animals Article Bicarbonate-Triggered In Vitro Capacitation of Boar Spermatozoa Conveys an Increased Relative Abundance of the Canonical Transient Receptor Potential Cation (TRPC) Channels 3, 4, 6 and 7 and of CatSper-γ Subunit mRNA Transcripts Estíbaliz Lacalle 1,2,† , César Consuegra 1,3,† , Cristina A. Martínez 1 , Manuel Hidalgo 3 , Jesús Dorado 3 , Felipe Martínez-Pastor 2,4 , Manuel Álvarez-Rodríguez 1,5, *,‡ and Heriberto Rodríguez-Martínez 1,‡ 1 Department of Biomedical & Clinical Sciences (BKV), BKH/Obstetrics & Gynecology, Faculty of Medicine and Health Sciences, Linköping University, SE-581-85 Linköping, Sweden; estibaliz.1302@gmail.com (E.L.); cconsuegra93@hotmail.com (C.C.); cristina.martinez-serrano@liu.se (C.A.M.); heriberto.rodriguez-martinez@liu.se (H.R.-M.) 2 Institute of Animal Health and Cattle Development (INDEGSAL), Universidad de León, 24071 León, Spain; felipe.martinez@unileon.es 3 Veterinary Reproduction Group, Department of Medicine and Animal Surgery, Faculty of Veterinary Medicine, University of Cordoba, 14071 Cordoba, Spain; mhidalgo@uco.es (M.H.); jdorado@uco.es (J.D.)  4 Department of Molecular Biology (Cell Biology), Universidad de León, 24071 León, Spain  5 Department of Animal Health and Anatomy, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain Citation: Lacalle, E.; Consuegra, C.; * Correspondence: manualvro@gmail.com or manuel.alvarez.rodriguez@uab.cat Martínez, C.A.; Hidalgo, M.; Dorado, † These authors contributed equally to this work. J.; Martínez-Pastor, F.; ‡ These authors contributed equally to this work. Álvarez-Rodríguez, M.; Rodríguez-Martínez, H. Simple Summary: The detection of sub-fertile boars has been a difficult task, and despite their Bicarbonate-Triggered In Vitro prevalence being low, its impact is very significant because it implies economic drawbacks for Capacitation of Boar Spermatozoa artificial insemination (AI) centers and farms. Unfortunately, some crucial reproductive processes Conveys an Increased Relative fall beyond the routine analysis performed in the porcine model, such as sperm capacitation, which Abundance of the Canonical is a necessary event for fertilization. A synergistic action of bicarbonate (HCO3 − ) with calcium Transient Receptor Potential Cation (Ca2+ ) is needed to achieve capacitation. The transport of Ca2+ is mediated by CatSper channels (TRPC) Channels 3, 4, 6 and 7 and of and Canonical Transient Potential Channels (TRPC). We quantified mRNA transcripts of different CatSper-γ Subunit mRNA subunits of CatSper (β, γ and δ) and TRPC (1, 3, 4, 6 and 7) before and after in vitro capacitation Transcripts. Animals 2022, 12, 1012. by HCO3 − ions. Our results showed that in vitro capacitation using HCO3 − increases the relative https://doi.org/10.3390/ ani12081012 abundance of mRNA transcripts of almost all subunits of Ca2+ channels, except CatSper-δ and TRPC1, which were significantly reduced. More studies are needed to elucidate the specific roles of the TRPC Academic Editor: Raquel Romar channels at a physiological and functional level. Received: 9 March 2022 Accepted: 11 April 2022 Abstract: Sperm capacitation is a stepwise complex biochemical process towards fertilization. It Published: 13 April 2022 includes a crucial early calcium (Ca2+ ) transport mediated by CatSper channels and Canonical Transient Potential Channels (TRPC). We studied the relative abundance of mRNA transcripts Publisher’s Note: MDPI stays neutral changes of the CatSper β, γ and δ subunits and TRPC-channels 1, 3, 4, 6 and 7 in pig spermatozoa, with regard to jurisdictional claims in published maps and institutional affil- after triggering in vitro capacitation by bicarbonate ions at levels present in vivo at the fertilization iations. site. For this purpose, we analyzedfive5 ejaculate pools (from three fertile adult boars) before (control- fresh samples) and after in vitro exposure to capacitation conditions (37 mM NaHCO3 , 2.25 mM CaCl2 , 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 ◦ C, 5% CO2 for 30 min. In vitro capacitation using bicarbonate elicits an increase in the relative abundance of mRNA Copyright: © 2022 by the authors. transcripts of almost all studied Ca2+ channels, except CatSper-δ and TRPC1 (significantly reduced). Licensee MDPI, Basel, Switzerland. These findings open new avenues of research to identify the specific role of each channel in boar This article is an open