Manipulation of bicarbonate concentration in sperm capacitation media improvesin vitro fertilisation output in porcine species

Background The in vivo concentration of bicarbonate (HCO3−), one of the essential sperm capacitating effectors, varies greatly in the different environments sperm go through from cauda epididymis to the fertilisation site. On the contrary, porcine in vitro sperm capacitation and fertilisation media usually contains a standard concentration of 25 mmol/L, and one of the main problems presented is the unacceptable high incidence of polyspermy. This work hypothesised that by modifying the HCO3− concentration of the medium, the output of in vitro sperm capacitation and fertilisation could be increased. Results Once exposed to the capacitation medium, the intracellular pH (pHi) of spermatozoa increased immediately even at low concentrations of HCO3−, but only extracellular concentrations of and above 15 mmol/L increased the substrates protein kinase A phosphorylation (pPKAs). Although with a significant delay, 15 mmol/L of HCO3− stimulated sperm linear motility and increased other late events in capacitation such as tyrosine phosphorylation (Tyr-P) to levels similar to those obtained with 25 mmol/L. This information allowed the establishment of a new in vitro fertilisation (IVF) system based on the optimization of HCO3− concentration to 15 mmol/L, which led to a 25.3% increment of the viable zygotes (8.6% in the standard system vs. 33.9%). Conclusions Optimising HCO3− concentrations allows for establishing an IVF method that significantly reduced porcine polyspermy and increased the production of viable zygotes. A concentration of 15 mmol/L of HCO3− in the medium is sufficient to trigger the in vitro sperm capacitation and increase the fertilisation efficiency in porcine. Electronic supplementary material The online version of this article (10.1186/s40104-019-0324-y) contains supplementary material, which is available to authorized users.


Background
Fertilisation is considered a multifactorial process in which many factors are directly and/or indirectly involved. The success rate of this physiological process depends to a high degree on the ability of spermatozoa to undergo capacitation and penetrate the oocyte vestments [1]. In vivo sperm capacitation is a gradual event that is actively and progressively coordinated within succeeding segments of the female genital tract [2]. The changes occurring in spermatozoa during in vivo capacitation begin as soon as they are ejaculated as a result of, among other reasons, the bicarbonate (HCO 3 − ) intake, subsequent activation of cAMP synthesis, intracellular pH alkalinisation (pH i ), increase in intracellular Ca 2+ concentration and motility activation (fast capacitation events) [3]. Once inside the female genital tract, other events occur in a sequential and parallel way for an extended period, such as hyperactivation, tyrosine phosphorylation (Tyr-P) and preparation to undergo acrosome reaction (AR) (slow capacitation events). Both fast and slow events depend on the HCO 3 − and Ca 2+ concentrations, but the slow ones also depend on the presence of cholesterol acceptors like albumin (reviewed by Visconti [3]). The inward transport of HCO 3 − from the surrounding medium during capacitation involves electrogenic Na + /HCO 3 − co-transporter (NBC) and Cl − /HCO 3 − anion exchangers [4]. It starts with induction of the atypical soluble adenylyl cyclase Adcy10 aka sAC (from now on Adcy10), increase in cAMP levels and activation of protein kinase A (PKA) [5]. Numerous studies have indicated that PKA phosphorylates several substrates (pPKAs) which supposes the initial point either for fast or slow events of the capacitation [3]. In porcine sperm, previous studies suggest that phosphorylation of pPKAs rapidly increases in~160 s through the action of HCO 3 − , after which the activity increases slowly to reach a maximum at 21 min [6]. The HCO 3 − acts as a key capacitating agent and its importance in mediating changes in spermatozoa has been demonstrated in multiple studies in mice and humans. Knock-out mice that fail to express Adcy10 were infertile [7,8] and females with defective HCO 3 − secretion in the uterus produced low sperm capacitation and fertility [9].
After maturing as they pass through the epididymis, the spermatozoa are stored in the epididymal cauda in low HCO 3 − conditions (~4 mmol/L [10]). During ejaculation, the spermatozoa come into contact with the seminal plasma, which contains higher HCO 3 − concentrations (~20 mmol/L) [11] but also decapacitating factors, mainly spermadhesins from accessory sex glands, that coat and stabilise the sperm surface to prevent premature capacitation [12]. Once in the female genital tract, sperm are subjected to different selection mechanisms that remove the immature or damaged ones. As they move forward in the uterus towards the oviduct, the spermatozoa have to swim through several environments increasingly rich in HCO 3 − (~25 mmol/L [7]) and the decapacitating factors are gradually removed, enabling membrane destabilisation and capacitation. Inside the oviduct, they remain attached to the epithelium after the utero-tubal junction, in the caudal isthmus, establishing the sperm reservoir. Once ovulation occurs, the spermatozoa are gradually released from the sperm reservoir and swim towards the fertilisation site at the ampullary-isthmic junction [13]. However, many of the ovulation signals and mechanisms of gradual sperm release from the reservoir are still unknown, and, in vitro conditions are far from ideal for gradually exposing spermatozoa to capacitating conditions. Currently, most of the in vitro capacitation and fertilisation (IVF) methods, in porcine and other species, expose all the spermatozoa to a fixed HCO 3 − concentration of 25 mmol/L at the same time [14][15][16][17][18][19], providing a static IVF system that little resembles the dynamic system of fluids to which gametes are exposed in vivo. Despite the development of chemically defined fertilisation media [15,20,21], different spermatozoa and oocytes co-incubation times [22], sperm capacitation methods [23] and the use of innovative IVF methods in straws or in devices in which both gametes are physically separated [24], polyspermy, one of the major problems in porcine IVF, persists. The microenvironment of in vitro capacitation and fertilisation currently used in laboratories is probably far from optimal for porcine species. The objective of this work was to study the effect of different concentrations of HCO 3 − on sperm capacitation, mimicking as far as possible the increase that takes place in vivo. For this purpose, different HCO 3 − concentrations were tested on PKAs activation, pH i and sperm motility, and on several subsequent events downstream of PKA activation in the cascade of capacitation, such as Tyr-P and the AR. Finally, the in vitro fertility parameters were studied. The results of this study could increase our knowledge of sperm physiology and lead to an improvement of in vitro sperm capacitation and oocyte fertilisation output in porcine species.

