermore, Furthermore,VAP VAPsignificantly significantly(p(p< < 0.05) increased 0.05) increased with patterns with 4 (94.4 patterns μm/sµm/s 4 (94.4 ± 4.6±μm/s), 2-2-2 (96.0 4.6 µm/s), 2-2-2 μm/s ± 4.7 μm/s), 3-3-3 (96.4 μm/s ± 5.6 μm/s), and 4-4-4 (94.8 μm/s ± 3.9 μm/s) (96.0 µm/s ± 4.7 µm/s), 3-3-3 (96.4 µm/s ± 5.6 µm/s), and 4-4-4 (94.8 µm/s ± 3.9 µm/s) with regard with regard to the to control (81.9(81.9 the control μm/s ± 4.7 µm/s μm/s; ± 4.7 seesee µm/s; Table 1).1). Table Finally, BCF Finally, BCFsignificantly significantly(p(p<<0.05) 0.05)decreased decreasedafter after the the application application of of patterns patterns 3-3-3 3-3-3 min min (8.4 (8.4 Hz Hz ±± 0.6 0.6 Hz) Hz) and and 5-1-5 5-1-5 (8.4 (8.4 Hz Hz ±±0.6 0.6Hz) Hz)when when compared compared to to the the control (9.7 Hz ± 0.6 Hz; Table control (9.7 Hz ± 0.6 Hz; Table 1). 1). (a) (b) Figure Percentagesofoftotal Figure 4. Percentages total(a;(a; TMOT) TMOT) andand progressively progressively motile motile spermatozoa spermatozoa (b; PMOT) (b; PMOT) in the in the control control and different and different irradiation irradiation patterns.patterns. No significant No significant differences differences between thebetween controlthe andcontrol and light- light-stimulation stimulation patterns patterns were wereData observed. observed. Data as are shown aremean shown± as mean SEM ± SEM of nine of nine independent independent experiments. experiments. Table 1. Kinetic parameters (mean ± SEM) of horse spermatozoa in the control and different Table 1. Kinetic parameters (mean ± SEM) of horse spermatozoa in the control and different irradiation patterns. irradiation patterns. Patterns VCL (µm/s) VSL (µm/s) VAP (µm/s) LIN (%) STR (%) WOB (%) ALH (µm) BCF (Hz) Patterns VCL (µm/s) VSL (µm/s) VAP (µm/s) LIN (%) STR (%) WOB (%) ALH (µm) BCF (Hz) a a a a 4.6 aa 77.6 2.1a a 2.7 0.1a a 9.79.7 a Control Control 107.2±±5.9 107.2 5.9 a 56.9±±2.5 56.9 2.5a a 81.9±± 4.7 81.9 55.6 ± 4.7aa 55.6 ± 4.7 71.0 ±± 4.6 4.7 aa 71.0 77.6 ±±2.1 2.7±±0.1 ± 0.6 ± 0.6 a Ph 1 116.0 ± 8.3 a a 58.9 ± 3.9 a 88.5 ± 4.7 a 52.2 ± 6.2 a 66.3 ± 5.7 aa 77.2 ± 2.8aa 2.9 ± 0.2a a 9.3 ± 0.4 a Ph 1 116.0 ± 8.3 58.9 ± 3.9 88.5 ± 4.7 52.2 ± 6.2 a 66.3 ± 5.7 a 77.2 ± 2.8 a 2.9 ± 0.2 a 9.3 ± 0.4 a a Ph 2 117.2 ± 4.8 a 62.3 ± 5.3 a 92.0 ± 4.9 a 53.6 ± 4.8 68.0 ± 4.2 78.1 ± 2.6 2.9 ± 0.1 9.1 ± 0.3 PhPh3 2 117.2±±7.0 113.2 4.8a a 62.3±±4.0 60.7 5.3a a 92.0±± 6.3 90.4 4.9aa 53.6 ± 4.2 54.5 ± 4.8 aa 68.0 68.2 ±± 4.2 4.5 a 78.1 a 79.6±±2.6 2.1 a 2.9 a 2.9±±0.1 0.2 a 9.19.1 a ± 0.3 a ± 0.2 a PhPh4 3 113.2±±5.2 117.7 7.0a a 62.5 4.0a a 60.7±±4.5 90.4±± 4.6 94.4 6.3ba 54.5 4.2 aa 68.2 ± 4.6 53.2 ± 4.8 aa 79.6 66.2 ±± 4.5 2.2aa 2.9 80.2±±2.1 0.3a a 9.19.2 3.0±±0.2 ± 0.2 a a ± 0.8 PhPh5 4 117.7±±6.6 111.3 5.2a a 58.9 4.5a a 62.5±±2.6 87.7 4.6aᵇ 53.2 94.4 ±± 4.3 54.8 ± 4.6 aa 66.2 ± 5.0 5.0 aa 80.2 68.6 ±± 4.8 2.0aa 3.0 79.2±±2.2 0.1a a 9.29.4 2.6±±0.3 a a ± 0.5 ± 0.8 PhPh 105 115.4 ± 6.9 a 62.6 ± 3.3 a 91.8 ± 6.3 a 55.4 ± 4.2 a 69.4 ± 4.3 a 79.5 ± 2.0 a 2.9 ± 0.1 a 9.3 ± 0.2 a 111.3 ± 6.6 a 58.9 ± 2.6 a 87.7 ± 4.3 a 54.8 ± 5.0 aa 68.6 ± 5.0 aa 79.2 ± 2.0 aa 2.6 ± 0.1 a a 9.4 ± 0.5 a a Ph 1-1-1 112.4 ± 8.5 a a 60.7 ± 2.5 a a 84.6 ± 5.7 aa 55.0 ± 5.6 a 70.6 ± 5.3 a 77.0 ± 2.3a 3.2 ± 0.3a 9.1 ± 0.7 Ph 10 115.4 ± 6.9 62.6 ± 3.3 91.8 ± 6.3 55.4 ± 4.2 a 69.4 ± 4.3 a 79.5 ± 2.0 a 2.9 ± 0.1 a 9.3 ± 0.2 a a Ph 2-1-2 116.3 ± 7.4 a 62.9 ± 3.3 a 90.6 ± 4.5 a 56.1 ± 5.5 70.6 ± 5.0 78.6 ± 2.4 3.2 ± 0.3 8.8 ± 0.6 Ph2-2-2 Ph 1-1-1 112.4±±7.3 121.6 8.5a a 60.7±±3.4 62.4 2.5a a 84.6±± 4.7 96.0 5.7ba 55.0 ± 5.0 53.5 ± 5.6 aa 70.6 65.9 ±± 5.3 4.6 a 77.0 a 79.6±±2.3 2.5 a 3.2 a 2.9±±0.3 0.3 a 9.18.6 a ± 0.7 a ± 0.8 a Ph3-1-3 Ph 2-1-2 116.3±±6.8 115.6 7.4a a 61.1 3.3a a 62.9±±3.5 4.5aa 56.1 90.6±± 6.2 91.4 5.5 aa 70.6 ± 3.5 53.7 ± 4.1 aa 78.6 68.2 ±± 5.0 1.8aa 3.2 78.6±±2.4 0.3a a 8.88.6 3.3±±0.3 ± 0.6 a a ± 0.6 Ph Ph3-3-3 2-2-2 121.6±±6.9 119.5 7.3a a 3.4b a 62.4±±4.8 65.7 4.7bᵇ 53.5 96.0±± 5.6 96.4 55.6 ± 5.0 aa 