access article sperm capacitation and elucidate the physiological meaning of the changes on sperm mRNA cargo. distributed under the terms and conditions of the Creative Commons Keywords: calcium channels; catSper subunits; TRPC; pig; capacitation Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Animals 2022, 12, 1012. https://doi.org/10.3390/ani12081012 https://www.mdpi.com/journal/animals Animals 2022, 12, 1012 2 of 14 1. Introduction Spermatozoa undergo different maturation steps while they interact with different environments throughout their life [1]. Although they have come to be mature and motile in the epididymis, these male gametes unable to fertilize oocytes upon ejaculation because they must reside in the reproductive female tract for a period of time and interact with its environment to be able to fertilize [2]. Sperm capacitation is a gradual and necessary event for fertilization, which occurs during the journey of the spermatozoa through the female reproductive tract in vivo [3,4]. This process is characterized by controlled biochemical changes, including destabilization of the plasma membrane [3]. These changes should ultimately prepare spermatozoa to reach and bind to the zona pellucida (ZP) of the oocyte, undergo acrosome reaction (AR), and eventually penetrate the oocyte [5]. The capacitation process in the porcine model occurs mainly in the ampulla, where increasing extracellular bicarbonate enters spermatozoa by the concentration gradient, resulting in initial destabilizing membrane modifications [4]. The synergistic action of HCO3 − with calcium (Ca2+ ) is needed to achieve capacitation since HCO3 − stimulates Ca2+ uptake in porcine spermatozoa [6]. Sperm capacitation occurs through two complementary pathways [7]. One of them is dependent on HCO3 − , which elevates cAMP levels. cAMP increase induces PKA, produc- ing tyrosine phosphorylation in the proteins (cAMP/PKA pathway). Boar spermatozoa require HCO3 − to capacitate [3], an ion that exists in a low concentration in the epididymis (3–4 mM), helping to maintain sperm quiescence [8]. Levels increase 9–10-fold in the seminal plasma (up to 20–30 mM in the sperm-rich fraction), which is known to stimulate but not capacitate spermatozoa (in physiological conditions) [8–10]. This concentration also varies among fractions, being 14–17 mM in the pre-sperm fraction and sperm-rich fraction while increasing to >30 mM in the post sperm-rich fraction [10]. However, in the oviduct, the HCO3 − concentration increases from the proximal to the distal portion, which is the up- per segment (where spermatozoa are closer to the fertilization place). Whereas bicarbonate is still low in the isthmus, it considerably increases to the ampulla (10 to 33 mM) according to in vivo measurements. In the ampulla, bicarbonate enters spermatozoa by the concen- tration gradient, resulting in changes in sperm plasma membrane [4], acting synergically with Ca2+ and inducing capacitation. HCO3 − stimulates Ca2+ uptake in porcine sperm [6], and together, they activate adenylate cyclase, increasing intracellular cyclic adenosine monophosphate [cAMP]i. The increase in [cAMP]i activates cAMP-dependent protein kinases (PKA), resulting in the phosphorylation of key effectors for capacitation [9–12]. Alternatively, spermatozoa can achieve capacitation by a c-Src-dependent pathway. c-Src, a member of the Src family kinase (SFK), decreases the activity of serin/threonine phosphatases and increasing the levels of tyrosine phosphorylation (SFK/phosphatase pathway) [7]. The capacitation events increase protein–tyrosine phosphorylation in the sperm tail [12,13], which appears to be related to the accompanying hyper-activation of sperm motility [13–15]. There is also an activation of protein kinases in the sperm head, increasing membrane fluidity and lipid reorganization [16,17]. These alterations in the sperm head allow cholesterol removal through serum albumin, a cholesterol acceptor that acts only on the subpopulation of spermatozoa sensitive to bicarbonate [16]. Albu- min extracts cholesterol from the plasma membrane, increasing the fluidity of the sperm membrane and ion permeability (Na+ entry and K+ output). Then, there is a sperm mem- brane hyperpolarization and an increase in Ca2+ levels inside spermatozoa, resulting in increased intracellular pH and protein–tyrosine phosphorylation [18]. In addition, there is a redistribution