Spermatozoa collection and preparation
Epididymes from mature Large White boars (Sus scrofa) were obtained from a slaughterhouse and transported to the laboratory within 30 min of slaughter. The cauda epididymis was dissected and the epididymal duct was isolated. A 24G BD Insyte™ catheter (381212, Becton Dickinson Infusion Therapy Systems, Inc., Sandy, Utah, USA) adapted to a syringe was inserted into the epididymal duct, and sperm samples were obtained by pressurised air through the deferens duct. Spermatozoa samples were incubated for up to 60 min (1,5,15,30 and 60 min, depending on the experiment) in modified Tyrode's albumin lactate pyruvate media (TALP) based on that described by Rath et al. [15], composed by 114.06 mmol/L NaCl, 3.20 mmol/L KCl, 0.50 mmol/L MgCl 2 •6H 2 O, 18.00 mmol/L sodium lactate, 0.35 mmol/ L NaH 2 PO 4 •H 2 O, 5.00 mmol/L glucose, 2.00 mmol/L caffeine, 8.00 mmol/L calcium lactate•5H 2 O, 1 mg/mL polyvinyl alcohol, 0.17 mmol/L kanamycin and 0.003 mmol/L phenol red, 3 mg/mL bovine serum albumin (BSA), 1 mmol/L sodium pyruvate, but containing different HCO 3 − concentrations: 0, 5, 15 or 25 mmol/L. Prior to sperm incubation, all media were adjusted to pH 7.4. The pH of the medium containing 0 mmol/L was adjusted using NaOH and HCl, the pH of the media containing 5, 15 and 25 mmol/L was adjusted in an incubator for nearly 3 h at 38.5°C, saturated humidity and 1.0%, 3.0% or 5.0% of CO 2 atmosphere in air, respectively, according to the Henderson-Hasselbalch equation. A group of spermatozoa incubated in phosphate buffer saline (PBS) was included as non-capacitating control (NCAP).

Spermatozoa intracellular pH (pH i )
The analysis of pH i was carried out in spermatozoa (30 × 10 6 cells/mL) loaded with 5 μmol/L of the pH-sensitive dye BCECF-AM (B1150, Sigma-Aldrich®, Madrid, Spain) for 30 min at 38.5°C. After that, the samples were centrifuged at 700×g for 3 min to remove the excess of dye and resuspended in PBS without Ca 2+ and Mg 2+ and incubated again for 15 min at 38.5°C for the de-esterification of the dye. Finally, the samples were centrifuged and resuspended in NCAP, 0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L of HCO 3 − for 1 and 60 min. The fluorescence was monitored using a spectrofluorometer (FP-6300, Jasco®, Cremella, Italy) every 2 s for a total time of 300 s. A calibration of the system was first performed using BCECF-AM stained and equilibrated spermatozoa at pH 6.0, 6.5, 7.0, 7.5 and 8.0 in the presence of 0.1% Triton X-100 by adjusting the pH with HCl and NaOH [27]. The emitted fluorescence ratio from the excitation at 490/440 nm was calculated and the regression line for extracellular pH (pH e ) vs. the 490/440 nm ratio was obtained (Additional file 1). The pH i of sperm cells was estimated from the regression line.

