biology Article Exogenous Albumin Is Crucial for Pig Sperm to Elicit In Vitro Capacitation Whereas Bicarbonate Only Modulates Its Efficiency Bruna Resende Chaves 1,2,3 , Ana Paula Pinoti Pavaneli 1,4 , Olga Blanco-Prieto 5 , Elisabeth Pinart 1,2 , Sergi Bonet 1,2 , Márcio Gilberto Zangeronimo 3 , Joan E. Rodríguez-Gil 5, *,† and Marc Yeste 1,2, *,† 1 Biotechnology of Animal and Human Reproduction (TechnoSperm), Institute of Food and Agricultural Technology, University of Girona, ES-17003 Girona, Spain; brunarufla@gmail.com (B.R.C.); anap.pavaneli@gmail.com (A.P.P.P.); elisabeth.pinart@udg.edu (E.P.); sergi.bonet@udg.edu (S.B.) 2 Unit of Cell Biology, Department of Biology, Faculty of Sciences, University of Girona, ES-17003 Girona, Spain 3 Department of Veterinary Medicine, Federal University of Lavras, BR-37200-000 Lavras, MG, Brazil; zangeronimo@ufla.br 4 Laboratory of Andrology and Technology of Swine Embryos, Department of Animal Reproduction, School of Veterinary Medicine and Animal Science, University of São Paulo, BR-13635-900 Pirassununga, SP, Brazil 5 Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, ES-08193 Bellaterra (Cerdanyola del Vallès), Spain;  obprieto@gmail.com  * Correspondence: juanenrique.rodriguez@uab.cat (J.E.R.-G.); marc.yeste@udg.edu (M.Y.); Tel.: +34-93-5811045 (J.E.R.-G.); +34-972-419514 (M.Y.) Citation: Chaves, B.R.; Pinoti † Joint senior authors. Pavaneli, A.P.; Blanco-Prieto, O.; Pinart, E.; Bonet, S.; Zangeronimo, M.G.; Rodríguez-Gil, J.E.; Yeste, M. Simple Summary: In this work, we addressed if the presence of exogenous bicarbonate required for Exogenous Albumin Is Crucial for Pig pig sperm capacitation, which is a necessary step to acquire fertilizing ability. While sperm incubated Sperm to Elicit In Vitro Capacitation in media without BSA or BSA/bicarbonate did not achieve in vitro capacitation, those incubated with Whereas Bicarbonate Only Modulates BSA reached that status under any bicarbonate concentration, even when bicarbonate was absent. Its Efficiency. Biology 2021, 10, 1105. Interestingly, there were differences related to the concentration of bicarbonate, since sperm incubated https://doi.org/10.3390/ in media with BSA and with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in biology10111105 achieving in vitro capacitation than those incubated in the presence of BSA and higher concentration of bicarbonate. Additionally, at the end of the experiment, sperm incubated in the presence of BSA Academic Editors: Pilar Santolaria and 38 mM bicarbonate showed lower motility and plasma membrane integrity than those incubated Blasco, Rosaura Pérez-Pé, Jessica in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is crucial in for Rickard and Paul S. Cooke pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome Received: 25 August 2021 exocytosis. In contrast, although exogenous bicarbonate does not appear to be indispensable, it Accepted: 22 October 2021 shortens the time needed to reach that capacitated status. Published: 26 October 2021 Abstract: This work sought to address whether the presence of exogenous bicarbonate is required for Publisher’s Note: MDPI stays neutral pig sperm to elicit in vitro capacitation and further progesterone-induced acrosome exocytosis. For with regard to jurisdictional claims in this purpose, sperm were either incubated in a standard in vitro capacitation medium or a similar published maps and institutional affil- medium with different concentrations of bicarbonate (either 0 mM, 5 mM, 15 mM or 38 mM) and BSA iations. (either 0 mg/mL or 5 mg/mL). The achievement of in vitro capacitation and progesterone-induced acrosomal exocytosis was tested through the analysis of sperm motility, plasma membrane integrity and lipid disorder, acrosome exocytosis, intracellular calcium levels, mitochondria membrane poten- tial, O2 consumption rate and the activities of both glycogen synthase kinase 3 alpha (GSK3α) and Copyright: © 2021 by the authors. protein kinase A (PKA). While sperm incubated in media without BSA or BSA/bicarbonate, they did Licensee MDPI, Basel, Switzerland. not achieve in vitro capacitation; those incubated in media with BSA achieved the capacitated status This article is an open access article under any bicarbonate concentration, even when bicarbonate was absent. Moreover, there were distributed under the terms and differences related to the concentration of bicarbonate, since sperm incubated in media with BSA and conditions of the Creative Commons with no bicarbonate or 5 mM bicarbonate showed lower overall efficiency in achieving in vitro capac- Attribution (CC BY) license (https:// itation than those incubated in the presence of BSA and 15 mM or 38 mM bicarbonate. Additionally, creativecommons.org/licenses/by/ at the end of the experiment, sperm incubated in the presence of BSA and 38 mM bicarbonate showed 4.0/). Biology 2021, 10, 1105. https://doi.org/10.3390/biology10111105 https://www.mdpi.com/journal/biology Biology 2021, 10, 1105 2 of 24 significantly (p < 0.05) lower values of motility and plasma membrane integrity than those incubated in media with BSA and lower concentrations of bicarbonate. In conclusion, BSA is instrumental for pig sperm to elicit in vitro capacitation and trigger the subsequent progesterone-induced acrosome exocytosis. Furthermore, while exogenous bicarbonate does not seem to be essential to launch sperm capacitation, it does modulate its efficiency. Keywords: sperm capacitation; pig; bicarbonate; bovine serum albumin (BSA); glycogen synthase kinase (GSK3); protein kinase A (PKA) 1. Introduction Mammalian sperm are unable to fertilize oocytes upon ejaculation because, despite being mature and motile, they need to reside within the female reproductive tract and interact with that environment [1]. Within the female reproductive tract, sperm undergo capacitation, which was first described in the 1950s [2,3]. Following this, sperm are able to interact with oocyte vestments, trigger acrosome exocytosis and subsequently fuse with the oocyte plasma membrane (reviewed in [4]). The capacitated spermatozoon undergoes a series of functional changes that include, amongst others, modifications in the composition of the sperm plasma membrane, acrosome remodeling, an increase in mitochondrial activity and sperm motility, a noticeable rise in intracellular Ca2+ and ROS levels, and the tyrosine-phosphorylation of certain sperm proteins [5–10]. At present, sperm