65.9 ± 4.8 4.7 aa 79.6 68.0 ±± 4.6 2.5aa 2.9 79.7±±2.5 0.3a a 8.68.4 3.1±±0.3 a b ± 0.6 ± 0.8 Ph ± a ± a ± a ± a ± a ± a ± a ± a Ph4-1-4 3-1-3 112.3 7.7 115.6 ± 6.8 a 63.9 2.8 61.1 ± 3.5 a 93.3 5.7 91.4 ± 6.2 a 59.6 4.5 72.6 5.0 81.9 1.4 2.8 0.1 53.7 ± 3.5 a 68.2 ± 4.1 a 78.6 ± 1.8 a 3.3 ± 0.3 a 8.6 ± 0.6 a a a a a a 9.6 0.3 Ph 4-4-4 112.0 ± 7.6 a a 64.4 ± 3.2 a 94.8 ± 3.9 b 59.0 ± 4.3 a 71.6 ± 4.7 a 82.2 ± 1.7a 2.6 ± 0.1a 9.0 ± 0.5 Ph 3-3-3 119.5 ± 6.9 65.7 ± 4.8 ᵇ 96.4 ± 5.6 ᵇ 55.6 ± 4.8 a 68.0 ± 4.7 a 79.7 ± 2.5 a 3.1 ± 0.3 a 8.4 ± 0.6 ᵇ b Ph 5-1-5 113.0 ± 6.8 a 64.0 ± 2.0 a 91.0 ± 4.1 a 58.1 ± 4.0 71.2 ± 3.8 81.1 ± 1.9 3.1 ± 0.3 8.4 ± 0.6 Ph5-5-5 Ph 4-1-4 112.4 ±±7.4 112.3 7.7a a 63.9 ± 2.8 66.7 ± 2.0 b a 93.3±± 4.3 92.7 5.7aa 59.6 ± 4.4 61.3 ± 4.5 aa 72.6 74.4 ±± 5.0 4.4 a 81.9 a 82.4±±1.4 1.5 a 2.8 a 2.9±±0.1 0.1 a 9.68.9 a ± 0.3 a ± 0.3 a PhPh 4-4-4 10-5-10 112.0±±8.3 105.8 7.6a a 64.4±±2.5 61.3 3.2a a 94.8 ±± 5.4 86.0 3.9 ᵇ 59.0 a ± 5.4 60.8 ± 4.3 71.6 aa 73.7 ±± 4.7 5.8 aa 82.2 81.9±±1.7 1.7a a 2.6 2.5±±0.1 0.1a a 9.09.1 ± 0.5 a a ± 0.3 PhPh 5-1-5 10-10-10 109.4 6.8a a 113.0± ±12.3 58.1 2.0a a 64.0±±5.1 4.1aa 58.1 91.0±± 8.8 84.0 4.0 aa 71.2 ± 6.5 57.3 ± 6.9 aa 81.1 73.0 ±± 3.8 3.4aa 3.1 77.4±±1.9 0.3a a 8.49.7 2.8±±0.3 ± 0.6 ᵇa ± 0.6 a a a a 6.1 aa 82.4 2.8aa 2.9 0.2a a 8.99.8 a PhPh15-5-15 5-5-5 112.4±±8.9 109.2 7.4 a 66.7±±3.4 60.5 2.0 ᵇ 92.7±± 4.9 83.6 58.6 ± 4.3 a 61.3 ± 6.2 74.3 ±± 4.4 4.4 a 74.4 77.4±±1.5 3.0±±0.1 ± 0.4 ± 0.3 a Ph 15-15-15 112.2 ± 9.4 a a 59.6 ± 6.1 a a 85.7 ± 5.1 aa 56.7 ± 7.4 aa 70.9 ± 7.5 aa 77.7 ± 3.7aa 2.8 ± 0.2a a 9.7 ± 0.3 a Ph 10-5-10 105.8 ± 8.3 61.3 ± 2.5 86.0 ± 5.4 60.8 ± 5.4 73.7 ± 5.8 81.9 ± 1.7 2.5 ± 0.1 9.1 ± 0.3 a Different 109.4 Ph 10-10-10 letters± (a, b) aindicate 12.3 58.1 ±significant 5.1 a differences 84.0 (p ≤ ±0.05) ± 8.8 a 57.3 6.5 abetween 73.0 ± 6.9thea control 77.4 ± and 3.4 airradiation 2.8 ± 0.3 patterns. a 9.7 ± 0.6 a Ph 15-5-15 109.2 ± 8.9 a 60.5 ± 3.4 a 83.6 ± 4.9 a 58.6 ± 6.2 a 74.3 ± 6.1 a 77.4 ± 2.8 a 3.0 ± 0.2 a 9.8 ± 0.4 a Ph 15-15-15 112.2 ± 9.4 a 59.6 ± 6.1 a 85.7 ± 5.1 a 56.7 ± 7.4 a 70.9 ± 7.5 a 77.7 ± 3.7 a 2.8 ± 0.2 a 9.7 ± 0.3 a Different letters (a, b) indicate significant differences (p ≤ 0.05) between the control and irradiation patterns. Biology 2020, 9, 254 10 of 17 Biology 2020, 9, x 10 of 18 3.4. Sperm Sperm Subpopulations Four different sperm subpopulations were identified following cluster analyses based on the analyzed individual kinematic parameters. Table Table 2 shows the kinematic parameters for these sperm subpopulations, which subpopulations, which were identified as SP1, SP2, SP1, SP2, SP3, SP3, and and SP4. SP4. SP1 SP1 was was the slowest sperm subpopulation, subpopulation, since since it showed showed lowlow VCL, VCL, VSL, VSL, VAP, VAP, andand ALH. ALH. SP2 SP2 presented presented intermediate intermediate values (higher than SP1 but lower than SP3) SP3) in in most most sperm sperm kinetic kinetic parameters parameters(VCL, (VCL,VSL, VSL,VAP, VAP, LIN, LIN, STR, STR, and WOB) WOB) andandexhibited exhibitedhigher higherALH ALHthanthan SP1SP1andand SP3.SP3. In addition, In addition, SP2SP2 alsoalso showed showed the highest the highest BCF. BCF. SP3thewas SP3 was mostthe most linear linear subpopulation, subpopulation, as displayed as the displayed the highest highest values in mostvalues kineticin most kinetic parameters (i.e., parameters VSL, (i.e.,STR, VAP, LIN, VSL,andVAP, LIN,Finally, WOB). STR, and SP4WOB). was theFinally, SP4 wasthat, subpopulation the subpopulation despite showingthat, the despite highest showing values the highest of VCL and ALH, values of VCL its VSL and ALH, was similar itsofVSL to that SP1,was whichsimilar to slowest was the that of SP1, which was subpopulation the (SP1), slowest subpopulation and its LIN, STR, and WOB (SP1), wasand itsthan lower LIN,inSTR, and three the other WOBsubpopulations was lower than in SP2, (SP1, the and other three SP3). subpopulations (SP1, SP2, and