of tyrosine-phosphorylated proteins localized in the acrosomal region [16]. Presently, knowing the composition of the female tract place where sperm capacitation occurs and the molecules involved in either of the two pathways of the capacitation process, in vitro capacitation can be performed in most of the mammalian species. It is accepted that a capacitation medium should contain ions, including HCO3 − and Ca2+ and bovine serum albumin (BSA) [19,20]. Animals 2022, 12, 1012 3 of 14 This chain of events occurring through the capacitation pathways induces membrane hyperpolarization, which later activates diverse calcium channels [21–23]. The regulation of Ca2+ influx is governed through sperm-specific Ca2+ channels (CatSper) and other ion channels such as canonical transient receptor potential channels (TRPC) [24]. The CatSper channel family is the most studied Ca2+ channel in spermatozoa. CatSper is a sperm-specific Ca2+ permeable channel located in the membrane of the flagellum principal piece, which helps the influx of this cation during capacitation [25] through changes in membrane potential and pH [26,27]. It has ten subunits with four pore-forming α subunits (CatSper 1–4) and, at least, six auxiliary subunits: β, γ, δ, ε, ζ and EFCAB9 [28], although other previous studies describe three [29] (β, γ and δ) or five (β, γ, δ, ε and ζ) accessory proteins [30]. The CatSper channel complex is organized in quadrilateral longitudinal nanodomains, running down the principal piece of the flagellum [24]. These channels can trigger signal transduction factors that are generally required for initiating the cAMP-PKA signaling pathway and subsequent steps in sperm capacitation [31]. CatSper mutations in humans have been associated to infertility [32,33], and it is established that they are essential for egg coat penetration and fertility in mammals [33], showing their importance for sperm function. In addition to mammals, these channels are also present in reptiles, tunicates, echinoderms and cnidarians [34]. Canonical Transient Receptors Potential channels (TRPC) also affect ion influx, such as Ca2+ (but also including K+ and Na+ ) [35]. TRPC can be activated by voltage, although they are not voltage-gated channels [36]. Up to seven TRPC (TRPC1-7) genes have been identified in mammals, being related with the phospholipase C (PLC)-dependent Ca2+ influx [37]. These seven TRPC subunits are functionally localized in spermatozoa [35], although their distribution varies between different species of mammals. TRPC3, one of the most studied TRPC subunits, appears in the acrosome of human, mouse and goat sperm but only in the midpiece of human and goat sperm. However, in mouse sperm, TRPC3 protein expression appears also in the distal segment of the flagellum [35,38,39]. The complete mapping of the ejaculate parameters could define fertility when ret- rospectively related to the observed sire fertility [40], including the omics of both sper- matozoa [40–42] and seminal plasma (SP) [43]. This combination of analyses should help determine suitable non-invasive molecular biomarkers to identify different fertility po- tentials in the pig using SP [43] or RNA present in spermatozoa [44]. The study of the proteins, genes and metabolites involved, as well as the impact of epigenetic modifications as biomarkers, may be used to diagnose infertility also in human andrology [45]. mRNA transcripts in spermatozoa have been proposed as a possible marker for male fertility [44,46]. Given the importance of Ca2+ channels for sperm functionality and fertility, our objective was to determine possible changes in the relative abundance of mRNA transcripts of sperm Ca2+ channels in pig spermatozoa, after in vitro capacitation was triggered by bicarbonate at levels present in vivo at the site of fertilization. Due to the relevance of this channels on calcium signaling, we studied the CatSper subunits (CatSper β, γ and δ subunits) and various TRPC channels (TRPC 1, 3, 4, 6 and 7) using commercially available primers for pig. We assessed other sperm variables such as motility, membrane fluidity and PKA and cAMP pathways to find a possible link with mRNA transcript abundance. 2. Materials and Methods All chemicals and reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) unless otherwise stated. 