biology Article Cryotolerance of Stallion Spermatozoa Relies on Aquaglyceroporins rather than Orthodox Aquaporins Ariadna Delgado-Bermúdez 1 , Federico Noto 1 , Sebastián Bonilla-Correal 2 , Estela Garcia-Bonavila 1 , Jaime Catalán 2 , Marion Papas 2 , Sergi Bonet 1 , Jordi Miró 2,† and Marc Yeste 1,†, * 1 Biotechnology of Animal and Human Reproduction (TechnoSperm), Unit of Cell Biology, Department of Biology, Institute of Food and Agricultural Technology, Faculty of Sciences, University of Girona, 17003 Girona, Spain; ariadna.delgado@udg.edu (A.D.-B.); fnoto83@gmail.com (F.N.); estela.garcia@udg.edu (E.G.-B.); sergi.bonet@udg.edu (S.B.) 2 Equine Reproduction Service, Department of Animal Medicine and Surgery, Faculty of Veterinary Sciences, Autonomous University of Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain; sebastian.bonilla@e-campus.uab.cat (S.B.-C.); dr.jcatalan@gmail.com (J.C.); papas.marion@gmail.com (M.P.); jordi.miro@uab.cat (J.M.) * Correspondence: marc.yeste@udg.edu † These authors contributed equally to this work.  Received: 2 October 2019; Accepted: 11 November 2019; Published: 12 November 2019  Abstract: Aquaporins (AQPs), a family of ubiquitous water channels divided into orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs, are present in stallion spermatozoa. The aim of this study was to elucidate the functional relevance of each group of AQPs during stallion sperm cryopreservation through the use of three different inhibitors: acetazolamide (AC), phloretin (PHL) and propanediol (PDO). Sperm quality and function parameters were evaluated in the presence or absence of each inhibitor in fresh and frozen–thawed samples. In the presence of AC, different parameters were altered (p < 0.05), but not in a concentration- or time-depending manner. PHL was found to decrease sperm motility, viability, acrosome integrity, and the percentages of spermatozoa with low membrane lipid disorder, high mitochondrial membrane potential (MMP) and high intracellular levels of calcium and superoxides (p < 0.05). Finally, the sperm motility, viability, acrosome integrity, the percentages of spermatozoa with low membrane lipid disorder, high MMP and high intracellular calcium levels were higher (p < 0.05) in PDO treatments than in the control. The sperm response to AC, PHL and PDO indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Furthermore, post-thaw sperm quality was higher in PDO treatments than in the control, suggesting that this molecule is a potential permeable cryoprotectant. Keywords: acetazolamide; aquaporins; phloretin; propanediol; sperm; stallion 1. Introduction The permeability of the plasma membrane to water and solutes is crucial for proper cell function and homeostasis. Because of the amphipathic nature of the plasma membrane, the simple diffusion of water molecules does not occur at high rates [1]. Aquaporins (AQPs) are a family of ubiquitous transmembrane proteins that allow for the facilitated diffusion of water. Some AQPs are also permeable to small solutes [2]. To date, 13 AQPs (AQP0–AQP12) have been identified in mammalian cells and have been classified into three different groups (orthodox AQPs, aquaglyceroporins (GLPs), and superAQPs), which differ in their sequence and solute permeability. The orthodox AQP group includes AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8, which are exclusively permeable Biology 2019, 8, 85; doi:10.3390/biology8040085 www.mdpi.com/journal/biology Biology 2019, 8, 85 2 of 18 to water. The group of GLPs that is permeable to water, glycerol, urea and other small electrolytes comprises AQP3, AQP7, AQP9 and AQP10. Finally, AQP11 and AQP12 are members of the superAQPs group, which are localized in the membranes of intracellular organelles and are involved in the transport of water and glycerol. Even if ubiquitous, the presence of AQPs varies between cell types and species. In mammalian spermatozoa, AQP1 has been identified in porcine [3]; AQP3, AQP7 and AQP11 in equine [4], porcine [5,6], murine [7–9], human [10,11] and bovine species [12,13]; AQP8 has been found in mouse [8] and human spermatozoa [10,11]; and AQP9 is present in boar spermatozoa [14]. In the male gamete, AQPs are involved in the regulation of cell volume and osmotic balance, which are crucial for spermatogenesis [15] and post-ejaculation events, including the activation of sperm motility upon ejaculation and sperm adaptation to the female environment (reviewed in [2]). Cryopreservation, which is the most efficient method for the long-term storage of stallion spermatozoa, causes a drastic impairment in sperm quality at both lethal and sub-lethal temperature levels. The hyperosmotic shock during freezing and the following hypotonic stress during thawing induce a dramatic modification of the sperm cell volume that injuries the cytoskeleton, the mitochondria and the plasma membrane (reviewed in [16]). Mitochondria are the most sensitive organelle to osmotic stress and, when damaged, these organelles are a major source of oxidative stress through the generation of reactive oxygen species (ROS) [17]. Moreover, alterations in the plasma membrane affect embedded and membrane-associated proteins. This, in turn, affects specific signaling pathways and may detrimentally impact sperm fertilizing ability [18]. It is worth mentioning that there is a high variability between and within stallions in the ability of their ejaculates to withstand cryopreservation, i.e., cryotolerance or freezability. This inter- and intra-individual variability has also been described in other mammalian species [19–21]. Good (GFE) and poor freezability ejaculates (PFE) differ in their post-thaw sperm quality and function parameters, such as sperm membrane integrity, motility [22], ROS production and mitochondrial membrane potential (MMP) [23], all of which may ultimately affect the fertilizing capacity [24]. During the last decade, the efforts to improve the efficiency of sperm cryopreservation protocols have been focused on the use of alternative cryoprotectant agents (CPAs) and antioxidants