capacitation can be performed in vitro in several (including pigs), although not all, mammalian species. Because effective in vitro sperm capacitation is required for successful in vitro fertilization (IVF), the main attempts to establish a proper capacitation medium have been related to the development of this assisted reproductive technique. Most previous studies have aimed to set a medium mimicking the oviductal environment, where the capacitation status is fully achieved (reviewed in [11]). It is widely accepted that a capacitation medium should contain a protein source, which is usually bovine serum albumin (BSA), and a variety of ions, including bicarbonate (HCO3 − ) and Ca2+ [5,12,13]. During capacitation, removal of cholesterol from sperm plasma membrane is induced by BSA, which leads to the displacement and rearrangement of phospholipids, increases membrane fluidity, primes acrosome exocytosis and prepares sperm for the penetration of oocyte vestments (including zona pellucida) prior to gamete fusion [14–17]. Furthermore, high levels of bicarbonate destabilize the sperm plasma membrane and trigger a signaling cascade that involves a sperm-specific soluble adenylyl cyclase (sAC), the increase in cAMP levels and the activation of protein kinase A (PKA; [18]). The activation of PKA is one of the most important signals that elicits sperm capacitation and is also involved in triggering acrosome exocytosis [1,19,20]. Additionally, the concentration of bicarbonate changes across epididymal regions and in the oviductal fluid [19]. These variations play a crucial role in the modulation of sperm function, as they are involved in events such as sperm maturation during the epididymal transit, the acquisition of motility upon ejaculation and the modulation of capacitation timing within the female reproductive tract [19]. After ejaculation, sperm encounter a high bicarbonate concentration in the female tract, ranging between 35 mM and 90 mM [5,21]. In vitro, these variations in the concentration of bicarbonate do not exist, but rather sperm are exposed to a standard concentration throughout all the process [22], this scenario likely being one of the limiting factors in the efficiency of in vitro capacitation. Previous research used different concentrations of bicarbonate and BSA to elicit in vitro capacitation in pig sperm. Whereas several studies used a medium contain- ing 25 mM NaHCO3 and 0.4% BSA [23], others used 36–38 mM NaHCO3 and 0.5% of BSA [9,24–28]. In addition, other authors even found that a medium with 5 mg/mL BSA but without NaHCO3 is able to induce in vitro capacitation in pig sperm [29,30]. These differences clearly indicate that while both BSA and NaHCO3 are important in the achieve- Biology 2021, 10, 1105 3 of 24 ment of in vitro capacitation in pig sperm, neither the precise role of these two effectors nor their particular relevance in the achievement of sperm capacitation are well known. Since, to the best of our knowledge, no previous study interrogated the specific importance of both exogenous bicarbonate and BSA during the achievement of in vitro capacitation, this work aimed to explain to which extent these two components are crucial for pig sperm to elicit in vitro capacitation and undergo progesterone-induced acrosome exocytosis. 2. Materials and Methods 2.1. Reagents Unless otherwise declared, all the reagents were purchased from Sigma–Aldrich (Saint-Louis, MO, USA) and Merck (Darmstadt, Germany). 2.2. Semen Samples Semen samples were obtained from 12 separate boars of proven fertility according to the farm records. Ejaculates were purchased from a local farm (Servicios Geneticos Porcinos, S.L., Roda de Ter, Spain), where boar semen was routinely collected twice a week through the gloved-hand method. Sperm-rich fractions were diluted in a commercial extender (Duragen® ; Magapor, S.L.; Ejea de los Caballeros, Spain) to a final concentration of 3.3 × 107 sperm/mL. Samples were cooled to 17 ◦ C, packed into 90 mL doses and finally transported in an insulated container at 17 ◦ C for approximately 45 min, which was the time required to arrive at the laboratory. Upon arrival, sperm quality was evaluated to confirm that conventional spermiogram parameters were above quality standards. All procedures involving animals were performed according to the EU Directive 2010/63/EU for animal experiments, the Animal Welfare Law issued by the Regional Government of Catalonia, and the current regulation on Health and Biosafety issued by the Department of Agriculture, Livestock, Food and Fisheries, Regional Government of Catalonia (Spain). In addition, the production of seminal doses by the farm followed the ISO certification (ISO-9001:2008). The authors of this study did not manipulate any animal but rather purchased the semen samples from a local farm, which operates under commercial standard conditions. For this reason, no approval from a local ethics committee was required. 2.3. In Vitro Capacitation and Progesterone-Induced Acrosome Exocytosis All media were freshly prepared on the day of use and consisted of Tyrode’s medium containing lactate and pyruvate, and different concentrations of bicarbonate and BSA. The composition of this basic medium was: 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 112 mM NaCl, 3.1 mM KCl, 5 mM glucose, 0.3 mM Na2 HPO4 , 0.4 mM MgSO4 , 4.5 mM CaCl2 , 21.7 mM sodium lactate and 1 mM sodium pyruvate. Eight different treatments were tested: (1) negative control (No BSA/No Bic), which was the basic medium without bicarbonate or BSA; (2) basic medium supplemented with 5 mg/mL BSA (BSA/No Bic); (3) basic medium supplemented with 5 mg/mL BSA and 5 mM NaHCO3 (BSA + 5 mM Bic); (4) basic medium supplemented with 5 mM NaHCO3 (No BSA + 5 mM Bic); (5) basic medium supplemented with 5 mg/mL BSA and 15 mM NaHCO3 (BSA + 15 mM Bic); (6) basic medium supplemented with 15 mM NaHCO3 (No BSA + 15 mM Bic); (7) basic medium supplemented with 5 mg/mL BSA and 38 mM NaHCO3 (BSA + 38 mM Bic); and (8) basic medium supplemented with 38 mM NaHCO3 (No BSA + 38 mM Bic). In all cases, pH was previously adjusted to 7.4 and media were equilibrated at 38.5 ◦ C and 5% CO2 for 60 min before use. Seminal doses were separated from the diluent through centrifugation at 600× g and 17 ◦ C for 10 min, washed with PBS, and resuspended with the previously described media to a final concentration of 2 × 107 sperm/mL. Sperm were then incubated at 38.5 ◦ C and 