2.1. Ethics Statement Animal husbandry and all experimental and analytical procedures were performed in compliance with European Community and Swedish legislation (Directive 2010/63/EU; Swedish SJVFS 2017:40), and approvals by the “Regional Committee for Ethical Approval Animals 2022, 12, 1012 4 of 14 of Animal Experiments” were obtained (Linköpings Djurförsöksetiska nämnd), Linköping, Sweden (Dnr 75-12; ID1400; 03416-2020). 2.2. Sperm Handling and In Vitro Capacitation Boar ejaculates were supplied as commercial AI doses (5 batches of 3 same boars per pool) from Svenska Köttföretagen AB, Hållsta, Sweden extended in Hampshire Longlife to 4.8 × 109 total spermatozoa/dose, each batch containing a pool of three different breeding boars of proven fertility and semen quality (>80% motility, <15% total sperm abnormalities), and stored at 17 ◦ C, as recommended for AI purposes. All incoming sperm samples were re-examined for motility (QualiSperm, Biophos AG, Pfäffikon, Switzerland) [47] and sperm membrane stability using YO-PRO-1 labeling. All samples yielding > 80% motility and viability were accepted. Spermatozoa that were used for in vitro capacitation group were washed twice in phosphate-buffered saline (PBS, 300× g, 5 min) and incubated in a capacitation medium (37 mM NaHCO3 , 2.25 mM CaCl2 , 2 mM caffeine, 0.5% bovine serum albumin and 310 mM lactose) at 38 ◦ C, 5% CO2 , for 30 min [48]. Following centrifugation (5000× g, 5 min), the sperm pellets of both control and in vitro capacitated samples were stored at −80 ◦ C until further analysis (Ultra Low Freezer; Haier Inc., Qingdao, China) for analyzing all param- eters: sperm evaluation (motility, viability and mitochondrial status) mRNA expression, cAMP and PKA. 2.3. Experimental Design Both capacitated and non-capacitated sperm samples were evaluated by measuring several sperm quality parameters: Motility, velocity and membrane fluidity assessment were evaluated in non-capacitated samples and just after the samples were capacitated. After that, pellets were frozen at −80 ◦ C. The other parameters, including PKA and cAMP levels and the relative expression of the different subunits from Ca2+ channels, were evaluated from these stored pellets at −80 ◦ C. 2.4. Sperm Evaluation A 5 µL drop of each sperm sample was placed in a pre-warmed (38 ◦ C) slide, cov- ered with an 18 × 18 mm coverslip and examined under phase-contrast microscopy by using a computerized system (QualispermTM software, AKYmed, Cheseaux-sur-Lausanne, Switzerland) [47], which was connected via a CMOS camera (UEye, IDS Imaging Devel- opment Systems GmbH, Ubersulm, Germany) to an upright Zeiss Axio Scope A1 light microscope using a 10X phase contrast objective (Carl Zeiss, Stockholm, Sweden). An algorithm calculates the number of fluctuations in each pixel by correlation function, which is used to calculate the speed (velocity) distribution. This system runs in high throughput mode (usually four fields per minute), analyzing > 2000 spermatozoa/field. The following sperm motility parameters were obtained from the speed (µm/s): total motility (%), fast progressive (%), slow progressive (%), non-progressive (%) and immotile (%) spermatozoa. 2.5. Capacitation Status Capacitation status was assessed using the fluorochrome M540 in a fluorescence microscope and the analysis of PKA and cAMP by specific kits. 2.5.1. Membrane Fluidity Assessment A combination of two fluorescent probes in PBS was used to evaluate cell physiology: YO-PRO-1 (75 nM) for viability and Merocyanine 540 (M540, 2 µM) for capacitation status. Merocyanine 540 (M540) is an impermeant fluorescent probe that preferentially binds to membranes with lower cholesterol: phospholipids ratio, i.e., under destabilization, and YO-PRO-1 stains the nuclei of apoptotic or dead sperm with increased plasma membrane permeability. After 15 min at 37 ◦ C in the dark, 200 spermatozoa of each single boar from each treatment (control and capacitated) were counted using a fluorescence microscope at Animals 2022, 12, 1012 5 of 14 40× magnification (Eclipse Ti; Nikon, Stockholm, Sweden). Three fluorescence patterns were identified: Spermatozoa showing green fluorescence were considered apoptotic or dead; those showing yellow fluorescence were considered viable and capacitated; sperma- tozoa without fluorescence were considered viable non-capacitated. We recorded the viable sperm (YO-PRO-1− ) and viable and capacitated (live-capacitated) sperm ratio (bright M540 fluorescence and negative YO-PRO-1 fluorescence/total sperm count × 100). 