to reduce osmotic and oxidative stresses, respectively (reviewed in [16]). In addition, the presence of damaged and non-viable sperm cells after thawing, which releases factors that have deleterious effects on viable spermatozoa, has evidenced the importance of selecting stallion spermatozoa after cryopreservation [25]. In spite of these advances, increasing cryopreservation efficiency in stallion spermatozoa still needs further optimization through the prediction of sperm cryotolerance (i.e., identification of GFE). In this context, AQPs are potential freezability biomarkers, since their permeability to water and small molecules are crucial for sperm’s response to osmotic stress. In fact, the AQP involvement in sperm cryopreservation has previously been confirmed in bull [12,13], boar [26,27] and stallion spermatozoa [4]. In effect, AQP3 and AQP7 are related to the cryotolerance of boar spermatozoa [28], AQP7 [5] and AQP11 [13] are associated with that of bull spermatozoa, and AQP3, AQP7, and AQP11 are related to stallion sperm freezability [4]. As one may assume that the inhibition of these AQPs may affect the sperm’s ability to withstand cryopreservation, the present study aimed to elucidate the functional relevance of orthodox AQPs and GLPs during stallion sperm cryopreservation by using three separate inhibitors (phloretin (AC), phloretin (PHL) and propanediol (PDO)). The sperm response to AQP-inhibition indicates that GLPs, rather than orthodox AQPs, play a crucial role during stallion sperm cryopreservation. Moreover, post-thaw sperm quality is higher in the presence of PDO than in the control, suggesting that this molecule could be added to cryopreservation media as a permeable cryoprotectant. Biology 2019, 8, 85 3 of 18 2. Materials and Methods 2.1. Stallions and Ejaculates A total of 12 ejaculates coming from different stallions (n = 12) were used. Animals were housed at the Equine Reproduction Service, Autonomous University of Barcelona (Spain), which is a European Union (EU)-approved equine semen collection center (Authorization code: ES09RS01E) that operates under strict protocols of animal welfare and health control. Since all stallions used in this study were semen donors and were housed at the Equine Reproduction Service, the local ethics committee at our University indicated that no further ethics authorization was required. Semen samples were collected using a Hannover-type artificial vagina (Minitüb GmbH, Tiefenbach, Germany) with an in-line nylon mesh filter to separate the gel fraction. Gel-free semen ere subsequently diluted 1:5 (v:v) in a Kenney extender [29], previously warmed at 37 ◦ C. Sperm concentration was assessed with a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Köningshofen, Germany), and each sample was split into two different fractions. The first one was used to assess the quality of the fresh semen, whereas the other was divided into nine different sub-fractions that were cryopreserved in the presence or absence of different concentrations of the three AQP-inhibitors. 2.2. AQP Inhibitors Prior to cryopreservation, three AQP inhibitors were added to semen samples: 1,3-propanediol (PDO, Sigma-Aldrich, St. Louis, MO, USA), acetazolamide (AC, Sigma-Aldrich), and phloretin (PHL, Sigma-Aldrich). PDO was diluted in the commercial freezing extender used for cryopreservation (see next section) to a working concentration of 100 mmol/L; AC was diluted in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to a working concentration of 450 mmol/L, and PHL was diluted in methanol (Fisher Chemical, ThermoFisher Scientific; Waltham, MA, USA) to a working concentration of 365 mmol/L. Each inhibitor was assayed at the following concentrations, which were chosen according to preliminary experiments conducted in our laboratory and previous studies [27]: 0.1, 1 and 10 mmol/L for PDO; 250, 500 and 1000 µmol/L for AC; and 350 and 800 µmol/L for PHL. It is worth mentioning that, in the case of treatments containing PDO and AC, samples were exposed to methanol or DMSO at concentrations lower than 0.5% (v/v). These concentrations showed no detrimental effects on sperm quality parameters (data not shown). 2.3. Stallion Sperm Cryopreservation The cryopreservation of stallion spermatozoa was performed in order to assess how the inhibition of AQPs from different groups affected sperm cryotolerance. The fraction of the ejaculate intended for cryopreservation was centrifuged in a programmable centrifuge (Medifriger BL-S; JP Selecta S.A., Barcelona, Spain) at 600× g and 20 ◦ C for 15 min, and the supernatants were discarded. Pellets were subsequently resuspended in 2 mL of a BotuCRYO™ commercial extender (Botupharma, Botucatu, Brazil), and sperm concentration, motility and membrane integrity were evaluated for the subsequent adjustment of sperm concentration to 2 × 106 viable spermatozoa per mL. In this step, eight aliquots of 1 mL of semen each were added with AQP inhibitors (see Section 2.2), and the remaining semen was used as a control. Treatments were as follows: AC at 250 µmol/L (AC250); AC at 500 µmol/L (AC500); AC at 1000 µmol/L (AC1000); PDO at 0.1 mmol/L (PDO0.1); PDO at 1 mmol/L (PDO1); PDO at 10 mmol/L (PDO10); PHL at 350 µmol/L (PHL350); and PHL at 800 µmol/L (PHL800). Samples were subsequently packaged into 0.5 mL straws (Minitüb) and frozen in a controlled-rate freezer (Ice-Cube 14S-B; Minitüb), using the following cooling rates: i) −0.25 ◦ C/min from 20 to 5 ◦ C (60 min), ii) −4.75 ◦ C/min from 5 to −90 ◦ C (20 min), and iii) −11.11 ◦ C/min from −90 to −120 ◦ C (2.7 min). Finally, straws were plunged into liquid nitrogen (−196 ◦ C) for storage. Biology 2019, 8, 85 4 of 18 Frozen–thawed sperm quality was evaluated after thawing. Two straws per ejaculate were thawed at 37 ◦ C by immersion in a water bath for 20 s. The content of these straws was then diluted 1:3 (v:v) in a pre-warmed Kenney medium. After that, samples were incubated at 37 ◦ C for 2 h, and sperm quality was assessed twice: at 10 min (0 h) and 2 h post-thaw. 2.4. Sperm Motility Sperm motility was