5% CO2 in humidified air and evaluated after 0 min, 120 min and 240 min of incubation, since 240 min has been reported to be enough time for pig sperm to become in vitro capacitated [16,30]. After 240 min of incubation, progesterone was added to a final Biology 2021, 10, 1105 4 of 24 concentration of 10 µg/mL to induce acrosome exocytosis [31,32]. In order to evaluate the response of capacitated sperm to progesterone, especially with regard to acrosome exocytosis, samples were further incubated under the same conditions and evaluated after 5 min, 30 min and 60 min of progesterone addition. At each relevant time point, separate aliquots were taken for the assessment of sperm motility, plasma membrane integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential and O2 consumption. Another aliquot was also taken for protein extraction and analysis. In this latter case, aliquots were centrifuged at 2400× g and 17 ◦ C for 5 min and supernatants were discarded. Pellets were then stored at −80 ◦ C until protein extraction. 2.4. Sperm Motility Evaluation of sperm motility was performed using a CASA system (Integrated Sperm Analysis System V1.0; Proiser, Valencia, Spain). Briefly, 5 µL of each sample was placed onto a Makler counting chamber (Sefi-Medical Instruments). Samples were observed under a negative phase-contrast microscope (Olympus BX41; Olympus, Hamburg, Germany) at 100× magnification. Three replicates with a minimum of 1000 sperm per replicate were evaluated before calculating the corresponding mean ± SEM. The recorded sperm motility parameters were those described in Yeste et al. [33], namely: curvilinear velocity (VCL), which is the mean path velocity of the sperm head along its actual trajectory (µm/s); straight-line velocity (VSL), which is the mean path velocity of the sperm head along a straight line from its first to its last position (µm/s); average path velocity (VAP), which is the mean velocity of the sperm head along its average trajectory (µm/s); linearity coefficient (LIN), (VSL/VCL) × 100 (%); straightness coefficient (STR): (VSL/VAP) × 100 (%); wobble coefficient (WOB): (VAP/VCL) × 100 (%); mean amplitude of lateral head displacement (ALH), which is the mean value of the extreme side-to-side movement of the sperm head in each beat cycle (µm); and frequency of head displacement (BCF), which is the frequency with which the actual sperm trajectory crosses the average path trajectory (Hz). Total motility was defined as the percentage of sperm showing VAP > 10 µm/s, and progressive motility as the percentage of sperm exhibiting STR > 45%. 2.5. Flow Cytometry Analyses Information about flow cytometry analyses is provided according to the recommen- dations of the International Society for Advancement of Cytometry (ISAC; [34]). Flow cytometry was used to evaluate plasma membrane integrity, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, and intracellular calcium levels in all samples and time-points. Prior to evaluation, sperm concentration was adjusted to 1 × 106 sperm/mL in a final volume of 0.5 mL [35]. Sperm were then stained with the appropriate combinations of fluorochromes, following the protocols described below. In all cases, a minimum of 10,000 events per replicate were evaluated through a Cell Lab QuantaSC Flow Cytometer (Beckman Coulter, Fullerton, CA, USA). Particles were excited through an argon ion laser (488 nm) set at a power of 22 mW and cell diameter/volume was directly measured employing the Coulter principle for volume assessment. The EV channel was periodically calibrated using 10-µm Flow-Check fluorospheres (Beckman Coulter) by positioning this size of the bead at channel 200 on the volume scale. To capture fluorochrome signals, three different optical filters were used: FL1 (green fluorescence)— Dichroic/Splitter, DRLP: 550 nm, BP filter: 525 nm, detection width 505–545 nm; FL2 (orange fluorescence)—DRLP: 600 nm, BP filter: 575 nm, detection width: 560–590 nm; FL3 (red fluorescence)—LP filter: 670 nm, detection width: 655–685 nm. Debris (particle diameter < 7 µm) and aggregates (particle diameter > 12 µm) were excluded from the analysis by gating out the particles based on EV/side scatter (SSC) plots. In all tests except SYBR14/PI, data were corrected following the procedure described by Petrunkina et al. [36]. Each assessment per sample and parameter was repeated three times in independent tubes and the corresponding mean ± SEM was calculated. Biology 2021, 10, 1105 5 of 24 2.5.1. Evaluation of Sperm Membrane Integrity Plasma membrane integrity was evaluated using the LIVE/DEAD Sperm Viability Kit (SYBR14/PI) following the protocol set by Garner and Johnson [37]. For this purpose, sperm samples were incubated with SYBR14 (final concentration: 100 nM) at 38 ◦ C in darkness for 10 min and then with propidium iodide (PI; final concentration: 10 µM) at the same temperature for 5 min. Three sperm populations were identified in flow-cytometry dot plots: (i) membrane-intact sperm (green-stained), which were positive for SYBR14 and negative for PI (SYBR14+ /PI− ); (ii) non-viable sperm (red-stained), which were negative for SYBR14 and positive for PI (SYBR14− /PI+ ); and (iii) non-viable sperm (orange-stained), which were stained both in green and red (SYBR14+ /PI+ ). 2.5.2. Evaluation of Sperm Membrane Lipid Disorder Lipid disorder of plasma membrane was determined with Merocyanine 540 (M540) and YO-PRO-1, following the procedure set by Harrison et al. [38]. Briefly, sperm were stained with M540 (final concentration: 2.6 µM) and YO-PRO-1 (final concentration: 25 nM) and incubated at 38 ◦ C for 10 min in the dark. A total of four sperm populations were identified: (i) non-viable sperm with low membrane lipid disorder (M540− /YO-PRO-1+ ); (ii) non-viable sperm with high membrane lipid disorder (M540+ /YO-PRO-1+ ); (iii) viable sperm with low membrane lipid disorder (M540− /YO-PRO-1− ); and (iv) viable sperm with high membrane lipid disorder (M540+ /YO-PRO-1− ). 