Spermatozoa motility
Sperm motion assay was carried out using a computer-assisted spermatozoa motility analysis (CASA), the ISAS® system (PROISER R + D S.L., Valencia, Spain), connected to a negative phase-contrast microscope (magnification × 100; Leica DMR, Wetzlar, Germany) and a digital camera (Basler Vision, Ahrensburg, Germany). Spermatozoa motility was determined after 1, 15, 30 and 60 min of incubation in the different TALP media previously described. For this purpose, 4 μL of samples were placed on a warmed (38.5°C) 20-μm SpermTrack® chamber (STP-21006, PROISER R + D S.L., Valencia, Spain) and motility parameters were determined into 3 different fields per sample and time of incubation: the percentage of total motile spermatozoa (Mot, %), motile progressive spermatozoa (MotPro, %), curvilinear velocity (VCL, μm/s), straight line velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity of the curvilinear trajectory (LIN, ratio of VSL/VCL, %), straightness (STR, ratio of VSL/VAP, %), wobble of the curvilinear trajectory (WOB, ratio of VAP/VCL, %), amplitude of lateral head displacement (ALH, μm) and beat cross-frequency (BCF, Hz). Moreover, the spermatozoa from each replicate were classified in 3 different clusters per experimental group and time of incubation to identify groups of spermatozoa with a high degree of similarity in their motility pattern: i) Cluster 1: spermatozoa with the poorest motility (lowest values in all CASA parameters); ii) Cluster 2: spermatozoa with the most linear trajectory (high values of VSL, LIN, STR and WOB); and iii) Cluster 3: spermatozoa with the most curvilinear trajectory (high values of VCL and ALH and low values of VSL, LIN and STR) (Additional file 2).

Spermatozoa tyrosine phosphorylation (Tyr-P)
Indirect immunofluorescence (IIF) was performed as described by Luño et al. [28] to study sperm Tyr-P. Samples processing was carried out at 4°C. After 60 min of incubation in TALP media containing different HCO 3 − concentrations, sperm samples were first washed with PBS and centrifuged at 270×g for 10 min and then fixed in 2% p-formaldehyde/PBS for 60 min. The sample blocking was carried out overnight in a wet chamber in 2% (w/v) BSA in PBS. After that, samples were re-centrifuged at 270×g for 10 min, resuspended in PBS and spread on glass slides. The primary antibody used for incubation was anti-phosphotyrosine (4G10, Millipore, CA, USA, 1:300 in 0.1% BSA in PBS). The slides were washed with PBS and then incubated with the secondary antibody, fluorescein-conjugated goat anti-mouse (Bio-Rad Laboratories, Madrid, Spain, 1:400 in 0.1% BSA in PBS). Finally, samples were washed again with PBS and mounted with coverslips using a fluorescent medium (Dako, Carpinteria, CA, USA). Two-hundred spermatozoa per sample were analysed under an epifluorescence microscope (magnification × 400; Leica® DMR, USA). The Tyr-P location of sperm proteins was classified and grouped into 4 different categories according to the 4 patterns described by Luño et al. [28]: i) Pattern I (low capacitation), which included spermatozoa without fluorescence or with phosphorylated acrosome or tail or acrosome and tail; ii) Pattern II (medium capacitation), which included spermatozoa with fluorescence in the equatorial subsegment with or without the presence of signal in the flagellum; iii) Pattern III (high capacitation), which included spermatozoa with signal in the equatorial subsegment and acrosome area and with or without the presence of signal in the flagellum. A fourth pattern was established related to the motility activation which included those spermatozoa with a signal in the flagellum regardless of other locations (Pattern IV) (Additional file 3).