evaluated before and after freeze–thawing through a computer-assisted sperm analysis (CASA) system that consisted of a phase-contrast microscope (Olympus BX41; Olympus, Tokyo, Japan) equipped with a video camera and ISAS software (Integrated Sperm Analysis System V1.0; Proiser SL, Valencia, Spain). The assessment of sperm motility in extended samples was performed after 15 min of incubation at 37 ◦ C; frozen–thawed samples were evaluated after 10 min (0 h) and 2 h of thawing. Three replicates of 1000 spermatozoa per sample and time points were evaluated using a pre-warmed (at 37 ◦ C) Makler counting chamber (Sefi-Medical Instruments, Haifa, Israel) and observed under a negative phase-contrast field (Olympus 10 × 0.30 PLAN objective, Olympus). For each motility assessment, the evaluation of the following parameters was performed: total motility (TMOT, %), progressive sperm motility (PMOT, %); curvilinear velocity (VCL, µm·s−1 ); straight line velocity (VSL, µm·s−1 ); average path velocity (VAP, µm·s−1 ); amplitude of lateral head displacement (ALH, µm); beat cross frequency (BCF, Hz); linearity (LIN, %), which was calculated assuming that LIN = VSL/VCL × 100; straightness (STR, %), resulting from VSL/VAP × 100; and motility parameter wobble (WOB, %), obtained from VAP/VCL × 100. A sperm cell was considered to be motile when its VAP was higher than 10 µm/s, and it was considered to be progressively motile when its STR was higher than 75%. For each parameter, the corresponding mean ± standard error of the mean (SEM) was calculated. 2.5. Flow Cytometry Analyses Flow cytometry analyses were performed in order to evaluate different sperm quality parameters in both fresh and frozen–thawed sperm samples: viability, acrosome integrity, membrane lipid disorder, MMP, intracellular calcium levels, and intracellular levels of superoxide (O2 − ) and peroxides (H2 O2 ). Samples were diluted to a final concentration of 1 × 106 sperm/mL with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline solution (10 mmol/L HEPES, 150 mmol/L NaCl, 10% BSA; pH = 7.4) prior to staining with the corresponding fluorochromes (ThermoFisher Scientific, Waltham, MA, USA). After that, samples were incubated at 38 ◦ C in the dark, and a total of three replicates per sample and parameter were evaluated. Flow cytometry analyses were performed using a Cell Laboratory QuantaSCTM cytometer (Beckman Coulter; Fullerton, CA, USA), and samples were excited with an argon ion laser (488 nm) set at a power of 22 mW. Cell diameter/volume was determined using the Cell Lab Quanta™ SC cytometer through the Coulter principle for volume assessment, which is based on the changes in electrical resistance produced in an electrolyte solution by suspended, non-conductive particles. In this system, forward scatter (FS) is replaced by electronic volume (EV). The EV-channel was calibrated using 10 µm flow-check fluorospheres (Beckman Coulter), and this size of beads was positioned at channel 200 on the EV scale. Three different optical filters were used: FL1 (Dichroic/Splitter, DRLP (dichroic long pass): 550 nm, BP (band pass) filter: 525 nm, detection width: 505–545 nm), FL2 (DRLP: 600 nm, BP filter: 575 nm, detection width: 560–590 nm) and FL3 (LP (long pass) filter: 670 nm/730 nm, detection width: 655–685 nm). While FL1 allowed for the detection of green fluorescence from SYBR14, Arachis hypogaea lectin—peanut agglutinin—conjugated with fluorescein isothiocyanate (PNA-FITC), YO-PRO-1, JC1 (5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethyl-benzimidazolylcarbocyanine iodide) monomers (JC1mon ), Fluo3 and 2’,7’-dichlorofluorescein (DCF); FL2 was used to detect orange fluorescence from JC1 aggregates (JC1agg ); and FL3 allowed for the detection of red fluorescence from propidium iodide (PI), merocyanine Biology 2019, 8, 85 5 of 18 540 (M540), Rhod5 and ethidium (E). Signals were logarithmically amplified, and the adjustment of photomultiplier settings was performed according to particular staining methods. The sheath flow rate was set at 4.17 µL/min, and EV and side scatter (SS) were measured and linearly recorded for all particles. The analyzer threshold on the EV channel was adjusted to exclude subcellular debris (particle diameter <7 µm) and cell aggregates (particle diameter >12 µm), and the sperm-specific events were positively gated on the basis of EV/SS distributions. Data obtained from flow cytometry evaluations were analyzed with Flowing Software (Ver. 2.5.1; University of Turku, Turku, Finland), and the recommendations of the International Society for Advancement of Cytometry (ISAC) were adhered. Following Petrunkina et al. [30], the events corresponding to the double negative-stained particles of all protocols except SYBR14/PI were corrected using the percentage of non-sperm debris particles of the SYBR14− /PI− population. The percentages of all the other sperm populations were recalculated. Finally, the corresponding mean ± SEM was calculated for each parameter. 2.5.1. Plasma Membrane and Acrosome Integrity Plasma membrane and acrosome integrity were evaluated through two different tests: SYBR14/PI and PNA-FITC/PI. On the one hand, the LIVE/DEAD sperm viability kit (Molecular Probes, Eugene, OR, USA) was used following the protocol of Garner and Johnson [31]. In brief, sperm were incubated with SYBR14 at a final concentration of 100 nmol/L for 10 min, and PI was added at a final concentration of 12 µmol/L prior to an additional incubation of 5 min. Three different sperm populations were observed: 1) viable, green-stained spermatozoa (SYBR14+ /PI− ); 2) non-viable, red-stained spermatozoa (SYBR14− /PI+ ); and 3) non-viable spermatozoa stained in both green and red (SYBR14+ /PI+ ). The remaining population in the dot-plots, which corresponded to unstained, non-sperm particles (SYBR14− /PI− ), was not included in the calculation of the final percentages of each sperm population (SYBR14+ /PI− , SYBR14− /PI+ , and SYBR14+ /PI+ ). The SYBR14 fluorescence spill over into the FL3 channel was compensated for (2.45%). In the second