2.5.3. Evaluation of Acrosome Integrity Acrosome integrity was determined through staining with the lectin from Arachis hypogaea (peanut agglutinin, PNA) conjugated with fluorescein isothiocyanate (FITC) and ethidium homodimer (3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide; EthD-1), following the protocol described by Rocco et al. [9]. Samples were incubated with EthD-1 (final concentration: 2.5 µg/mL) at 38 ◦ C for 5 min in the dark. Following centrifugation at 2000× g and 17 ◦ C for 30 s, sperm were resuspended with PBS containing 4 mg/mL BSA. Samples were again centrifuged under the same conditions and subsequently added to 100 µL of ice-cold methanol (100%) for 30 s. After centrifugation at 2000× g and 17 ◦ C for 30 s, samples were resuspended with 250 µL PBS before adding 0.8 µL PNA- FITC (final concentration: 2.5 µM). Samples were incubated at 25 ◦ C for 15 min in the dark and then centrifuged at 2000× g and 17 ◦ C for 30 s. Pellets were resuspended with 0.6 µL PBS and centrifuged at 2000× g and 17 ◦ C for 30 s. This step was repeated twice. Sperm were evaluated with the flow cytometer and four sperm populations were identified: (i) viable sperm with an intact acrosome (PNA+ /EthD-1− ); (ii) viable sperm with an exocytosed acrosome (PNA− /EthD-1− ); (iii) non-viable sperm with an intact acrosome (PNA+ /EthD-1+ ); and (iv) non-viable sperm with an exocytosed acrosome (PNA− /EthD-1+ ). Fluorescence of EthD-1 was detected through FL3, whereas that of PNA was detected through FL1. 2.5.4. Evaluation of Intracellular Calcium Levels Intracellular calcium levels were evaluated through two different fluorochromes (Fluo3 and Rhod5; [7]). For Fluo3, sperm were incubated at 38 ◦ C in the dark for 10 min with Fluo3-AM (final concentration: 1 µM) and PI (final concentration: 12 µM), which were detected through FL1 and FL3, respectively. Four sperm populations were identified: (i) viable sperm with low levels of intracellular calcium (Fluo3− /PI− ), (ii) viable sperm with high levels of intracellular calcium (Fluo3+ /PI− ), (iii) non-viable sperm with low levels of intracellular calcium (Fluo3− /PI+ ), and (iv) non-viable sperm with high levels of intracellular calcium (Fluo3+ /PI+ ). The geometric mean of Fluo3 intensity was recorded for all sperm populations. With regard to Rhod5, sperm were incubated at 38 ◦ C for 10 min in the dark with Rhod5-N (final concentration: 5 µM) and YO-PRO-1 (final concentration: 25 nM). Filters used to detect the fluorescence from Rhod5 and YO-PRO-1 were FL3 and FL1, respectively. Biology 2021, 10, 1105 6 of 24 A total of four sperm populations were identified: (i) viable sperm with low levels of intracellular calcium (Rhod5− /YO-PRO-1− ), (ii) viable sperm with high levels of intracel- lular calcium (Rhod5+ /YO-PRO-1− ), (iii) non-viable sperm with low levels of intracellular calcium (Rhod5− /YO-PRO-1+ ), and (iv) non-viable sperm with high levels of intracellular calcium (Rhod5+ /YO-PRO-1+ ). The geometric mean of Rhod5 intensity was recorded for all sperm populations. 2.5.5. Evaluation of Mitochondrial Membrane Potential Mitochondrial membrane potential (MMP) was determined through JC1 (5,50 ,6,60 - tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) fluorochrome, following the protocol described by Guthrie and Welch [39]. For this purpose, sperm samples were incubated with JC1 (final concentration: 0.3 µM) at 38 ◦ C in the dark for 30 min, and two sperm populations were distinguished: (i) sperm with high mitochondrial membrane potential, and (ii) sperm with low mitochondrial membrane potential. When MMP was high, JC1 inside mitochondria formed orange aggregates that were detected through FL2. Ratios between orange (FL2) and green (FL1) fluorescence in the sperm population with high MMP (JC1agg ) were also calculated. 2.6. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS–PAGE) Sperm pellets stored at −80 ◦ C were thawed and resuspended in 400 µL ice-cold lysis buffer and maintained at 4 ◦ C for 30 min under constant agitation. The lysis buffer was made up of 2% SDS, 1% Triton-X-100, 8 M urea, 2 mM dithiothreitol (DDT), 0.5% Tween 20 and 50 mM Tris-HCl; the pH was adjusted to 7.4. On the day of use, the lysis buffer was mixed with 1% commercial protease inhibitor cocktail (Sigma–Aldrich), 1% phenylmethanesulfonyl fluoride (PMSF), and 0.15% sodium orthovanadate. Following this, samples were homogenized by sonication (50% amplitude; 10 long-lasting pulses; Bandelin Sonopuls HD 2070; Bandelin Electronic GmbH and Co., Heinrichstrasse, Berlin), and then centrifuged at 10,000× g and 4 ◦ C for 15 min. Supernatants were carefully collected and total protein was quantified in triplicate with a detergent-compatible protein assay (DC Protein Assay; BioRad, Hercules, CA, USA). Standard curves were produced with different concentrations of BSA (Quick Start Bovine Serum Albumin Standard; Bio-Rad). For SDS–PAGE separation, 10 µg protein of each sample was diluted with 2× Laemmli reducing buffer containing 5% (v:v) β-mercaptoethanol (Bio-Rad Laboratories). Samples were boiled at 90 ◦ C for 5 min and then loaded onto 1-mm SDS–PAGE gels, together with a molecular-weight marker (All Blue Precision Plus Protein Standards; Bio-Rad Laboratories). The separating gel contained 12% (w:v) acrylamide, whereas the stacking gel contained 5% (w:v) (Bio-Rad Laboratories). Subsequently, gel electrophoresis was run at constant voltage (80 V for stacking gels and 120 V for separating gels). Proteins were transferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore, Darmstadt, Germany) at 120 mA for 120 min. Thereafter, membranes were incubated with blocking solution, consisting of 5% BSA (w:v) in TBST at 4 ◦ C overnight with agitation. 2.7. Immunoblotting of Tyr-P-GSK3 Membranes were incubated with a primary anti-phospo-GSK3 (Tyr279/Tyr216) anti- body (Tyr-P-GSK3; clone 5G-2F, 05- 413, Millipore) diluted (1:1000; v:v) in blocking solution at room temperature and agitation for 60 min. After washing membranes four times (5 min each) in TBST, they were incubated with a secondary horseradish peroxidase (HRP)- conjugated anti-mouse antibody (P0260; Dako Denmark A/S, Glostrup, Denmark) diluted 1:5000 (v:v) in blocking solution at room temperature with agitation for 60 min. Membranes were subsequently washed eight times (5 min each) in TBST. Reactive bands were visualized with a chemiluminescent substrate (Immobilon West- ern Chemiluniniscent HRP Substrate; Millipore) and scanned using G:BOX Chemi XL 1.4 (SynGene, Frederick, MT, USA) and GeneSys image acquisition software v1.2.8.0 (SynGene). Protein bands from scanned images were quantified through Quantity One v4.6.9 software Biology 2021, 10, 1105 7 of 24 package (Bio-Rad Laboratories, Inc.). Protein quantification was expressed as the total signal intensity inside the boundary of a band measured in pixel intensity units (density, square millimeter) minus the