In vitro fertilisation (IVF)
Porcine oocytes were isolated from ovaries obtained from 6-to 7-month-old animals at a local slaughterhouse and transported to the laboratory in saline solution at 38.5°C. Once in the laboratory, the ovaries were washed in 0.04% cetrimide solution (w/v) and saline solution. Cumulus-oocytes complexes (COCs) were collected from antral follicles (3-6 mm diameter) and washed twice with Dulbecco's PBS. Groups of 50 COCs with complete and dense cumulus oophorus were in vitro matured in 500 μL NCSU-37 medium based on that described by Petters and Wells [29] composed by 108.73 mmol/L NaCl, 25 mmol/L NaHCO 3 , 4.78mmol/L KCl, 1.19 mmol/L KH 2 PO 4 , 1.19 mmol/L MgSO 4 •7H 2 O, 1.70 mmol/L CaCl 2 •2H 2 O, 5.55 mmol/L glucose, 1.00 mmol/L glutamine, 12.00 mmol/L sorbitol, 0.18 mmol/L penicillin and 0.07 mmol/L streptomycin, for 42 h, as previously described by Funahashi et al. [30]. After IVM, the COCs were gently pipetted to remove the cumulus. Groups of 50 oocytes were transferred to a 4-well multidish containing 500 μL per well of TALP media containing the different HCO 3 − concentrations: 0, 5, 15 or 25 mmol/L described above. Each insemination dose comprised a pool of epididymal spermatozoa from 3 different boars. The spermatozoa were directly inseminated without being previously capacitated (monophasic IVF) or pre-incubated in capacitating media for 60 min with different HCO 3 − concentrations and then inseminated (biphasic IVF), in both cases giving a final concentration of 5 × 10 4 cells/mL.

Statistical analysis
All statistical analyses were performed using IBM SPSS v.19 (SPSS Inc. Chicago, IL, USA) and data were analysed by one-way ANOVA. In the motility experiment, 2 types of analysis were carried out per field: i) data were analysed using the general linear repeated-measures procedure, and ii) data were grouped by a non-hierarchical k-means clustering for each time of incubation and a repeated-measures procedure was followed for each cluster. Three different clusters centers where established per motility parameter to classify each spermatozoa (Additional file 2). In both sets of analyses, the obtained results were compared by one-way ANOVA. Data are expressed as the mean ± standard error of the mean (SEM). Differences were considered statistically significant at P < 0.05, and when these analyses revealed a significant effect, values were compared using the least significant difference pairwise multiple comparison post-hoc test (Tukey). A statistical tendency was considered when P≥ 0.05 and ≤ 0.75.

Effect of HCO 3 − concentration on spermatozoa functionality
Among the ions existing along the route that spermatozoa must follow from the epididymis, HCO 3 − has an important role in two main sperm functions: i) collaborating in the functional maturation of spermatozoa by affecting membrane dynamics and ii) buffering the environment [32][33][34]. Moreover, HCO 3 − is a key factor in inducing sperm capacitation by direct stimulation of Adcy10 before triggering the cascade of fast and slow events of capacitation. Here, the effect of different HCO 3 − concentrations on these capacitating events was investigated.

Spermatozoa protein kinase A substrates phosphorylation (pPKAs)
The effect of HCO 3 − concentration on pPKAs after 60 min in NCAP, 0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L groups are shown by WB in Fig. 1. The results showed that 15 mmol/L and 25 mmol/L produced a significantly higher degree of pPKAs compared with the rest of the groups (P < 0.05). Figure 1A showed that low concentrations of HCO 3 − (0 and 5 mmol/L) produced low levels of pPKAs, similar to those produced in NCAP group. However, the addition of a higher HCO 3 − concentration (of and above 15 mmol/L) induced a significant increment in pPKAs. When comparing experimental replicates, 15 mmol/L and 25 mmol/L produced a significantly higher degree of pPKAs compared with the rest of the groups (P < 0.05) (Fig. 1B). Although it has been described that the pPKAs is time-dependent, as shown in Fig. 2, it is also concentration-dependent. When low HCO 3 − concentrations were used, like 0 or 5 mmol/L, the highest level of pPKAs was achieved since the beginning of incubation ( Fig. 2A and C). On the other hand, when higher levels of HCO 3 − were used, 15 and 25 mmol/L, pPKAs are progressively increasing ( Fig. 2E and G), being faster as HCO 3 − concentration is higher (25 mmol/L). Through semi-quantification it could be determined that inexistence or low concentrations of HCO 3 − (0 or 5 mmol/L) could not provoke pPKAs similar to control (25 mmol/L) after 60 min of incubation ( Fig. 2B and D). Higher concentrations of HCO 3 − produce the highest pPKAs in a concentration-dependent way. A HCO 3 − concentration of 15 mmol/L produced the highest pPKAs after 60 min of incubation (P < 0.05) and 25 mmol/L after 15 min (P < 0.05) (Fig. 2H).