SP3). Table 2. Descriptive parameters (mean ± SEM; range) of the four sperm subpopulations (SP1, SP2, SP3, and SP4) Table identified inparameters 2. Descriptive stallion fresh semen. (mean ± SEM; range) of the four sperm subpopulations (SP1, SP2, SP3, and SP4) identified in stallion fresh semen. SP1 SP2 SP3 SP4 N SP1 11,893 SP2 10,098 SP3 11,210 SP4 8142 N 11,893 10,098 11,210 8142 Mean ± Mean ± Mean ± Mean ± Parameter Mean ± Range Mean ± Range Mean ± Range Mean ± Range Parameter SEM Range SEM Range SEM Range SEM Range SEM SEM SEM SEM VCL (µm/s) VCL (μm/s) 72.7 ± 72.7 ± 0.1 0.1 0.0–120.0 0.0–120.0 109.1 ± 109.1 ± 0.1 0.1 71.6–181.9 71.6–181.9 120.1 ± 0.2 120.1 ± 0.2 87.2–213.9 87.2–213.9 158.3 ± 0.3 158.3 ± 0.3 83.1–372.2 83.1–372.2 VSL (µm/s) 45.9 ± 0.1 0.0–84.6 64.7 ± 0.2 0.9-105.6 94.5 ± 0.2 43.4–199.0 47.2 ± 0.3 0.6–221.2 VSL (μm/s) 45.9 ± 0.1 0.0–84.6 64.7 ± 0.2 0.9-105.6 94.5 ± 0.2 43.4–199.0 47.2 ± 0.3 0.6–221.2 VAP (µm/s) 59.9 ± 0.1 0.0–112.9 85.1 ± 0.1 34.4–129.3 112.7 ± 0.2 83.9–224.8 109.5 ± 0.2 24.1-282.5 VAP (μm/s) 59.9 ± 0.1 0.0–112.9 85.1 ± 0.1 34.4–129.3 112.7 ± 0.2 83.9–224.8 109.5 ± 0.2 24.1-282.5 LIN (%) 63.2 ± 0.2 0.0–100.0 60.7 ± 0.2 0.8–97.7 79.4 ± 0.1 33.8–99.3 30.0 ± 0.2 0.4–84.4 LIN (%) 63.2 ± 0.2 0.0–100.0 60.7 ± 0.2 0.8–97.7 79.4 ± 0.1 33.8–99.3 30.0 ± 0.2 0.4–84.4 STR (%) 72.1 ± 0.2 0.0–100.0 76.6 ± 0.2 1.4–99.7 84.3 ± 0.1 35.4–100.0 43.7 ± 0.2 0.5–98.3 STR WOB(%)(%) 72.1 ± 0.2 80.6 ± 0.1 0.0–100.0 0.0–100.0 76.6 78.6± 0.2 ± 0.1 1.4–99.7 28.1–100.0 84.3 93.8 ±±0.1 0.1 35.4–100.0 43.7±±0.2 71.2–100.0 69.7 0.2 17.6–100.0 0.5–98.3 WOB ALH(%)(µm) 80.62.2± 0.1 ± 0.1 0.0–100.0 0.0–5.9 78.63.5± ± 0.10.1 28.1–100.0 0.9–6.6 93.8 2.5 ±± 0.1 0.1 71.2–100.0 0.4–5.30 69.7± ±0.1 5.7 0.2 17.6–100.0 2.0–16.9 ALHBCF(μm) (Hz) 2.27.7± 0.1 ± 0.1 0.0–5.9 0.0–21.0 3.5 ± 0.1 11.0 ± 0.1 0.9–6.6 3.6–22.0 2.5 7.9 ±±0.1 0.1 0.4–5.30 0.0–20.0 5.7±±0.1 8.4 0.1 2.0–16.9 0.0–22.0 BCF (Hz) 7.7 ± 0.1 0.0–21.0 11.0 ± 0.1 3.6–22.0 7.9 ± 0.1 0.0–20.0 8.4 ± 0.1 0.0–22.0 Data were obtained after classifying sperm cells into motile subpopulations through principal component and Data clusterwere obtained after classifying sperm cells into motile subpopulations through principal analyses. component and cluster analyses. Figure 5a Figure 5a shows shows the the percentages percentages of of spermatozoa spermatozoa belonging belonging to to SP1. These percentages SP1. These percentages were were significantly (p < significantly (p 0.05) higher < 0.05) higher in in the the control (39.8% ±± 5.8%) control (39.8% 5.8%) than than inin the the following following irradiation irradiation patterns: patterns: (23.3% ±± 5.1%), 22 (23.3% 5.1%), 2-2-2 (24.6%±±4.4%), 2-2-2(24.6% 4.4%),3-3-3 (25.5%±±5.1%), 3-3-3(25.5% 5.1%),and and4-1-4 4-1-4(26.3% (26.3% ± 5.8%). ± 5.8%). Percentages Percentages of of spermatozoa spermatozoa belonging belonging to SP3 to SP3 significantly significantly (p < 0.05) (p < 0.05) increased increased following following light-stimulation light-stimulation for for 2 min (27.2% ± 6.9%±vs. 2 min (27.2% 6.9% 15.8% ± 3.4%±in3.4% vs. 15.8% the in the control; control; FigureFigure 5c). In5c). In contrast, contrast, no significant no significant differences differences (p > (p > 0.05) between the control and irradiation patterns were observed in SP2 0.05) between the control and irradiation patterns were observed in SP2 and SP4 (Figure 5b,d).and SP4 (Figure 5b,d). (a) (b) Figure 5. Cont. Biology 2020, 9, x 11 of 18 Biology 2020, 9, 254 11 of 17 Biology 2020, 9, x 11 of 18 (c) (d) (c) Figure 5. Box plots showing (d) the percentages of sperm belonging to (a) Subpopulation 1 (SP1, which was the slowest subpopulation based on VCL, VSL, and VAP); (b) Subpopulation 2 (SP2, which Figure 5. Box plots plots showing showingthe thepercentages percentagesofof sperm sperm belonging belonging to (a) to (a) Subpopulation Subpopulation 1 (SP1, 1 (SP1, whichwhich was presented intermediate values, higher than SP1 but lower than