background signal, considered as 0 (white). Data were normalized with α-tubulin following the stripping of the same membranes. In brief, membranes were stripped through two incubations at room temperature under agitation for 10 min with a stripping buffer containing 0.02 M glycine, 3% (w:v) SDS and 1% (v:v) Tween 20 (pH 2.2) in 100 mL Mili-Q water. Thereafter, membranes were washed twice in TBS at room temperature for 10 min and twice with TBST for 5 min. Following this, stripped membranes were incubated with an anti-α-tubulin mouse antibody (anti- α-tubulin antibody clone DM1A, MABT205; Millipore) diluted 1:5000 (v:v) in blocking solution at room temperature under agitation for 60 min. After washing with blocking solution twice (5 min each), membranes were incubated with a HRP-conjugated anti- mouse antibody (P0260; Dako Denmark A/S) diluted 1:15,000 (v:v) in blocking solution (v:v) at room temperature under agitation for 60 min. Reactive bands were visualized and band density calculated as previously described. Relative content of Tyr-P-GSK3α was normalized, dividing the intensity of Tyr-P-GSK3α by that of α-tubulin. Data were corrected to a basal arbitrary value of 100 for the control point, which corresponded to the incubation in the medium containing BSA and 38 mM bicarbonate at 0 min. 2.8. Immunoblotting of DARPP-32 (PKA) The procedure was similar to that carried out to determine the Tyr-P-GSK3α/α- tubulin ratio. Thus, after the SDS-PAGE/membrane transference procedure conducted as described above, membranes were incubated with an anti-phospho-DARPP-32 (Thr75) primary antibody (Cell Signaling Technology; Leyden; The Netherlands) at 4 ◦ C overnight. The dilution factor was 1:1000 (v:v). After three washes, membranes were incubated with a horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (Dako; Glostrup, Denmark) at a dilution of 1:5000 (v:v) in blocking solution for 60 min. Membranes were washed six times and then revealed using a chemiluminescent HRP substrate (ImmunoCruz Western Blotting Luminol Reagent; Santa Cruz Biotechnology, Dallas, TX, USA). Protein quantification was expressed as the total signal intensity inside the boundary of a band measured in pixel intensity units (density, square millimeter) minus the background signal, considered as zero (white). Afterwards, the same membranes were reprobed with a specific total PKAα antibody. For this purpose, membranes were stripped through two incubations at room temperature under agitation for 10 min with a stripping buffer containing 0.02 M glycine, 3% (w:v) SDS and 1% (v:v) Tween 20 (pH 2.2) in 100 mL Mili-Q water. Afterwards, membranes were incubated with the specific anti-PKAα primary antibody (Cell Signaling Technology; final dilution: 1:1000, v:v) at 4 ◦ C overnight. After three washes, membranes were incubated with a horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (Dako; Glostrup, Denmark) at a dilution of 1:5000 (v:v) in blocking solution for 60 min. Membranes were washed six times and were then revealed using a chemiluminescent HRP substrate (ImmunoCruz Western Blotting Luminol Reagent; Santa Cruz Biotechnology, Dallas, TX, USA). Reactive bands were visualized and their densities were calculated as previously described. At this point, the DARPP-32/PKA ratio was calculated by dividing the intensity of DARPP-32 by that of PKA. Data were corrected to a basal arbitrary value of 100 for the control point, which corresponded to the incubation in the medium containing BSA and 38 mM bicarbonate at 0 min. 2.9. Determination of O2 Consumption Rate Determination of O2 consumption rate was carried out through a SensorDish® Reader (SDR) system (PreSens Gmbh; Regensburg, Germany). Briefly, 1 mL of each sample was incubated in four separate media (No BSA/No Bic, BSA/No Bic, BSA + 38 mM Bic, and No BSA + 38 mM Bic) for 0 min or 240 min. Following this, samples were transferred onto Oxodish® OD24 plates (24 wells/plate) specifically designed for this device. Plates were sealed with Parafilm® , loaded into the SDR system, and incubated at 38.5 ◦ C (controlled Biology 2021, 10, 1105 8 of 24 atmosphere) for 120 min. During that period, O2 concentration was recorded in each well at a rate of one reading/min. Results were exported to an Excel file and O2 consumption rate was normalized against the total number of viable sperm per sample, determined through flow cytometry (SYBR14+ /PI− sperm) using another aliquot. For this experiment, only a single concentration of bicarbonate (38 mM) was used. This was because, from an operative point of view, the analysis of all bicarbonate concentrations at all incubation times was not feasible. Thus, the authors decided to focus on the highest concentration (38 mM) and on the outcomes after 240 min of incubation at 38.5 ◦ C and at a 5% CO2 atmosphere, as this is the time needed for pig sperm to achieve the capacitated status. 2.10. Statistical Analyses Data were analyzed with a statistical package (IBM SPSS for Windows, ver. 25.0; Armonk, NY, USA) and are shown as mean ± standard error of the mean (SEM). Data were first checked for normality (Shapiro–Wilk test) and homogeneity of variances (Levene test). Following this, a linear mixed model (repeated measures) was run with the treatment as the fixed-effects inter-subject factor and the incubation time as the intra-subjects factor. Sidak’s post-hoc test was run for pair-wise comparisons. The level of significance was set at p ≤ 0.05. 3. Results 3.1. Effects of Different Concentrations of Bicarbonate and BSA on Plasma Membrane Integrity Plasma membrane integrity during in vitro capacitation and after the addition of progesterone was evaluated through SYBR14/PI, as shown in Figure 1 (mean ± SEM). A significant (p < 0.05) reduction in the percentages of sperm with an intact plasma membrane (SYBR14+ /PI− ) was observed in treatments containing 38 mM of bicarbonate, with or without BSA, after 120 min of starting the experiment. After 120 min and 240 min of incubation, all media containing either no bicarbonate or bicarbonate at 5 mM presented Biology 2021, 10, x FOR PEER REVIEWsignificantly (p < 0.05) higher percentages of sperm with an intact plasma membrane 9 of 24 than treatments containing 15 mM or 38 mM bicarbonate. After 300 min of incubation, the medium without BSA and without bicarbonate had a significantly (p < 0.05) higher percentage of sperm with an intact plasma membrane than the other treatments. Figure 1. Percentages