Spermatozoa motility
One of the first events related with Adcy10/PKA pathway activation by HCO 3 − is sperm motility stimulation. Thus, the effect of HCO 3 − on sperm motility and trajectory was studied in capacitating conditions in 0 mmol/L, 5 mmol/L, 15 mmol/L and 25 mmol/L (Figs. 4, 5 and 6). The results showed that the HCO 3 − concentration did not affect Mot (ranged from 84.9% to 92.9%), MotPro (from 83.2% to 92.4%) or VCL (from 138.2 to 165.0 μm/s) but stimulated straight and average path velocities (VSL and VAP, P < 0.05) (Fig. 4). A HCO 3 − concentration of 15 mmol/L produced significantly higher LIN, STR and WOB than 0 mmol/L and 5 mmol/L but 25 mmol/L did not induce any extra improvement (Fig. 4). There were no statistical differences in ALH and BCF between groups. A visual representation of the effect of HCO 3 − on the trajectories of spermatozoa is shown in Fig. 5. An increasingly straight trajectory was directly proportional to the HCO 3 − concentration in the medium. Moreover, a cluster analysis revealed that the proportion of spermatozoa with poorer motility (cluster 1) was similar between groups, regardless of the HCO 3 − concentration and length of incubation (Fig. 6). The proportion of spermatozoa with the most rectilinear trajectory (cluster 2) was significantly higher (P < 0.05) in the 5 mmol/L, 15 mmol/L and 25 mmol/L groups than in 0 mmol/L. In addition, and despite a certain tendency (P = 0.051) to decrease in the presence of HCO 3 − , there were no statistical differences in the proportion of spermatozoa with the most curvilinear trajectory (cluster 3) between groups.

Spermatozoa tyrosine phosphorylation (Tyr-P)
The pH i seems to favour the cascade of slow and fast events in spermatozoa, including the activation of tyrosine kinases and the subsequent Tyr-P of proteins. In fact, the capacitation status was described to be strongly correlated with Tyr-P [5] as a direct consequence of the action of HCO 3 − , whose omission not only inhibits Tyr-P [5], but also diminishes the ability of spermatozoa to bind to ZP [35]. This slow capacitation response must be preceded by lipid scrambling and cholesterol removal by a cholesterol acceptor, normally albumin. Different compartments have been described as undergoing a specific sequence of phosphorylation events during both capacitation and bind-ing to ZP [36]. The present experiment tried to evaluate the effect of HCO 3 − concentration on Tyr-P immunolocation in epididymal porcine spermatozoa (Fig. 7). At 1 min of incubation, all the experimental groups had the same proportion of spermatozoa with patterns I and III (low and high capacitation status, respectively). The 25 mmol/L concentration had a significantly reduced proportion of spermatozoa with pattern II (medium capacitation status) (17.4 ± 3.7%) compared with NCAP (29.2 ± 1.4%) (Fig. 7A). At 60 min of incubation, the proportion of pattern I and II (low and medium capacitation status, respectively) did not show any statistical difference between the media used (P > 0.05). However, the proportion of pattern III (high capacitation status) was significantly higher in 15 mmol/L and 25 mmol/L (45.2 ± 3.0% and 45.5 ± 4.3%, respectively; P < 0.05) (Fig. 7B). As regards pattern IV, 15 mmol/L and 25 mmol/L produced the highest level of Tyr-P in the spermatozoa tail, which differed significantly from the rest of the groups (68.0 ± 4.7% and 65.9 ± 4.8%, respectively; P < 0.05) (Fig. 7C). Taken together, these results showed that a 15 mmol/L concentration of HCO 3 − in the capacitation medium is sufficient to attain a high level of Tyr-P and probably motility activation.

Effect of HCO 3 − concentration on IVF
As described above, the concentration of HCO 3 − affects sperm functionality and modulates sperm capacitation, a regulatory event that prepares spermatozoa for fertilisation prior to encountering the oocyte. Based on this, the present study tried to determine the fertility of spermatozoa and the interaction between gametes in the same conditions.

Monophasic IVF
The IVF output for penetration, monospermy, SPZ/O, SPZ/ZP and efficiency are shown in Table 1A. In the absence of HCO 3 − (0 mmol/L) no oocyte was penetrated, which confirms that HCO 3 − is an indispensable component of the IVF medium for the spermatozoa to bind to ZP and to penetrate the oocyte. When 5 mmol/L was used, all the studied parameters were significantly lower than when 15 mmol/L or 25 mmol/L was used. The 25 mmol/L concentration had a lower efficiency (8.6 ± 1.7%) than 15 mmol/L because, although penetration was high, monospermy was low. The use of 15 mmol/L significantly increased the efficiency of the technique to 26.2 ± 3.1%, with a lower penetration but a higher monospermy (P < 0.05) than at 25 mmol/L. In light of these results, it is clear that sperm fertility was affected by the mere presence of HCO 3 − and also its concentration. This study then turned to the task of identifying an IVF method that increased the efficiency of obtaining potentially viable zygotes by optimizing the HCO 3 − concentration to which gametes are gradually exposed.