SP3); (c) Subpopulation 3 (SP3, which was the slowest the slowest subpopulation subpopulation based based on VCL, onVSL, VCL,and VSL, and(b) VAP); VAP); (b) Subpopulation Subpopulation 2 (SP2, 2 (SP2, which which presented was the one with the highest VSL, VAP, and LIN); and (d) Subpopulation 4 (SP4, which was the one presented intermediateintermediate values, values, higher higher than thanlower SP1 but SP1 but lower than SP3);than SP3); (c) Subpopulation (c) Subpopulation 3 (SP3,was 3 (SP3, which which the with the highest VCL and ALH but with the lowest LIN, STR, and WOB) in the control and different was the one with the highest VSL, VAP, and LIN); and (d) Subpopulation 4 (SP4, which was the one one with the highest VSL, VAP, and LIN); and (d) Subpopulation 4 (SP4, which was the one with the irradiation patterns. The superscript (*) means significant differences (p ≤ 0.05) between the control highest with theVCL and VCL highest ALHand but ALH with the butlowest LIN, with the STR,LIN, lowest and WOB) STR, andin the control WOB) andcontrol in the different andirradiation different and irradiation patterns. Data are shown as mean ± SEM of nine independent experiments. patterns. The irradiation superscript patterns. The (*) means significant superscript (*) meansdifferences significant ≤ 0.05) between (p differences (p ≤ the 0.05)control betweenandthe irradiation control patterns. and Data are irradiation shownData patterns. as mean ± SEMas are shown of mean nine independent ± SEM of nine experiments. independent experiments. 3.5. Effects of Red-Light Stimulation on Intracellular ATP Levels 3.5. Effects of 3.5. Effects of Red-Light Stimulation Stimulation on Intracellular Intracellular ATP ATP Levels Figure 6Red-Light shows intracellular on Levelsand control samples. Samples irradiated ATP levels in irradiated for 4Figure min showed Figure 6 showssignificantly (p intracellular < 0.05) ATP higher levels in intracellular 6 shows intracellular ATP levels in irradiated and irradiated andATP levels control control (3.8 nmol/mg samples. samples. Samples Samplesprotein ± 0.3 irradiated irradiated nmol/mg for for 44 min protein) min showed than showedsignificantly significantly (p(p<<0.05) the control (2.5 nmol/mg 0.05) higher higher protein ± ATP 0.2ATP intracellular intracellular nmol/mg levels levels protein). (3.8(3.8 No nmol/mg nmol/mg significant protein protein ± ± 0.3 differences 0.3 nmol/mg between the protein) control than the and controlthe other (2.5 light-stimulation nmol/mg protein ± patterns 0.2 nmol/mgwere observed. protein). nmol/mg protein) than the control (2.5 nmol/mg protein ± 0.2 nmol/mg protein). No significant No significant differences between differences between the the control control and and the the other other light-stimulation light-stimulation patterns patterns were were observed. observed. Figure 6. Intracellular ATP levels in control and irradiated samples. The superscript (*) means Figure 6. Intracellular ATP levels in control and irradiated samples. The superscript (*) means significant differences (p ≤ 0.05) between the control and different light-stimulation patterns. Data are significant differences (p ≤ 0.05) between the control and different light-stimulation patterns. Data are Figure shown as6. mean Intracellular ± SEM ofATP ninelevels in control independent and irradiated samples. The superscript (*) means experiments. shown as mean ± SEM of nine independent experiments. significant differences (p ≤ 0.05) between the control and different light-stimulation patterns. Data are 3.6. Effects shownof asRed-Light mean ± SEMStimulation on Oxygenexperiments. of nine independent Consumption 3.6. Effects of Red-Light Stimulation on Oxygen Consumption Figure 7a shows O2 consumption rates, which did not differ (p > 0.05) between irradiated 3.6. andEffects Figureof7a control Red-Light shows O samples. Stimulation However, on Oxygen 2 consumption when, rates, Consumption which in