of sperm with an intact plasma membrane (SYBR14+ /PI− ) following incubation Figure 1. Percentages with different of sperm concentrations ofwith an intact(Bic; bicarbonate plasma 0 mM,membrane 5 mM, 15(SYBR14 +/PI−) following incubation mM, 38 mM) and BSA (0, 5 mg/mL) with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) was for 300 min. The black arrow indicates the time at which 10 µg/mL of progesterone and added BSA (0, 5 (i.e., mg/mL) for 300 min. The black arrow indicates the time at which 10 μg/mL of progesterone was 240 min) to induce acrosome exocytosis. Different superscripts (a–e) indicate significant differences added (i.e., 240 min) to induce acrosome exocytosis. Different superscripts (a–e) indicate significant (p < 0.05) between treatments within the same time point. Data are shown as mean ± SEM for 12 differences (p < 0.05) between treatments within the same time point. Data are shown as mean ± independent SEM experiments. for 12 independent experiments. 3.2. Effects of Different Concentrations of Bicarbonate and BSA on Membrane Lipid Disorder The impact of different bicarbonate and BSA concentrations on the lipid disorder of sperm membrane is shown in Figure 2. Percentages of viable sperm with low lipid mem- − − Figure 1. Percentages of sperm with an intact plasma membrane (SYBR14+/PI−) following incubation with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) and BSA (0, 5 mg/mL) for 300 min. The black arrow indicates the time at which 10 μg/mL of progesterone was added (i.e., 240 min) to induce acrosome exocytosis. Different superscripts (a–e) indicate significant Biology 2021, 10, 1105 9 of 24 differences (p < 0.05) between treatments within the same time point. Data are shown as mean ± SEM for 12 independent experiments. 3.2. Effects of Different Concentrations of Bicarbonate and BSA on Membrane Lipid Disorder 3.2. Effects of Different Concentrations of Bicarbonate and BSA on Membrane Lipid Disorder The impact The impact of of different different bicarbonate bicarbonateand andBSA BSAconcentrations concentrationsononthe lipid the disorder lipid of disorder sperm membrane is shown in Figure 2. Percentages of viable sperm of sperm membrane is shown in Figure 2. Percentages of viable sperm with low lipid with low lipid mem- brane disorder membrane (M540 disorder −/YO-PRO-1−; Figure 2a) after 120 min of incubation were signifi- (M540 − /YO-PRO-1− ; Figure 2a) after 120 min of incubation were cantly higher (p < 0.05) in the significantly higher (p < 0.05) in treatments without the treatments bicarbonate, without eithereither bicarbonate, with or without with BSA, or without than in those containing bicarbonate at 5 mM, 15 mM and 38 mM. BSA, than in those containing bicarbonate at 5 mM, 15 mM and 38 mM. Additionally, Additionally, percent- ages of viable percentages ofsperm viablewith sperm low membrane with lipid disorder low membrane in the medium lipid disorder without BSA in the medium and without with bicarbonate at 38 mM were significantly (p < 0.05) lower than in the BSA and with bicarbonate at 38 mM were significantly (p < 0.05) lower than in the other other treatments throughoutthroughout treatments the entire period of incubation. the entire period of incubation. Figure 2. (a) Percentages of viable sperm with low membrane lipid disorder (M540− /YO-PRO-1 − ), and (b) percentages of Figure 2. (a) Percentages of viable sperm with low membrane lipid disorder (M540−/YO-PRO-1 −), and (b) percentages of sperm with high membrane lipid disorder considering the viable sperm population only (M540+ /viable sperm) following sperm with high membrane lipid disorder considering the viable sperm population only (M540 /viable sperm) following + incubation with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) and incubation with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) and BSA (0, 5 mg/mL) BSA (0, 5 mg/mL) for for 300 300 min. The black arrow indicates the time at which 10 µg/mL of progesterone was added (i.e., 240 min. The black arrow indicates the time at which 10 μg/mL of progesterone was added (i.e., 240 min) to induce acrosome min) to induce acrosome exocytosis. exocytosis. Different superscripts Different superscripts (a–e)significant (a–e) indicate indicate significant differencesdifferences (p < 0.05)treatments (p < 0.05) between between treatments within the within the same time same point.time Datapoint. Data are are shown shown± as as mean SEM for ± mean 12SEM for 12 independent independent experiments.experiments. On the other hand, when percentages of sperm with high membrane lipid disorder (M540+ ) were calculated considering the viable sperm population only (YO-PRO-1− ), the media containing BSA, with or without bicarbonate, showed significantly (p < 0.05) higher values of this parameter than those without BSA, from 240 min and until the end of the incubation period (Figure 2b). Remarkably, treatments containing BSA and bicarbonate at 5 mM, 15 mM or 38 mM showed significantly (p < 0.05) higher percentages of M540+ /viable sperm than those containing BSA and no bicarbonate throughout all the incubation time. 3.3. Effects of Different Concentrations of Bicarbonate and BSA on Acrosome Integrity As shown in Figure 3a, percentages of viable sperm with an intact acrosome (PNA+ / EthD-1− ) in the medium containing BSA and 38 mM were significantly (p < 0.05) lower than in the other treatments throughout the entire incubation period. In contrast, media without bicarbonate, either with or without BSA, showed significantly (p < 0.05) higher percentages of viable sperm with an intact acrosome than the other treatments. In addition, it was observed that the higher the concentration of bicarbonate, the lower the percentages of viable sperm with an intact acrosome. With regard to the percentages of viable sperm with an exocytosed acrosome (PNA− /EthD-1− ), the presence of BSA in the media containing the same concentration of bicarbonate (i.e., 0 mM, 5 mM, 15 mM and 38 mM) led to significantly (p < 0.05) higher percentages of this sperm population after 240 min, 245 min, 270 min and 300 min of incubation (Supplementary Table S1). higher percentages of viable sperm with an intact acrosome than the other treatments. In addition, it was observed that the higher the concentration of bicarbonate, the lower the percentages of viable sperm with an intact acrosome. With regard to the percentages of viable sperm with an exocytosed