Biphasic IVF (spermatozoa pre-incubation + monophasic IVF)
The pre-incubation of sperm in a capacitating medium affected all the parameters studied (Table 1B). Penetration increased proportionally to the concentration of HCO 3 − in sperm pre-incubation, although monospermy was more closely related to the HCO 3 − concentration in the IVF medium, reaching between 21.5% and 25.4% when IVF was carried out at 25 mmol/L and between 36.2% and 48.7% when it was carried out at 15 mmol/L. Thus, the higher efficiency was achieved when both pre-incubation and IVF was performed in 15 mmol/L (33.9 ± 3.7%, P < 0.05).

Discussion
Spermatozoa maturation in the epididymis comprises sequential modifications that have been demonstrated to be essential for the acquisition of motility and fertility [37]. Once in the epididymal cauda, where the HCO 3 − concentration is minimal (~4 mmol/L), matured spermatozoa remain in a quiescent state. After ejaculation, spermatozoa are activated and changes in the sperm membrane begin as they progress through the uterus, where they find increasing concentrations of HCO 3 − . Subsequently, sperm capacitation is completed in the female isthmus and spermatozoa are again progressively exposed to increasing amounts of the capacitating effector HCO 3 − from the isthmus to ampulla [38]. In the present study we have elucidated how epididymal spermatozoa respond to different HCO 3 − concentrations during capacitation and in vitro fertilisation.
This study was carried out using mature spermatozoa that had never been in contact with high concentrations of HCO 3 − or with decapacitant agents from accessory sex glands. Epididymal spermatozoa were incubated in a capacitating medium containing 0, 5, 15 or 25 mmol/L of HCO 3 − and in non-capacitating medium. The results showed that the highest degree of PKAs activation was achieved by incubation in a capacitating medium containing 15 mmol/L of HCO 3 − or above, although the 25 mmol/L concentration did not perform any better than 15 mmol/L. In spite of the presence of other capacitating agents such as calcium and albumin, concentrations lower than 15 mmol/L (for example the 5 mmol/L, which is similar to that existing in the epididymis) produced a low pPKAs, similar to that produced in the absence of HCO 3 − (0 mmol/L), quantitatively matching the NCAP conditions which lack other capacitation effectors as albumin and calcium. This fact reflects that not only HCO 3 − is necessary to initiate the capacitation cascade but also albumin and calcium, as has been previously described. It is known that defective HCO 3 − secretion into the uterus may be related to impaired sperm capacitation and a low female fertility [9]. Therefore, a 15 mmol/L concentration of HCO 3 − is postulated as being sufficient to achieve a high degree of PKAs activation that is essential in the sperm capacitation process. The differences in the results obtained with different HCO 3 − concentrations were not dependent on the viability of the cells analysed (Additional file 4). Therefore, although this study showed the dose-dependent effect of HCO 3 − on sperm capacitation, above 15 mmol/L there was no extra effect. However, the kinetic of PKAs activation allowed us to understand that the  [6]. Furthermore, PKAs was highly phosphorylated after 30 min of incubation with 25 mmol/L and after 60 min with 15 mmol/L, demonstrating that a concentration of 15 mmol/L can produce a high degree of pPKAs, although it takes longer. The absence (0 mmol/L) or low concentrations of HCO 3 − (5 mmol/L), similar to that existing in the cauda epididymis, did not enable PKAs activation even after 60 min of incubation, which shows that not only is the presence of HCO 3 − essential in this pathway but also a minimum concentration is required. HCO 3 − is a key activator of a cascade of events in capacitation and a minimum concentration and incubation time is necessary to trigger the capacitation process and produce a high pPKAs, despite the presence of other capacitating agents like calcium and albumin. 15 mmol/L of HCO 3 − seems to be a sufficient concentration to allow epididymal sperm capacitation. Nevertheless, the importance of HCO 3 − not only lies in the activation of PKAs but also the fact that it produces another series of events essential for sperm physiology. The increase in intracellular pH conditions, a phenomenon during capacitation (reviewed by Nishigaki et al. [39]) and the Adcy10/cAMP/PKA pathway are not an exception.
Epididymal spermatozoa in the cauda epididymis remain in a quiescent state favoured by low HCO 3 − content and acidic pH [10]. The present study showed that, in the absence of HCO 3 − and the presence of other capacitating agents like calcium and albumin, the pH i remained at 6.7-6.8. In the presence of HCO 3 − , the pH i increased 0.3-0.4 units, confirming that the increase depends exclusively on HCO 3 − . This increase of around 0.3-0.4 units in pH i may seem small, but, it has been demonstrated that an increase of 0.2 units of pH may modify enzymatic activity more than 20-fold [40]. It is known that the activity of intra-and extra-cellular enzymes taking part in the cellular metabolism is pH-sensitive.
The effect of HCO 3 − has been widely studied and there is evidence that extracellular HCO 3 − influences the motility of epididymal spermatozoa of several species [10,41,42]. In the present study, HCO 3 − concentration did not affect the proportion of motile spermatozoa during incubation in a capacitating medium, or their progressivity, but did affect parameters related to sperm trajectory. Holt and Harrison [43] showed in ejaculated, preserved and selected spermatozoa by Percoll gradient that the straight and average velocities, straightness and linearity significantly increased with the addition of HCO 3 − to the incubation medium. The results of the present study presented evidence that HCO 3 − activates motility in epididymal spermatozoa and produces a change in their trajectory which becomes more linear (see Fig. 5) and rapid. These data suggest that HCO 3 − is not only important for motility activation in a balanced salt solution but also that the type of movement produced is concentration-dependent. A concentration of 15 mmol/L HCO 3 − during sperm capacitation produced a more linear movement, which has previously been related with the state of capacitation in boar [43,44].