order did not individual to remove differ (p > 0.05) between variability, O2irradiated consumptionand control samples. ratesFigure in irradiated However, 7a showssamples when, in order were normalized O2 consumption to remove individual againstdid rates, which their variability, notcorresponding O consumption controls differ (p > 0.05) 2 between(Figure rates in 7b), those irradiated and irradiated standardized control samples rates samples. were in samples However, normalized irradiated when, against for 4tomin in order their corresponding wereindividual remove significantly controls (p < 0.05) higher variability, (Figure 7b), those than in therates O2 consumption control. in standardized rates in samples irradiated for 4 min were significantly (p < 0.05) irradiated samples were normalized against their corresponding controls (Figure 7b), those higher than in the control. standardized rates in samples irradiated for 4 min were significantly (p < 0.05) higher than in the control. Biology 2020, 9, 254 12 of 17 Biology 2020, 9, x 12 of 18 (a) (b) Figure 7. O2 consumption rates in control and irradiated samples (a), and rates in irradiated samples Figure 7. O2 consumption rates in control and irradiated samples (a), and rates in irradiated samples normalized against the corresponding control (b). The superscript (*) means significant differences normalized against the corresponding control (b). The superscript (*) means significant differences (p (p ≤ 0.05) between the control and irradiation patterns. Data are shown as mean ± SEM of nine ≤ 0.05) between the control and irradiation patterns. Data are shown as mean ± SEM of nine independent experiments. independent experiments. 3.7. Effects of Red-Light Stimulation on DNA Fragmentation 3.7. Effects of Red-Light Stimulation on DNA Fragmentation As shown in Table 3, percentages of DNA fragmentation (% DFI), mean fluorescence intensity of As shown in single-stranded Table DNA 3, percentages (mean of DNA fragmentation DFI), and percentages of high DNA(%stainability DFI), mean(% fluorescence intensity HDS) did not differ of single-stranded DNA (mean DFI), and percentages (p > 0.05) between the control and irradiated samples. of high DNA stainability (% HDS) did not differ (p > 0.05) between the control and irradiated samples. Table 3. DNA fragmentation parameters of horse sperm in control and irradiated samples. Data Table ±3.SEM) (mean DNAare fragmentation parameters given as percentages of horse of sperm withsperm damaged in control chromatinand(%irradiated DFI), mean samples. Data fluorescence (mean ± SEM) intensity are given of ssDNA (meanas DFI), percentages of sperm with and percentages damaged of high chromatin stainability (% DFI), mean fluorescence (% HDS). intensity of ssDNA (mean DFI), and percentages of high stainability (% HDS). Patterns % DFI Mean DFI % HDS Patterns % DFI Mean DFI % HDS Control 15.4 ± 3.0 322.9 ± 30.1 21.1 ± 5.1 Photo 1 Control 15.4 ± 15.4 0.8± 3.0 322.9335.5 ± 30.1 21.1 ± 5.1 22.8 ± 8.9 ± 33.3 Photo 2 Photo 1 15.4 ± 15.4 1.2± 0.8 335.5332.1 ± 33.3 22.8 ± 8.9 22.8 ± 7.6 ± 19.5 Photo 3 Photo 2 15.4 15.9 ± 2.9± 1.2 332.1 ± 19.5 22.8 ± 7.6 22.2 ± 5.1 327.5 ± 25.4 Photo 4 Photo 3 15.9 14.2 ± 1.4± 2.9 327.5 ± 25.4 22.2 ± 5.1 18.1 ± 5.6 319.0 ± 21.3 Photo 5 Photo 4 15.8 ± 14.2 1.4± 1.4 319.0326.9 ± 21.3 18.1 ± 5.6 19.7 ± 8.9 ± 29.9 Photo 10 Photo 5 14.6 ± 0.9± 1.4 326.9319.7 15.8 ± 25.4 ± 29.9 19.7 ± 8.9 19.3 ± 8.5 Photo 1-1-1 Photo 10 17.0 ± 1.4± 0.9 319.7326.0 14.6 ± 34.8 ± 25.4 19.3 ± 8.5 25.3 ± 8.1 Photo 2-1-2 Photo 1-1-1 15.6 ± 0.7± 1.4 326.0319.7 17.0 ± 29.0 ± 34.8 25.3 ± 8.1 21.1 ± 5.8 Photo 2-2-2 Photo 2-1-2 17.1 ± 1.7± 0.7 319.7332.6 15.6 ± 30.1 ± 29.0 21.1 ± 5.8 25.9 ± 6.1 Photo 3-1-3 Photo 2-2-2 16.4 ± 2.9± 1.7 332.6351.7 17.1 ± 46.2 ± 30.1 25.9 ± 6.1 22.7 ± 8.9 Photo 3-3-3 15.8 ± 2.9 330.4 ± 30.2 21.8 ± 3.1 Photo 3-1-3 16.4 ± 2.9 351.7 ± 46.2 22.7 ± 8.9 Photo 4-1-4 14.3 ± 1.0 332.5 ± 26.4 18.7 ± 8.3 Photo 3-3-3 15.8 ± 2.9 330.4 ± 30.2 21.8 ± 3.1 Photo 4-4-4 16.8 ± 2.9 357.7 ± 44.1 32.3 ± 7.2 Photo 5-1-5 Photo 4-1-4 14.3 17.5 ± 3.9± 1.0 332.5323.7 ± 26.4 18.7 ± 8.3 28.2 ± 12.2 ± 28.1 Photo 5-5-5 Photo 4-4-4 16.8 17.5 ± 2.7± 2.9 357.7350.0 ± 44.1 32.3 ± 7.2 31.6 ± 6.0 ± 37.5 Photo 10-5-10 Photo 5-1-5 17.5 18.4 ± 3.8± 3.9 323.7341.4 ± 28.1 28.2 ± 12.2 31.9 ± 7.0 ± 27.0 Photo 10-10-10 Photo 5-5-5 17.5 18.4 ± 3.3± 2.7 350.0324.6 ± 37.5 31.6 ± 6.0 31.4 ± 10.4 ± 27.4 Photo 15-5-15Photo 10-5-10 18.4 19.3 ± 4.5± 3.8 341.4313.7 ± 27.0 31.9 ± 7.0 29.7 ± 9.2 ± 24.3 Photo 15-15-15 Photo 10-10-1018.3 ± 4.3± 3.3 324.6314.5 18.4 ± 19.3 ± 27.4 31.4 ± 10.4 25.1 ± 9.6 Photo No significant differences between 15-5-15 control 19.3 ± 4.5 and irradiated 313.7were samples ± 24.3 29.7(p± >9.2 observed 0.05). DFI: DNA fragmentation Photo 15-15-15 18.3 ± 4.3 314.5 ± 19.3 25.1 ± 9.6 index. HDS: High DNA stainability. No significant differences between control and irradiated samples were observed (p > 0.05). DFI: DNA fragmentation index. HDS: High DNA stainability. Biology 2020, 9, 254 13 of 17 4. Discussion Our results clearly show that irradiation with LED-based red-light modifies some kinetic parameters and the structure of motile sperm subpopulations. These changes occurred together with an increase in mitochondrial activity of horse spermatozoa, without affecting the integrity of plasma membrane or DNA. Interestingly, our data suggest that the effects of exposing horse spermatozoa to red-light heavily rely upon the specific utilized light-dark interval. These findings are similar to those observed in previous studies based on the use of both laser and LED-based light sources in species such as dogs [17], buffalos [34], and humans [35]. In addition, our data are consistent with those reported by other authors, who have suggested that irradiation effects depend on the precise rhythm and intervals of light-darkness regardless of the light source (LED or laser) used [7,36,37]. In fact, distinct wavelengths have different effects and, although the optimal wavelength varies between species, mounting evidence indicates that blue- or green-light is detrimental for mammalian spermatozoa [11,13,38]. Related with this, it is important to emphasize that, in previous studies carried out in other mammalian species, red-light has been demonstrated to be the one that mostly improves motility and other functional parameters in both human and animal spermatozoa (see [14] for review). The results shown herein suggest that stallion sperm exposed to short patterns of red-light stimulation, both single or combining light with dark regimens, exhibit better response on motility parameters than the long ones. This is very apparent when looking at the changes in kinetic parameters (patterns 4, 2-2-2, 3-3-3, 4-4-4, 5-1-5, and 5-5-5), and in the sperm subpopulation structure (patterns 2, 2-2-2, 3-3-3, and 4-1-4). In this context, it is worth mentioning that, in a study conducted using liquid-stored boar semen irradiated with LED red-light, the shortest irradiation patterns were the ones that exerted the most intense effects [7]. A similar result was also observed in a study conducted with human sperm, in which irradiation with a single exposition to red LED light for 50 s, 100 s, and 200 s increased sperm motility, whereas the exposure for 400 s had inhibitory effects [13]. Therefore, all these results, including those of our study, would be consistent with the fact that the two-phase response to light-doses follows the Arndt–Schultz curve [38,39]. In fact, these data also suggest that low levels of irradiation have greater but variable effect on tissues than higher levels of irradiation. This variable response, including the impact upon sperm motility, has already been described elsewhere [12,13,35,37,40,41] although one study with human sperm suggested a linear relationship between the dose of red-light irradiation and sperm motility [42]. However, taking into account the