case, spermatozoa were co-stained with PNA-FITC and PI, following the procedure described by Nagy et al. [32] with minor modifications. Briefly, spermatozoa were incubated with PNA-FITC (final concentration: 2.5 µg/mL) and with PI (12 µmol/L) for 10 min. Flow cytometry dot-plots allowed for the identification of four different populations: 1) spermatozoa with an intact plasma membrane (PNA-FITC− /PI− ); 2) spermatozoa with a damaged plasma membrane (PNA-FITC+ /PI− ); 3) spermatozoa with a damaged plasma membrane and a partially altered outer acrosome membrane (PNA-FITC+ /PI+ ); and 4) spermatozoa with a damaged plasma membrane and a lost outer acrosome membrane (PNA-FITC− /PI+ ). Thereafter, spermatozoa were classified into two different categories: (a) spermatozoa with an intact plasma membrane and acrosome (Population 1; PNA-FITC− /PI− ); and (b) spermatozoa with acrosome and/or plasma membrane damage, which included Populations 2, 3 and 4. Events appearing in the PNA-FITC− /PI− quadrant were corrected using the percentages of non-sperm debris particles found in the SYBR14− /PI− quadrant; the percentages of the other three populations were recalculated. PNA-FITC fluorescence spill over into the FL3 channel was compensated for (2.45%). 2.5.2. Sperm Membrane Lipid Disorder The evaluation of sperm membrane lipid disorder was performed according the protocol from Rathi et al. [33] with minor modifications [34], using M540 and YO-PRO-1. M540 detected the decrease in packing order of phospholipids in the outer monolayer of the plasma membrane. Samples were incubated with M540 (final concentration: 2.6 µmol/L) and YO-PRO-1 (final concentration: 25 nmol/L) for 10 min. Four populations were observed in flow cytometry dot-plots: 1) non-viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1+ ); 2) non-viable spermatozoa with high membrane lipid disorder (M540+ /YO-PRO-1+ ); 3) viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1− ); and 4) viable spermatozoa with high membrane lipid disorder Biology 2019, 8, 85 6 of 18 (M540+ /YO-PRO-1− ). The percentages of viable spermatozoa with low membrane lipid disorder (M540− /YO-PRO-1− ) were corrected using proportions of non-sperm debris particles found in the SYBR-14− /PI− quadrant; the percentages of the other three populations were recalculated. Data were not compensated for. 2.5.3. Mitochondrial Membrane Potential (MMP) JC1-staining was used for the assessment of MMP, following the protocol of Ortega-Ferrusola et al. [35] with minor modifications. In brief, samples were incubated in the presence of JC1 at a final concentration of 0.3 µmol/L for 30 min. JC1 molecules form aggregates (JC1agg ) in the presence of high MMP, whereas JC1 molecules remain monomers in the presence of low MMP (JC1mon ). Three different populations were identified in flow cytometry dot-plots: 1) spermatozoa with low MMP (JC1mon ; FL1+ /FL2− ); 2) spermatozoa with high MMP (JC1agg ; FL1− /FL2+ ; and 3) spermatozoa with heterogeneous mitochondria (JC1agg and JC1mon ; FL1+ /FL2+ ) in the same cell. The percentages of double-negative particles (FL1− /FL2− ) were corrected using the proportions of SYBR14− /PI− ; the percentages of the other populations were recalculated. Spermatozoa with high MMP resulted from the sum of Populations 2 and 3. FL1 spill-over into the FL2 channel was compensated for (70.29%). 2.5.4. Intracellular Calcium Levels For the evaluation of intracellular calcium levels, two different co-staining tests were performed: Fluo3-AM (acetoxymethyl, ester form)/PI and Rhod5-N/YO-PRO-1. In the first test, intracellular calcium levels were evaluated with Fluo3-AM, which penetrates cell membranes and has more affinity for the calcium residing in the sperm mid-piece [36]. This test was performed according to the protocol of Harrison et al. [37] that was modified by Kadirvel et al. [38]. In brief, spermatozoa were incubated in the presence of 1 µmol/L of Fluo3-AM and 12 µmol/L of PI for 10 min. Four different sperm populations were identified in dot-plots: 1) non-viable spermatozoa with low intracellular calcium levels (Fluo3− /PI+ ); 2) non-viable spermatozoa with high intracellular calcium levels (Fluo3+ /PI+ ); 3) viable spermatozoa with low intracellular calcium levels (Fluo3− /PI− ); and 4) viable spermatozoa with high intracellular calcium levels (Fluo3+ /PI− ). The percentages of viable spermatozoa with low intracellular calcium levels (Fluo3− /PI− ) were corrected using proportions of non-sperm debris particles found in the SYBR14− /PI− quadrant; the percentages of the other three populations were recalculated. The spillover of PI into the FL1-channel and Fluo3 spill over into the FL3-channel were compensated for (28.72% and 2.45%, respectively). Intracellular calcium levels were also assessed through Rhod-5N staining following the protocol described by Yeste et al. [36], in which Rhod-5N was found to have more affinity for the calcium residing in the sperm head. Briefly, samples were incubated with Rhod-5N at a final concentration of 5 µmol/L and YO-PRO-1 at a final concentration of 25 nmol/L for 10 min. Four different populations were identified in flow cytometry dot-plots: 1) non-viable spermatozoa with low levels of intracellular calcium (Rhod5− /YO-PRO-1+ ); 2) non-viable spermatozoa with high levels of intracellular calcium (Rhod5+ /YO-PRO-1+ ); 3) viable spermatozoa with low levels of intracellular calcium (Rhod5− /YO-PRO-1− ); and 4) viable spermatozoa with high levels of intracellular calcium (Rhod5+ /YO-PRO-1− ). The percentages of viable spermatozoa with low intracellular calcium levels (Rhod5N− /YO-PRO-1− ) were corrected using the percentages of non-sperm debris particles found in the SYBR14− /PI− quadrant; the percentages of the other three populations were recalculated. Rhod5 spill over into the FL1-channel was compensated for (3.16%). Biology 2019, 8, 85 7 of 18 2.5.5. Intracellular Superoxide Levels (O2 − ) The evaluation of intracellular superoxide (O2 − ) radical levels was performed following a modification of the protocol from Guthrie and Welch [39] through hydroethidine (HE) and YO-PRO-1 co-staining. HE is able to penetrate the sperm plasma membrane, and it is oxidized by O2 − to ethidium (E+ ) and other products at the intracellular environment. In brief, samples were incubated with HE (final concentration: 4 µmol/L) and YO-PRO-1 (final concentration: 40 nmol/L) for 20 min. Four different sperm populations were identified in flow cytometry dot-plots: 1) non-viable spermatozoa with low superoxide levels (E− /YO-PRO-1+ ); 2) non-viable spermatozoa with high superoxide levels (E+ /YO-PRO-1+ ); 3) viable spermatozoa with low superoxide levels (E− /YO-PRO-1− ); and 4) viable spermatozoa with high superoxide levels (E+ /YO-PRO-1− ). The percentages of viable spermatozoa with low superoxide levels (E− /YO-PRO-1− ) were corrected using the percentages of non-sperm debris particles found in the SYBR14− /PI− quadrant; the percentages of the other three populations were recalculated. YO-PRO-1 spill over into the FL3-channel was compensated for (5.06%). 2.5.6. Intracellular Hydrogen Peroxide Levels (H2 O2 ) The determination of the intracellular levels of hydrogen peroxide (H2 O2 ) through co-staining with 2’,7’-dichlorodihydrofluorescein diacetate (H2 DCFDA) and PI fluorochromes was performed following the protocol from Guthrie and Welch [39] with minor modifications. H2 DCFDA is a non-fluorescent probe that penetrates the sperm plasma membrane and is intracellularly de-esterified and converted into highly fluorescent, 2’,7’-dichlorofluorescein (DCF+ ) upon oxidation. In brief, samples were incubated with H2 DCFDA (final concentration: 200 µmol/L) and PI (final concentration: 12 µmol/L) for 30 min. Four different populations were identified in flow cytometry dot-plots: 1) viable spermatozoa with high peroxide levels (DCF+ /PI− ); 2) non-viable spermatozoa with high peroxide levels (DCF+ /PI+ ); 3) viable spermatozoa with low peroxide levels (DCF− /PI− ); and 4) non-viable spermatozoa with low peroxide levels (DCF− /PI+ ). The percentages of viable spermatozoa with low peroxide levels (DCF− /PI− ) were corrected using the percentages of non-sperm debris particles found in the SYBR14− /PI− quadrant; the percentages of the other three populations were recalculated. DCF-spill over into the FL3-channel was compensated for (2.45%). 2.6. Statistical Analyses All data were analyzed using a statistical package (IBM SPSS Statistics 25.0; Armonk, New York, NY, USA). Data were first tested for normal distribution (Shapiro–Wilk test) and homogeneity of variances (Levene test). Following this, the effects of each inhibitor and cryopreservation step (i.e. fresh, frozen–thawed at 0 h, and frozen–thawed at 2 h) were tested through a mixed model followed by the post-hoc Sidak test for pair-wise comparisons. The intra-subjects factor (i.e., repeated measures) was the cryopreservation step (i.e., fresh, frozen–thawed at 0 h, frozen–thawed at 2 h), and the inter-subjects factor was the treatment (C, and different concentrations of PDO, AC or PHL). The level of significance was set at p ≤ 0.05, and data are shown as mean ± SEM. 3. Results As previously mentioned, sperm quality and function parameters were evaluated in both fresh and frozen–thawed samples in order to determine the effects of AQP inhibition during sperm cryopreservation. Regarding fresh samples, the absence of differences between controls and treated samples in any sperm parameter was due to the fact that inhibitors were added immediately before cryopreservation (p > 0.05; Table 1). Biology 2019, 8, 85 8 of 18 Table 1. Sperm quality and function parameters (mean ± SEM) in fresh stallion semen. SYBR14+ /propidium iodide (PI− ) spermatozoa, viable cells; peanut agglutinin conjugated with fluorescein isothiocyanate (PNA-FITC− )/PI− spermatozoa, cells with an intact plasma membrane and acrosome; merocyanine 540 (M540− )/YO-PRO-1− spermatozoa, live cells with low mitochondrial membrane disorder; JC1agg spermatozoa, cells with high mitochondrial membrane potential (MMP); Fluo3+ /PI− spermatozoa, live cells with high intracellular calcium levels, mainly in the sperm mid-piece; Rhod5+ /YO-PRO-1− spermatozoa, live cells with high intracellular calcium levels, mainly in the sperm head; E+ /YO-PRO-1− spermatozoa, live cells with high superoxide (O2 − ) levels; 2’,7’-dichlorofluorescein (DCF+ )/PI− spermatozoa, live cells with high peroxide (H2 O2 ) levels. Sperm Parameter Mean ± SEM % Total motile spermatozoa (TMOT) 78.4 ± 3.6 Biology 2019, 8, x FOR%PEER REVIEW motile spermatozoa (PMOT) Progressively 57.9 ± 2.4 8 of 18 % SYBR14+ /PI− spermatozoa 80.5 ± 3.2 3.1. Sperm Motility % PNA-FITC− /PI− spermatozoa 79.1 ± 3.0 % M540− /YO-PRO-1− spermatozoa 76.2 ± 3.2 The total and progressive % JC1motilities of frozen–thawed spermatozoa agg spermatozoa are 73.6 shown in Figure 1. The ± 3.1 percentages of total motile% Fluo3 + /PI− spermatozoa spermatozoa were significantly (p < 0.05) higher 14.3 ± in 0.9PDO treatments than + and 2 h/YO-PRO-1 − spermatozoa in the control at both%0 Rhod5 post-thaw (Figure PHL± concentrations 1A), whereas the two20.8 1.4 caused a % E + /YO-PRO-1 − spermatozoa 8.7 ± 0.7 significant (p < 0.05) decrease in+total motility at both post-thaw time points; the presence of AC did % DCF /PI− spermatozoa 4.5 ± 0.4 not cause any significant effect (p > 0.05). Progressive sperm motility (Figure 1B) significantly (p < 0.05) increased in the presence of both 3.1. AC Sperm and PDO Motility immediately after thawing, whereas PHL did not have any significant effect (p > 0.05). TwoThehours afterand total thawing, there were progressive no significant motilities differences spermatozoa of frozen–thawed between controlsare and samples shown treated in Figure 1. withpercentages The either AC orofthe lowest total concentration motile spermatozoa ofwere PDOsignificantly (p > 0.05). In (p < 0.05)progressive contrast, sperm higher in PDO motility treatments in theintreatments than the control containing at both 0 1andand2 10 mmol/L of (Figure h post-thaw PDO was significantly 1A), whereas the(p two < 0.05) PHLhigher