The HCO 3 − induction of an increase in LIN in porcine is controversial, since it has been previously reported by Satake et al. [45] using 15 mmol/L HCO 3 − , but it is in clear contrast to other studies [46] in which hyperactivation of ejaculated preserved spermatozoa was related to a decreased LIN. However, sperm motility in capacitating conditions seems to be species-specific, for instance in rodents as rat and hamster the hyperactivated motility is characterised by a decreased LIN [47,48]. In our study, HCO 3 − concentrations lower than 15 mmol/L produced a more curvilinear trajectory, while higher concentrations (25 mmol/L) did not produce any extra improvement over the value obtained at 15 mmol/L. However, each individual spermatozoon responds differently to HCO 3 − [45] and the clustering analysis identified 3 different sperm subpopulations according to their trajectory in response to HCO 3 − . In this case, the proportion of spermatozoa with more linear trajectory was higher in the presence of HCO 3 − . While a concentration of HCO 3 − from 15 mmol/L stimulated sperm motility and produced a change in the type of trajectory, no additional effect was obtained with 25 mmol/L. The role of HCO 3 − in sperm capacitation is not only restricted to the immediate effects on spermatozoa (increased pH i , activation of motility and pPKAs) but is also important in slow capacitation events such as the increase in Tyr-P and AR. In vitro sperm capacitation is accompanied by a time-dependent increase in the Tyr-P of a subset of proteins, as one of the final steps of the PKA-dependent cascade of capacitation [5]. Sperm incubated in a medium lacking HCO 3 − fails to undergo capacitation, but the process can be recovered by adding HCO 3 − in a concentration-dependent way [5]. The present study showed that a concentration of 15 mmol/L is sufficient to produce an intracellular alkalinisation and a high degree of Tyr-P in spermatozoa but no extra effect was observed when 25 mmol/L was used. Something similar occurred with Tyr-P in the flagellum of spermatozoa. A high degree of pattern IV was observed at 15 mmol/L and above, although no significantly improvement was observed in 25 mmol/L. Overall, this study agrees with those of other authors [49] who showed that HCO 3 − is required to activate Adcy10, which increases intracellular cAMP, promotes PKA activity and subsequent Tyr-P. The above-mentioned observations concerning the role of HCO 3 − concentration on sperm functionality have shown the modulating power of HCO 3 − on sperm physiology in capacitating conditions. However, whether this modulating effect influences the sperm fertilising ability during IVF has not been elucidated. In this study, the effect of HCO 3 − concentration on sperm fertility was investigated (Table 1A). We hypothesised that varying the HCO 3 − concentration in the porcine sperm capacitation process regulate their functionality and probably affect IVF output. Indeed, IVF was directly and strongly affected by the HCO 3 − concentration in the medium. The importance of HCO 3 − in the fertilisation process was confirmed by the fact that no oocyte was penetrated in the absence of HCO 3 − (0 mmol/L). This agrees with Suzuki et al. [50], who described how the absence of HCO 3 − in fertilisation media makes oocyte fertilisation impossible. In our study, we found that HCO 3 − exerts a concentration-dependent impact on fertilisation as penetration increased with increasing HCO 3 − concentrations.
In addition, the presence of HCO 3 − also upregulated the SPZ/O resulting in monospermy reduction.
The high incidence of polyspermy in porcine has been widely related with an unacceptably low efficiency of IVF [51]. Hunter and Nichol [52] hypothesised that the incidence of polyspermy is a direct consequence of the number of capacitated spermatozoa in the immediate vicinity of ovulated porcine oocytes in vivo. Later, Abeydeera and Day [21] also described this correlation under in vitro conditions. Results from this manuscript suggest that the effect on IVF of a specific HCO 3 − concentration is related with the timing of the capacitation events and their influence in monospermic fertilisation success. At the molecular level, our results indicate that 5 mmol/L did not activate PKAs while 25 mmol/L produce faster phosphorylation compared to 15 mmol/L. The slower PKAs activation of 15 mmol/L could be responsible for the lower penetration and the higher monospermy than observed with 25 mmol/L in the fertilisation experiments. Moreover, Funahashi [53] described that one of the strategies to reduce polyspermy in porcine is to induce full AR in partially reacted spermatozoa. However, the results of the AR assay (Additional file 5) showed that polyspermy is not related with the proportion of acrosome-reacted spermatozoa surrounding the egg. Concentrations of 15 and 25 mmol/L HCO 3 − in the capacitating media produce the same proportion of acrosome-reacted sperm after 60 min of incubation.
In addition to its effects in sperm penetration and monospermy parameters, HCO 3 − had a determining action in the Results are expressed as mean ± SEM. Different superscripts ( a-d ) in the same column indicate significant differences (P < 0.05) Pen, % penetration percentage of total oocytes used, Mon, % monospermy percentage calculated from penetrated oocytes, SPZ/O number of spermatozoa penetrating each oocyte, SPZ/ZP number of sperm bound to ZP, Efficiency, % final number of putative zygotes (monospermic) in each group per 100 penetrated oocyte primary recognition between gametes, since practically no spermatozoon was able to bind to ZP in the absence of HCO 3 − . As Flesch et al. [54] suggested, it is probable that the absence of HCO 3 − hindered certain capacitation events that resulted in reduced binding affinity of spermatozoa for ZP. One important question is the extent by which the influence of HCO 3 − in IVF is exerted on spermatozoa during capacitation or on gamete co-incubation during the fertilisation process. After sperm pre-incubation with 15 mmol/L, which produced a moderate penetration, when IVF was also carried out at 15 mmol/L, monospermy was significantly higher and the SPZ/O lower than in the rest of groups, which resulted in a higher final efficiency (33.9%). What it is clear is that oocyte penetration directly increased with the HCO 3 − concentration in the sperm pre-incubation medium suggesting that the action of HCO 3 − is exerted more on spermatozoa than on oocytes. Very few studies have described the effect of HCO 3 − on in vitro matured oocytes, but Suzuki et al. [50] suggested that, although HCO 3 − was apparently affecting gamete interaction, it is highly probable that HCO 3 − affects fertilisation through its effect on spermatozoa rather than through any effect on the oocytes.