2.5.2. cAMP and PKA Assessment Each capacitated/non capacitated semen pellet (100 × 106 spermatozoa) was resus- pended in 1 mL of PBS. Then, each sample was split into two 1.5 mL tubes with 500 µL each (50 × 106 spermatozoa). One of them was directed to cAMP analysis by Direct Im- munoassay, and the other one was directed to PKA analysis by Kinase Activity assessment. For cAMP evaluation, the cAMP direct immunoassay fluorometric kit (abcam, Cambridge, United Kingdom; ab138880) was used, following the manufacturer’s speci- fications. In brief, all samples (50 × 106 spermatozoa), control and in vitro capacitated samples were centrifuged at 3000× g for 5 min at room temperature (RT); then, the supernatant was discarded, and the pellet was resuspended by adding 100 µL of Cell Lysis Buffer in each well of the plate and incubated at RT for 10 min. Samples were centrifuged for 5 min at top speed, and the supernatant was collected and transferred to a new tube that was kept on ice. A volume of 75 µL of standard and sample was added to the wells of the anti-cAMP coated 96-well plate and incubated at RT for 5–10 min. After this time, 25 µL/well of 1X HRP-cAMP conjugate was added to each standard and sample well. Then, the plate was incubated at RT for 2 h on a plate shaker and washed 4 times with 200 µL Wash Solution. Finally, 100 µL AbRed Working Solution was added into each standard and sample well, incubating the plate at RT for 1 h protected from light. Fluorescence change was measured in a microplate reader set to top read mode at Ex/Em = 540/590 nm (cutoff 570 nm). For PKA evaluation, we used the PKA Kinase Activity Assay Kit (abcam, Cambridge, United Kingdom; ab139435) according to the manufacturer’s instructions. In brief, all samples (50 × 106 spermatozoa), control and in vitro capacitated sperm were centrifuged at 3000× g for 5 min at RT, and the pellet was resuspended in 100 µL of RIPA. After sonication of the samples (10 s twice) and 5 s vortexing, the samples were kept on ice for 30 min. Then, the lysate was centrifuged 13,000× g for 15 min at 4 ◦ C. After recovery of a clear supernatant, the samples were kept on ice. Wells were previously prepared with 50 µL Kinase Assay Dilution Buffer at RT for 10 min. Then, 30 µL Kinase dilution buffer was added to the blank wells, samples (30 µL) and controls (30 µL) to the appropriate wells. An amount of 10 µL of diluted ATP to each well (except blank) initiates the reaction, after incubation at 30 ◦ C for 90 min. Then, 40 µL of phospho-specific antibody was added to each well, except blank, and the plate was incubated at RT for 60 min. All wells were washed 4 times with 100 µL 1X Wash Buffer. Afterward, 40 µL of diluted anti-rabbit IgG-HRP conjugated was added to each well except blank, and the plate was incubated at RT for 30 min and washed 4 times with wash buffer. Thereafter, 60 µL TMB solution was added and incubated for 45 min at RT. Finally, 20 µL stop solution was added to each well to stop the reaction, and the optical density (OD) at 450 nm was measured. 2.6. RNA Extraction and qPCR Analyses of Calcium Channels Total RNA was extracted from the sperm samples using an RNeasy micro kit (Qiagen, Venlo, The Netherlands) following the manufacturer’s instructions. The RNA concentration of the extracts was determined from the absorbance of 260 nm with Thermo Scientific NanoDropTM 2000 (Fisher Scientific, Gothenburg, Sweden). The High-Capacity RNA-to- cDNA™ Kit (Fisher Scientific, Gothenburg, Sweden) was used to synthesize the first-strand cDNA for quantitative polymerase chain reaction (qPCR) analyses (CFX96™; Bio-Rad Laboratories, Inc; Hercules, CA, USA). The gene relative abundance levels were quantified using the Pfaffl method, ∆∆Ct method (Pfaffl, 2001). The GAPDH gene and commercial Animals 2022, 12, 1012 6 of 14 gene-specific PCR primers for porcine samples were used (glyceraldehyde-3-phosphate dehydrogenase) (PrimePCR™SYBR® Green Assay: GAPDH, Pig; Bio-Rad Laboratories, Inc; Hercules, CA, USA). We included NTC controls in all plates, obtaining in all cases an indetectable presence of the products after qPCR analysis (>40 cycles). PowerUp SYBR Green Master Mix (2×) (Applied Biosystems, Foster City, CA, USA) was used for PCR reactions. The final reaction volume was 10 µL (2 µL of cDNA (25 ng), 5 µL of 2X Master mix, 1 µL of each primer (500 nM) and 1 µL of dH2O). The