acrosome (PNA−/EthD-1−), the presence of BSA in the media containing the same concentration of bicarbonate (i.e., 0 mM, 5 mM, 15 mM and 38 Biology 2021, 10, 1105 10 of 24 mM) led to significantly (p < 0.05) higher percentages of this sperm population after 240 min, 245 min, 270 min and 300 min of incubation (Supplementary Table S1). Figure 3. (a) Percentages of viable sperm with an intact acrosome (PNA++ /EthD-1 − ), and (b) percentages of sperm with Figure 3. (a) Percentages of viable sperm with an intact acrosome (PNA /EthD-1 − −), and (b) percentages of sperm with an an exocytosed acrosome considering the viable sperm population only (PNA /viable sperm) following incubation with exocytosed acrosome considering the viable sperm population only (PNA−/viable sperm) following incubation with dif- different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) and BSA (0, 5 mg/mL) for 300 min. The black ferent concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 mM) and BSA (0, 5 mg/mL) for 300 min. The black arrow arrow indicates indicates the the time time at at which which 10 10 µg/mL of progesterone μg/mL of was added progesterone was added (i.e., (i.e., 240 240 min) min) to to induce induce acrosome acrosome exocytosis. exocytosis. Different Different superscripts (a–e) indicate significant differences (p<0.05) between treatments within the same time superscripts (a–e) indicate significant differences (p<0.05) between treatments within the same time point. point. Data Data are are shown shown as mean ± as mean SEM for ± SEM for 12 12 independent independent experiments. When − When percentages percentages of of sperm sperm with withananexocytosed exocytosedacrosome acrosome(PNA (PNA−)) were were calculated calculated considering − considering the viable sperm population (EthD-1 ), no significant differences between the viable sperm population (EthD-1 − ), no significant differences between treatments treatmentswere wereobserved observedatat00minminand and120 120min min(Figure (Figure 3b). 3b).However, However, treatments containing treatments contain- BSA, withwith ing BSA, or without bicarbonate, or without showed bicarbonate, showedsignificantly significantly (p <(p0.05) higher < 0.05) percentages higher percentagesof viable sperm with an exocytosed acrosome (PNA − /viable sperm) than treatments without of viable sperm with an exocytosed acrosome (PNA /viable sperm) than treatments with- − BSA, rightright out BSA, afterafter the addition of progesterone the addition (245(245 of progesterone min), andand min), after 270270 after minminandand 300300minmin of incubation. In addition, percentages of viable sperm with an exocytosed of incubation. In addition, percentages of viable sperm with an exocytosed acrosome were acrosome were significantly significantly (p(p < 0.05) higher < 0.05) higher inin the the medium medium with with BSA BSA and and 3838 mM mM than than inin that that with with BSA BSA but without bicarbonate, from 245 min to 300 min of but without bicarbonate, from 245 min to 300 min of incubation. incubation. 3.4. Effects of Different Concentrations of Bicarbonate and BSA on Intracellular Calcium Levels Percentages of viable sperm with high intracellular calcium levels (%Fluo3+ /PI− and %Rhod5+ /YO-PRO-1− sperm) are shown in Figure 4. Significantly (p < 0.05) higher percentages of Fluo3+ /PI− sperm were observed in the medium containing BSA and 38 mM bicarbonate from 0 min to 300 min of incubation, with maximum values being observed after 5 min of progesterone addition (i.e., 245 min; Figure 4a). On the other hand, the medium without BSA and with 5 mM bicarbonate showed the lowest percentage of Fluo3+ /PI− sperm when compared to the other experimental conditions. In a similar fashion to that observed for Fluo-3/PI staining, percentages of viable sperm with a positive Rhod5 signal (Rhod5+ /YO-PRO-1− ) progressively increased when sperm were incubated in media containing BSA (p < 0.05), especially at 38 mM bicarbonate (Figure 4b). The addition of progesterone induced a rapid increase in these percentages, which reached the highest values 5 min after progesterone addition (i.e., 245 min of incubation). Moreover, the lowest percentages of Rhod5+ /YO-PRO-1− sperm from 0 min to 300 min of incubation were observed in the media without BSA, especially when containing 5 mM, 15 mM and 38 mM bicarbonate. Percentages of viable sperm with high intracellular calcium levels (%Fluo3 /PI and %Rhod5+/YO-PRO-1− sperm) are shown in Figure 4. Significantly (p < 0.05) higher percent- ages of Fluo3+/PI− sperm were observed in the medium containing BSA and 38 mM bicar- bonate from 0 min to 300 min of incubation, with maximum values being observed after 5 min of progesterone addition (i.e., 245 min; Figure 4a). On the other hand, the medium Biology 2021, 10, 1105 11 of 24 without BSA and with 5 mM bicarbonate showed the lowest percentage of Fluo3+/PI− sperm when compared to the other experimental conditions. Figure 4. Percentages of viable sperm with high intracellular calcium levels evaluated through Fluo3+ /PI − (a) and Figure+ 4. Percentages − of viable sperm with high intracellular calcium levels evaluated through Fluo3+/PI− (a) and Rhod5 + /YO-PRO-1 (b) following incubation with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, Rhod5 /YO-PRO-1− (b) following incubation with different concentrations of bicarbonate (Bic; 0 mM, 5 mM, 15 mM, 38 38 mM) and BSA (0, mM) and BSA (0, 5 mg/mL) 5 mg/mL) for min. for 300 300 min. The black The black arrow arrow indicates indicates the at the time time at which which 10 µg/mL 10 μg/mL of progesterone of progesterone was was added added (i.e., 240 min) to induce acrosome exocytosis. Different superscripts (a–e) indicate significant differences (p (i.e., 240 min) to induce acrosome exocytosis. Different superscripts (a–e) indicate significant differences (p < 0.05) between < 0.05) between treatments treatments within within the samethe same time time point. point. Data are Data shownareasshown mean as meanfor ± SEM ±12SEM for 12 independent independent experiments. experiments. In a similar 3.5. Effects fashion of Different to that observed Concentrations for Fluo-3/PI of Bicarbonate and BSAstaining, percentages of viable on Mitochondrial Membrane sperm withPotential a positive Rhod5 signal (Rhod5+/YO-PRO-1−) progressively increased when sperm were incubated Incubation in media containing of pig spermatozoa with BSA BSA and(p38< mM 0.05),bicarbonate especially significantly at 38 mM bicarbonate (p < 0.05) (Figure 4b). The addition of progesterone induced a rapid increase increased the percentage spermatozoa with high mitochondrial membrane potential in these percentages, which reached (MMP) from 0 minthe highest values to 300 min 5 min after of incubation progesterone (Figure 5a). Theaddition addition (i.e., 245 min of incu- of progesterone after bation). 