than in the concentrations control.aMoreover, caused significantthe(p 0.05). (p < 0.05) lower than in the control. Figure 1. Sperm motility of samples after cryopreservation with a standard freezing medium (control) Figure 1. Sperm motility of samples after cryopreservation with a standard freezing medium (control) or with a freezing medium supplemented with acetazolamide (AC) at three different concentrations or with a freezing medium supplemented with acetazolamide (AC) at three different concentrations (250, 500, and 1000 µmol/L), with phloretin (PHL) at two different concentrations (350 and 800 µmol/L), (250, 500, and 1000 µmol/L), with phloretin (PHL) at two different concentrations (350 and 800 or with 1,3-propanediol (PDO) at three concentrations (0.1, 1, and 10 mmol/L). (A) The percentages of µmol/L), or with 1,3-propanediol (PDO) at three concentrations (0.1, 1, and 10 mmol/L). (A) The total motile spermatozoa (TMOT). (B) The percentages of progressively motile spermatozoa (PMOT). percentages of total motile spermatozoa (TMOT). (B) The percentages of progressively motile Data, shown as mean ± SEM, correspond to 0 and 2 h post-thaw. Different letters (a–d) indicate spermatozoa (PMOT). Data, shown as mean ± SEM, correspond to 0 and 2 h post-thaw. Different significant differences (p < 0.05) between treatments within a given time point. letters (a–d) indicate significant differences (p < 0.05) between treatments within a given time point. Progressive sperm motility (Figure 1B) significantly (p < 0.05) increased in the presence of both 3.2. Sperm AC and PDO Viability: SYBR14/PI immediately afterTest thawing, whereas PHL did not have any significant effect (p > 0.05). Two hours after thawing, there were no significant The percentages of viable spermatozoa (SYBR14 differences +/PI−) did between not differcontrols betweenand ACsamples treated treatments and the control immediately after thawing (0 h), but the presence of this inhibitor induced a significant (p < 0.05) decrease at 2 h post-thaw (Figure 2A). While the percentages of viable spermatozoa in PHL treatments were significantly (p < 0.05) lower than in the control at 0 and 2 h post-thaw, all PDO treatments showed significantly (p < 0.05) higher percentages of viable spermatozoa than the control. Biology 2019, 8, 85 9 of 18 with either AC or the lowest concentration of PDO (p > 0.05). In contrast, progressive sperm motility in the treatments containing 1 and 10 mmol/L of PDO was significantly (p < 0.05) higher than in the control. Moreover, the progressive sperm motility in the treatments containing PHL was significantly (p < 0.05) lower than in the control. 3.2. Sperm Viability: SYBR14/PI Test The percentages of viable spermatozoa (SYBR14+ /PI− ) did not differ between AC treatments and the control immediately after thawing (0 h), but the presence of this inhibitor induced a significant (p < 0.05) decrease at 2 h post-thaw (Figure 2A). While the percentages of viable spermatozoa in PHL treatments were significantly (p < 0.05) lower than in the control at 0 and 2 h post-thaw, all PDO treatments Biology 2019, showed REVIEW (p < 0.05) higher percentages of viable spermatozoa than the control. significantly 8, x FOR PEER 9 of 18 Figure 2. Sperm viability (SYBR14/PI) and acrosome integrity (PNA-FITC/PI) after cryopreservation Figure 2. Sperm viability (SYBR14/PI) and acrosome integrity (PNA-FITC/PI) after cryopreservation with a standard freezing medium (control) or with a freezing medium supplemented with acetazolamide with a standard freezing medium (control) or with a freezing medium supplemented with (AC) at three different concentrations (250, 500 and 1000 µmol/L), with phloretin (PHL) at two different acetazolamide (AC) at three different concentrations (250, 500 and 1000 µmol/L), with phloretin (PHL) concentrations (350 and 800 µmol/L), or with 1,3-propanediol (PDO) at three concentrations (0.1, 1atand two different concentrations 10 mmol/L). (350 of (A) The percentages and 800 spermatozoa viable µmol/L), or (SYBR-14 with 1,3-propanediol (PDO) (B) + /PI− spermatozoa). at three The concentrations (0.1, 1 and 10 mmol/L). (A) The percentages of viable spermatozoa percentage of viable spermatozoa with an intact acrosome (PNA-FITC /PI spermatozoa). Data, shown − − (SYBR-14 +/PI− asspermatozoa). mean ± SEM, (B) The percentage correspond to 0 and 2ofh post-thaw. viable spermatozoa with an Different letters intact (a–d) acrosome indicate (PNA-FITC significant −/PI− differences (pspermatozoa). Data, < 0.05) between shown aswithin treatments mean a± given SEM, correspond time point. to 0 and 2 h post-thaw. Different letters (a–d) indicate significant differences (p < 0.05) between treatments within a given time point. 3.3. Acrosome Integrity: PNA-FITC/PI Test 3.3. Acrosome Integrity: PNA-FITC/PI Test The PNA-FITC/PI test was carried out to determine the integrity of both acrosome and plasma The PNA-FITC/PI membranes, and PNA-FITC − /PI−carried test was out to determine spermatozoa were thosethe integrity that had anof bothacrosome intact acrosomeand andplasma plasma membranes, membrane and PNA-FITC (Figure 2B). Samples−/PItreated − spermatozoa with ACwere those did not showthat had an intact significant acrosome differences to theand plasma control at membrane any (Figure concentration 2B). Samples immediately treated after with thawing > 0.05), (p AC did but notthere showwas significant (p < 0.05) differences a significant todecrease the control in at any the concentration percentage immediately of spermatozoa after thawing that presented (p plasma an intact > 0.05),and butacrosome there was a significant membrane in the(psamples < 0.05) decrease treated in the lowest with percentage of spermatozoa concentrations of AC that presented at 2 h post-thaw. anThe intact plasma and percentages acrosome membrane of spermatozoa with an in theplasma intact samples andtreated withmembrane acrosome the lowestwereconcentrations < 0.05) significantlyof(pAC at 2 lower h post-thaw. in PHL The percentages treatments of than in spermatozoa the with0 an