considerable difference in the response to light between species, a more in-depth study is needed to establish better the mechanisms underlying the observed variability. Focusing on sperm motility, our findings in some kinetic patterns were similar to those observed in other species. These parameters were VSL (patterns: 3-3-3 min and 5-5-5 min), with similar effects to those found in humans [41], dogs [17], buffalos [34], and pigs [7]; and VAP (patterns: 4 min, 2-2-2 min, 3-3-3 min, and 4-4-4 min), with a similar impact to that reported in dogs [16,17], bulls [43], buffalos [34], and pigs [7]. In addition, we observed a significant decrease in BCF (patterns 3-3-3 min and 5-1-5 min) compared to the control, which matched with previous reports in humans [42], but differed from what found in dogs [16,17] and pigs [7]. Another important point to highlight is the lack of differences in total and progressive motility between control and light-stimulated samples. While these results are in agreement with those previously reported in dogs [16,17] and cattle [43], irradiation of sperm with red-light has been found to increase total and progressive motility in humans [35,37,41,42,44], buffalos [34], sheep [45], and pigs [7]. Therefore, the aforementioned differences also support that the effects of light-dose on sperm function rely upon species [11]. When evaluating the presence of sperm motile subpopulations in horse ejaculates, we identified four separate subpopulations. These results are similar to those reported previously in horses [46], donkeys [47], cattle [48], and goats [49,50]. Remarkably, we observed that percentages of spermatozoa belonging to SP1, which was the slowest subpopulation based on VCL, VSL, and VAP, were significantly Biology 2020, 9, 254 14 of 17 higher in the control than after the following light-stimulation patterns: 2 min, 2-2-2 min, 3-3-3 min, and 4-1-4 min. In addition, percentages of sperm belonging to SP3, which showed the highest values in most kinetic parameters, including VSL, VAP, and LIN, were significantly lower in the control than following light-stimulation for 2 min. Therefore, our data suggest that irradiation of horse sperm with red-light modifies the structure of motile sperm subpopulations by decreasing the percentage of the slowest sperm subpopulation and increasing the percentage of the most linear and fastest one. It is worth mentioning that these results are in agreement with a previous study conducted with dog semen, in which stimulation with laser red-light significantly increased the proportions of the fastest sperm subpopulation [17]. These changes in the characteristics of motile sperm subpopulations, in addition to those observed in sperm kinetic parameters, indicate that not only does irradiation with red-light increase sperm velocity, but it also modifies the way through which sperm move. At this moment, there is no clear explanation of how these effects occur, since the exact mechanism(s) through which red-light stimulates sperm remain(s) unclear. However, it has been hypothesized that a mechanism related to the activation of sperm mitochondria could be essential to explain those effects [14]. In fact, our results from the analysis of mitochondrial membrane potential agree with this possibility, since irradiation with patterns 4, 3-3-3, 4-4-4, 5-1-5, 5-5-5, 15-5-15, and 15-15-15 increased the percentages of spermatozoa with high ∆Ψm. A similar increase on ∆Ψm has been described in pig sperm [7,43]. Thus, these data suggest that stimulation with red-light could increase mitochondrial activity through photosensitizers that are present in the electronic chain, such as cytochrome C [7,51], which would result in higher sperm motility and greater fertilization potential [52]. Recent studies have concurred that oxygen consumption is an alternative way to measure mitochondrial activity, which could be better than the use of markers of mitochondrial membrane