240 min Moreover, of incubation theinduced lowest percentages of Rhod5 a rapid increase in this/YO-PRO-1 + percentage,sperm − whichfrom was 0followed min to 300 by min of incubation a decrease were at 270 min. Onobserved the otherinhand, the media while without incubation BSA, especially of sperm when containing in a medium without 5BSA mM, and15without mM andbicarbonate 38 mM bicarbonate. (No BSA/No Bic) prevented the increase in the percentage of sperm with high MMP from 0 min to 240 min of incubation, the addition of progesterone to 3.5. this Effects medium of Different Concentrations after 240 min of incubation of Bicarbonate and that also increased BSApercentage on Mitochondrial (FigureMembrane 5a). Remark- Potential ably, percentages of sperm with high MMP did not differ between the medium without BSA/bicarbonate Incubation of(No pig BSA/No spermatozoa Bic) with and the BSA medium and 38 with BSA and 38 mM mM bicarbonate bicarbonate significantly (p < (BSA + 38 mM Bic) after 270 min of incubation. At 300 min, sperm 0.05) increased the percentage of spermatozoa with high mitochondrial membrane poten-incubated with BSA and 38 mM tial (MMP)bicarbonate from 0 min showed to 300a significantly (p < 0.05) min of incubation higher (Figure 5a).percentage The addition of sperm with high of progesterone MMP than in the other experimental conditions. Furthermore, the addition after 240 min of incubation induced a rapid increase in this percentage, which was fol- of progesterone to the medium lowed with BSA by a decrease andmin. at 270 15 mM bicarbonate On the afterwhile other hand, 240 min of incubation incubation also in of sperm showed a me- an increased percentage of sperm with high MMP. However, dium without BSA and without bicarbonate (No BSA/No Bic) prevented the increase inthe extent of that increase was significantly the percentage (p < 0.05) of sperm lower with highthan MMP that observed from 0 min toin240themin medium containing of incubation, theBSA and addition 38 mM bicarbonate. Media without BSA showed, in all cases, significantly (p < 0.05) lower of progesterone to this medium after 240 min of incubation also increased that percentage percentages of sperm with high MMP than those containing both BSA and bicarbonate (Figure 5a). Remarkably, percentages of sperm with high MMP did not differ between the (Figure 5a). medium without BSA/bicarbonate (No BSA/No Bic) and the medium with BSA and 38 Figure 5b shows the FL2:FL1 ratio in the sperm population with high MMP. After mM bicarbonate (BSA + 38 mM Bic) after 270 min of incubation. At 300 min, sperm incu- 240 min of incubation, these ratios were significantly (p < 0.05) higher in the media with bated with BSA and 38 mM bicarbonate showed a significantly (p < 0.05) higher percent- BSA than in those without this protein, especially when exogenous bicarbonate was present. age of sperm with high MMP than in the other experimental conditions. Furthermore, the Following the addition of progesterone, the treatments containing BSA and bicarbonate at addition of progesterone to the medium with BSA and 15 mM bicarbonate after 240 min 15 mM (BSA + 15 mM Bic) or 38 mM (BSA + 38 mM Bic) showed significantly (p < 0.05) higher FL2:FL1 ratios than the other media. In spite of this, after 270 min of incubation, the media containing BSA either with (38 mM) or without bicarbonate, showed significantly (p < 0.05) higher FL2:FL1 ratios than the media without BSA. At 300 min, no significant differences between treatments were observed (Figure 5b). of incubation also showed an increased percentage of sperm with high MMP. However, the extent of that increase was significantly (p < 0.05) lower than that observed in the me- dium containing BSA and 38 mM bicarbonate. Media without BSA showed, in all cases, Biology 2021, 10, 1105 12 of 24 significantly (p < 0.05) lower percentages of sperm with high MMP than those containing both BSA and bicarbonate (Figure 5a). Figure 5. (a) Percentages of sperm with high mitochondrial membrane potential (MMP; JC1agg ), and (b) FL2:FL1 (or- Figure 5. (a) Percentages of sperm with high mitochondrial membrane potential (MMP; JC1agg), and (b) FL2:FL1 (or- ange:green) ratios in the sperm population with high MMP (JC1agg ) following incubation with different concentrations of ange:green) ratios in the sperm population with high MMP (JC1agg) following incubation with different concentrations of bicarbonate bicarbonate (Bic; (Bic; 00 mM, mM, 55 mM, mM, 15 15 mM, mM, 38 38 mM) mM) and and BSABSA(0, (0,55mg/mL) mg/mL)for for300 300min. min.The The black black arrow arrow indicates indicates thethe time time at at which 10 µg/mL of progesterone was added (i.e., 240 min) to induce acrosome exocytosis. Different superscripts which 10 μg/mL of progesterone was added (i.e., 240 min) to induce acrosome exocytosis. Different superscripts (a–d) (a–d) indicate indicate significant significant differences differences (p (p<< 0.05) 0.05) between between treatments treatmentswithin withinthe thesame sametime timepoint. point.Data Dataare areshown shownas mean±± SEM asmean SEM for 12 12 independent independent experiments. experiments. Figureof5b 3.6. Effects shows Concentrations Different the FL2:FL1 ratio in the sperm of Bicarbonate population and BSA on Sperm with high MMP. After Motility 240 min of incubation, these ratios were significantly (p < 0.05) In all media, total sperm motility decreased throughout incubation time,higher in the media with reaching min- BSA than in those without this protein, especially when exogenous bicarbonate imal values at the end of the experiment (Figure 6a). In addition, percentages of total was pre- motile sent. Following spermatozoa the addition incubated of progesterone, in media containing BSA theand treatments bicarbonatecontaining at either BSA 0 mMand or 5bicar- mM bonate at 15 mM (BSA + 15 mM Bic) or 38 mM (BSA + 38 mM Bic) showed significantly were significantly (p < 0.05) higher than in the other treatments. In contrast, incubation of (p