Conclusion
Altogether our results suggest that 15 mmol/L HCO 3 − is sufficient for IVF media because it produces a high degree of sperm capacitation and maximises IVF efficiency in porcine. In conclusion, adjusting the HCO 3 − concentration of the medium during in vitro epididymal sperm capacitation and fertilisation allowed us to increase the monospermy efficiency of these in vitro processes in porcine, which are currently suboptimal. In addition, a concentration of 15 mmol/L HCO 3 − in the capacitating medium is also sufficient to activate the sperm capacitation cascade and induce the slower progression of the sAC/cAMP/PKA pathway. A concentration of 15 mmol/ L produces a sufficient level of activation of sperm motility, pH i increase and Tyr-P, maintaining high levels of fertilizing ability and increased rates of monospermy.

Additional files
Additional file 1: Effect of HCO 3 − on boar epididymal sperm intracellular pH (pH i ). A) Calibration of the system in which fluorescence emission ratio 490/440 nm was detected when stained spermatozoa with BCECF-AM were exposed to different extracellular pH (pH e ) in the presence of 0.1% Triton X-100 and excited both at 490 and 440 nm. The regression line for pH e vs. the 490/440 nm ratio was obtained (y = 3.048x + 15.534) and pH i of sperm cells was estimated from that regression line.