control at both andintact plasma and 2 h post-thaw. acrosome In contrast, allmembrane treatments were significantly containing (p < 0.05) PDO showed lower in significantly < 0.05) (pPHL treatments than in the control higher percentages at both 0with of spermatozoa and 2anhintact post-thaw. plasma Inand contrast, all treatments acrosome membranecontaining at both 0 PDO and 2 hshowed significantly (p < 0.05) higher percentages of spermatozoa with an intact plasma and post-thaw. acrosome membrane at both 0 and 2 h post-thaw. 3.4. Membrane Lipid Disorder: M540/YO-PRO-1 Test 3.4. Membrane Lipid Disorder:test The M540/YO-PRO-1 M540/YO-PRO-1 allowed for theTestevaluation of sperm membrane lipid disorder, the population of M540 /YO-PRO-1 The M540/YO-PRO-1 − − test allowedspermatozoa corresponding for the evaluation of spermtomembrane those viable cells lipid with low disorder, the membrane lipid disorder (Figure 3A). While right after thawing, samples treated with population of M540 /YO-PRO-1 spermatozoa corresponding to those viable cells with low membrane - - 1000 µmol/L AC showed significantly lipid disorder < 0.05) (Figure(p3A). While lower rightpercentages of viable after thawing, spermatozoa samples treated withwith 1000low membrane µmol/L lipid AC showed significantly (p < 0.05) lower percentages of viable spermatozoa with low membrane lipid disorder than the control, the treatments containing 250 and 500 µmol/L AC also showed reduced (p < 0.05) percentages of viable spermatozoa with low membrane lipid disorder than the control at 2 h post- thaw. The percentages of viable spermatozoa with low membrane lipid disorder at the highest PHL concentration were significantly (p < 0.05) lower than the control immediately after thawing. At 2 h Biology 2019, 8, 85 10 of 18 disorder than the control, the treatments containing 250 and 500 µmol/L AC also showed reduced (p < 0.05) percentages of viable spermatozoa with low membrane lipid disorder than the control at 2 h post-thaw. The percentages of viable spermatozoa with low membrane lipid disorder at the highest PHL concentration were significantly (p < 0.05) lower than the control immediately after thawing. At 2 h post-thaw, both 350 and 800 µmol/L concentrations showed significantly (p < 0.05) lower percentages of viable spermatozoa with low membrane lipid disorder than the control. Finally, the percentages of viable spermatozoa with low membrane lipid disorder were significantly (p < 0.05) higher than8,the Biology 2019, control x FOR in all PDO treatments, both at 0 and 2 h post-thaw. PEER REVIEW 10 of 18 Figure 3. Sperm membrane lipid disorder (M540/YO-PRO-1) and mitochondrial membrane potential Figure 3. Sperm membrane lipid disorder (M540/YO-PRO-1) and mitochondrial membrane potential (JC1) after cryopreservation with a standard freezing medium (control) or with a freezing medium (JC1) after cryopreservation with a standard freezing medium (control) or with a freezing medium supplemented with acetazolamide (AC) at three different concentrations (250, 500 and 1000 µmol/L), supplemented with acetazolamide (AC) at three different concentrations (250, 500 and 1000 µmol/L), with phloretin (PHL) at two different concentrations (350 and 800 µmol/L), or with 1,3-propanediol with phloretin (PHL) at two different concentrations (350 and 800 µmol/L), or with 1,3-propanediol (PDO) at three concentrations (0.1, 1 and 10 mmol/L). (A) The percentages of viable spermatozoa with (PDO) at three concentrations (0.1, 1 and 10 mmol/L). (A) The percentages of viable spermatozoa with low membrane lipid disorder (M540−−/YO-PRO-1−− spermatozoa). (B) The percentages of spermatozoa low membrane lipid disorder (M540 /YO-PRO-1 spermatozoa). (B) The percentages of spermatozoa with high mitochondrial membrane potential (JC1agg spermatozoa). Data, shown as mean ± SEM, with high mitochondrial membrane potential (JC1agg spermatozoa). Data, shown as mean ± SEM, correspond to 0 and 2 h post-thaw. Different letters (a–e) indicate significant differences (p < 0.05) correspond to 0 and 2 h post-thaw. Different letters (a–e) indicate significant differences (p < 0.05) between treatments within a given time point. between treatments within a given time point. 3.5. Mitochondrial Membrane Potential (MMP): JC1 Test 3.5. Mitochondrial Membrane Potential (MMP): JC1 Test The percentages of spermatozoa with high MMP were significantly (p < 0.05) lower in the treatment The percentages containing AC at 1000 of spermatozoa µmol/L withcontrol than in the high MMP were significantly immediately after thawing (p and < 0.05) lower in the in the treatment treatment containing AC at 1000 µmol/L than in the control immediately containing AC at 250 µmol/L at 2 h post-thaw (Figure 3B). When treated with PHL, samples showed after thawing and in the a treatment containing AC at 250 µmol/L at 2 h post-thaw (Figure 3B). When treated decrease in MMP at any time point after thaw (p < 0.05). In contrast, the percentages of spermatozoa with PHL, samples showed with highaMMPdecrease intreatment in the MMP at any time point containing after thaw 1 mmol/L (p < 0.05). PDO were In contrast, significantly the percentages (p < 0.05) higher than in of spermatozoa with high MMP the control both at 0 and 2 h post-thaw. in the treatment containing 1 mmol/L PDO were significantly (p < 0.05) higher than in the control both at 0 and 2 h post-thaw. 3.6. Intracellular Calcium of Spermatozoa: Fluo3/PI Test 3.6. Intracellular Calcium of Spermatozoa: Fluo3/PI Test Intracellular calcium levels were assessed through the Fluo3/PI test, and the population of + /PI− spermatozoa Fluo3Intracellular calcium levels weretoassessed corresponded through viable cells the intracellular with high Fluo3/PI test, and the calcium population levels (Figure 4A).of Fluo3+/PI While, − spermatozoa corresponded to viable cells with high intracellular calcium levels (Figure compared to the control, samples treated with any concentration of AC showed a significant (p < 0.05) 4A). While, compared decrease in thetopercentage the control, samples of viable treated