potential, such as JC1 [53,54]. In addition to this, the rate of oxygen consumption also provides an indirect measure of ATP produced by oxidative phosphorylation in sperm [54]. The results obtained in this study agree with this possibility, since they showed an increase in intracellular ATP levels and O2 consumption rate (O2 consumption rate normalized against the corresponding control) in samples irradiated for 4 min, compared with the non-irradiated control. Furthermore, these results agree with those obtained in the evaluation of the percentages of viable sperm with high MMP with this stimulation pattern. In this context, it is important to take into account that the increase in the potential of the mitochondrial membrane is associated with changes in the consumption of ATP and the activity of the enzymes of the respiratory chain [55,56]. Related with this, Iaffaldano et al. [45] observed that light stimulation of frozen-thawed ram sperm with a He-Ne laser increased ATP content and the activity and affinity of cytochrome C oxidase (CCO) for their substrate (cytochrome C). Interestingly, these authors found that CCO activity and ATP content were positively correlated with each other and with sperm motility, supporting the hypothesis that the effects of red light upon sperm are mediated by mitochondria. 5. Conclusions In conclusion, this work has shown, for this first time, that irradiation of horse sperm with red-light modifies the structure of motile sperm subpopulations, increases some kinetic parameters, intracellular ATP levels, the rate of O2 consumption, and mitochondrial membrane potential, without affecting the integrities of DNA and plasma membrane. Therefore, we suggest that the effects of light on sperm are related to mitochondrial function. These effects, however, rely upon the specific light-stimulation pattern. While these changes could have a beneficial impact upon the fertility ability of horse spermatozoa, further research investigating whether such a positive effect exists is much warranted. In addition to contemplating in vivo or in vitro fertility assays, future studies should also address which mechanism(s) underlie(s) this sperm response to red light. Author Contributions: Conceptualization, J.E.R.-G., J.M. and M.Y.; Methodology, J.C., M.P., S.G. and Y.M.-O.; Validation, J.E.R.-G., J.M. and M.Y.; Formal analysis, J.C.; Investigation, J.C., M.P., S.G. and Y.M.-O.; Resources, J.E.R.-G., J.M. and M.Y.; Data curation, J.C.; Writing—original draft preparation, J.C.; Writing—review and editing, Biology 2020, 9, 254 15 of 17 J.E.R.-G., J.M. and M.Y.; Supervision, J.E.R.-G., J.M. and M.Y.; Project administration, J.E.R.-G., J.M. and M.Y.; Funding acquisition, J.E.R.-G., J.M. and M.Y. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the Ministry of Science, Innovation and Universities, Spain (Grants: RYC-2014-15581 and AGL2017-88329-R), and the Regional Government of Catalonia (2017-SGR-1229). JC was funded by the National Agency for Research and Development (ANID), Ministry of Education, Chile (Scheme: Becas Chile Doctorado en el Extranjero, PFCHA; Grant: 2017/72180128). Acknowledgments: The authors would like to thank the support from Sebastián Bonilla-Correal from Autonomous University of Barcelona, Spain, and from Marc Llavanera, Ariadna Delgado-Bermúdez, Sandra Recuero, Beatriz Fernandez-Fuertes, Sergi Bonet, and Isabel Barranco from University of Girona for their technical support. Conflicts of Interest: J.E.R.-G. and M.Y. are inventors of a patent entitled ‘Method and apparatus for improving the quality of mammalian sperm’ (European Patent Office, no. 16199093.2; EP-3-323-289-A1), which is owned by Instruments Utils de Laboratori Geniul, SL (Barcelona, Spain). References 1. Brinsko, S.P.; Varner, D.D. Artificial insemination and preservation of semen. Vet. Clin. N. Am. Equine Pract. 1992, 8, 205–218. [CrossRef] 2. Aurich, J.E. 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