following PCR conditions were used: initial UDG activation of 50 ◦ C for 2 min and a previous denaturation of 95 ◦ C for 2 min. Those steps were followed by 40 cycles of 5 s of denaturation at 95 ◦ C and 30 s of extension and annealing at 60 ◦ C. qPCRs were run in duplicate for each gene per sample. All primers (TRPC1, TRPC3, TRPC4, TRPC6, TRPC7, CatSper-β, CatSper-δ and CatSper-γ) were commercial from BIORAD (PrimePCR™SYBR® Green Assay, Pig; Bio-Rad Laboratories, Inc; Hercules, CA, USA). Target relative gene abundance was normalized with the reference gene GAPDH. By examining the results of the number of cycles in qPCR, we verified that there are no significant differences in the GAPDH cycles before and after sperm in vitro capacitation. In addition, the number of cycles increased in all TRPCs and CatSper subunits, although without significant differences. Moreover, additional validation was the same concentration of total sperm cells in both measurement time points before and after capacitation. 2.7. Statistical Analysis Normal distribution and homoscedasticity of the data were analyzed using the Shapiro– Wilk normality test and Levene’s test. Non-normal data distribution was reached using arcsin(x) transformation prior to analysis. R version 3.6.1 [49] was used to conduct statistical analyses, with nlme [50] to perform linear mixed effects (LME) models and multcomp [51] to perform pairwise comparisons adjusted by Tukey’s test. The threshold for significance was set at p < 0.05. Data are presented as mean ± SEM, unless otherwise stated. Our LME model included in vitro capacitation (control, in vitro capacitation) as fixed effects and the pool of samples as the random part of the model. 3. Results 3.1. In Vitro Capacitation Increases the Live-Capacitated Sperm Ratio In vitro exposure of commercial AI doses from a pool of ejaculated spermatozoa to elevated bicarbonate levels, under the assayed capacitation conditions (CAP; 38 ◦ C, 5% CO2 , 30 min), caused a significant reduction in total motility (96.0% ± 1.8 vs. 24.2% ± 1.9; p < 0.001; Figure 1A), fast progressiveness (48.7% ± 4.0 vs. 8.6% ± 2.1; p < 0.001; Figure 1B) and sperm velocity (26.1 ± 1.3 vs. 15.5 ± 1.5 µm/s; p < 0.001; Figure 1C) (CONTROL vs. CAP in all cases). In addition, the capacitating conditions for 30 min did not affect the membrane’s integrity, as depicted by YO-PRO-1− spermatozoa (CONTROL, 89.0% ± 3.8 vs. CAP, 69.8% ± 1.5; p = 0.16; Figure 1D). The ratios of capacitated sperm within the live population (viable sperm, YO-PRO-1− population) after in vitro capacitation were significantly higher than in the control group (p < 0.0083) (46.7% ± 3.3 vs. 66.7% ± 4.9; p = 0.0083; CONTROL vs. CAP; Figure 1E). Animals 2022, 12, x FOR PEER REVIEW 7 of 14 Animals 2022, 12, 1012 7 of 14 Figure 1. Total motility (TM), progressive motility (PM), velocity (VEL), membrane integrity (VIAB) and Figure 1. Total motility (TM), progressive motility (PM), velocity (VEL), membrane integrity (VIAB) ratio of capacitated taking into account only the viable sperm (RATIO+) in control (CONTROL) and and ratio of capacitated taking into account only the viable sperm (RATIO+) in control (CONTROL) in vitro and capacitated in vitro (CAP) capacitated boar ejaculated (CAP) spermatozoa boar ejaculated (n = 5).(nThe spermatozoa results = 5). are shown The results as meanas±mean are shown SEM. ±* SEM. depicts significant * depicts differences significant betweenbetween differences CONTROL and CAP CONTROL and (* pCAP < 0.05; (* p*** p < 0.001). < 0.05; ***p < 0.001). 3.2. In Vitro Capacitation for 30 min Increases the Sperm Protein Kinase A Content 3.2. In Vitro Capacitation for 30 Min Increases the Sperm Protein Kinase A Content Capacitated boar samples tend to lower its intracellular cAMP levels compared to Capacitated boar samples tend to lower its intracellular cAMP levels compared to control doses (54.2 ± 15.9 vs. 8.2 ± 3.2 nM; CONTROL vs. CAP; p > 0.05) although without control doses significant (54.2 ± 15.9 differences (p vs. 8.2 ±Figure = 0.07; 3.2 nM; CONTROL 2A). vs. CAP;PKA On the contrary, p > 0.05) although increased without in the sperm significant doses subjected to 30 min of capacitation; in this case, it increased significantly (6.36 sperm differences (p = 0.07; Figure 2A). On the contrary, PKA increased in the ± 0.13 doses subjected vs. 7.14 to 30 minCONTROL ± 0.16 nM/mL; of capacitation; in this vs. CAP, p