with spermatozoa withany highconcentration of AC showed levels of intracellular calciuma significant (pafter immediately < 0.05) decrease thawing, noin significant (p > 0.05) the percentage of viable spermatozoa differences betweenwith high levelsand AC treatments of intracellular the control calcium immediately after thawing, no significant (p > 0.05) differences between were found after 2 h of thawing. Treatments containing any concentration of PHL showed significantly AC treatments and the< control (p werepercentages 0.05) lower found after of 2 hspermatozoa of thawing. Treatments containing with high levels any concentration of intracellular calcium atofboth PHL0 showed and 2 h significantly post-thaw. In (p < 0.05) the contrast, lower percentages percentages of spermatozoa of viable spermatozoa with high with levels high of of levels intracellular intracellularcalcium calciumat were significantly (p < 0.05) higher in all PDO concentrations than in the control right after thawing both 0 and 2 h post-thaw. In contrast, the percentages of viable spermatozoa with high levels of intracellular calcium were significantly (p < 0.05) higher in all PDO concentrations than in the control right after thawing (0 h) and were significantly (p < 0.05) higher than the control at the two highest PDO concentrations after 2 h of thawing. Biology 2019, 8, 85 11 of 18 (0 h) and were significantly (p < 0.05) higher than the control at the two highest PDO concentrations Biology2 2019, after h of 8,thawing. x FOR PEER REVIEW 11 of 18 Figure 4. Intracellular calcium levels (Fluo3/PI and Rhod5/YO-PRO-1) after cryopreservation with a Figure 4. Intracellular calcium levels (Fluo3/PI and Rhod5/YO-PRO-1) after cryopreservation with a standard freezing medium (control) or with a freezing medium supplemented with acetazolamide standard freezing medium (control) or with a freezing medium supplemented with acetazolamide (AC) at three different concentrations (250, 500 and 1000 µmol/L), with phloretin (PHL) at two different (AC) at three different concentrations (250, 500 and 1000 µmol/L), with phloretin (PHL) at two concentrations (350 and 800 µmol/L), or with 1,3-propanediol (PDO) at three concentrations (0.1, 1 and different concentrations (350 and 800 µmol/L), or with 1,3-propanediol (PDO) at three concentrations 10 mmol/L). (A) The percentages of viable spermatozoa with high levels of intracellular calcium levels (0.1, 1 and 10 mmol/L). (A) The percentages of viable spermatozoa with high levels of intracellular (Fluo3+ /PI− spermatozoa). (B) The percentages of viable spermatozoa with high levels of intracellular calcium levels (Fluo3++/PI− spermatozoa). (B) The percentages of viable spermatozoa with high levels calcium levels (Rhod5 /YO-PRO-1− spermatozoa). Data, shown as mean ± SEM, correspond to 0 and of intracellular calcium levels (Rhod5+/YO-PRO-1− spermatozoa). Data, shown as mean ± SEM, 2 h post-thaw. Different letters (a–d) indicate significant differences (p < 0.05) between treatments correspond to 0 and 2 h post-thaw. Different letters (a–d) indicate significant differences (p < 0.05) within a given time point. between treatments within a given time point. 3.7. Intracellular Calcium of Spermatozoa: Rhod5/YO-PRO-1 Test 3.7. Intracellular Calcium of Spermatozoa: Rhod5/YO-PRO-1 Test The Rhod5/YO-PRO-1 test was also performed to determine intracellular calcium levels, and the population of Rhod5+ /YO-PRO-1 The Rhod5/YO-PRO-1 test was also performed − spermatozoa to determine corresponded tointracellular calcium viable cells with highlevels, and the intracellular population of Rhod5 +/YO-PRO-1− spermatozoa corresponded to viable cells with high intracellular calcium levels (Figure 4B). When AC was present, no significant (p > 0.05) differences with regard calcium to levelswere the control (Figure 4B). When observed ACatwas either 0 orpresent, no significant 2 h post-thaw. (p > 0.05) differences The treatments containingwithPHLregard showed to the control were observed either at 0 or 2 h post-thaw. The treatments containing no significant differences compared to the control at 0 h post-thaw, but those containing the lowest PHL showed no significant PHL differences concentration compared showed to the control significantly at 0lower (p > 0.05) h post-thaw, but those percentages containing of viable the lowest spermatozoa PHL with high concentration showed significantly (p > 0.05) lower percentages of viable spermatozoa intracellular calcium levels after 2 h of thawing. In contrast, the percentages of viable spermatozoa with high intracellular calcium levels after 2 h of thawing. In contrast, the percentages of viable with high intracellular calcium levels were significantly (p < 0.05) higher in PDO treatments than in the spermatozoa with high control at 0intracellular h but not at calcium levels were significantly (p < 0.05) higher in PDO treatments than in 2 h post-thaw. the control at 0 h but not at 2 h post-thaw. 3.8. Intracellular Superoxide Levels (O2 − ): HE/YO-PRO-1 Test 3.8. Intracellular Superoxide Levels (O2−): HE/YO-PRO-1 Test Intracellular O2 − levels were evaluated through the HE/YO-PRO-1 test, in which the population of E+Intracellular /YO-PRO-1−Ospermatozoa 2− levels werecorresponded evaluated through the HE/YO-PRO-1 to viable cells with hightest, in which the intracellular of O2 − . population levels of Epercentages The +/YO-PRO-1−of spermatozoa corresponded viable spermatozoa to viable with high cells with intracellular highof levels intracellular differof(pO>2−.0.05) O2 − did notlevels The percentages between AC of viable spermatozoa treatments with either and the control, high intracellular at 0 or at 2 levels of O2 (Figure h post-thaw − did not 5A). differIn(pcontrast, > 0.05) between the AC treatments treatment containing and the control, 800 µmol/L PHL either showed at significantly (p < 0.05) (Figure 0 or at 2 h post-thaw 5A). In contrast, lower percentages the of viable treatment containing spermatozoa with high800 µmol/L PHL intracellular showed levels − of O2 significantly both at 0 and(p2