In vivo measurement of pH, CO2 and HCO3- levels in the uterus of non-anesthetized sows through the estrous cycle and after insemination

The pH-CO 2 -HCO 3− system is a ubiquitous biological regulator with important functional implications for reproduction. Knowledge of the physiological values of its components is relevant for reproductive biology and the optimization of Assistant Reproductive Techniques (ARTs). In vivo pH of the oviduct and uterus has been estimated by direct in situ measurements in a few species. However, regarding the levels of CO 2 and HCO 3− , information is very scarce and, when available, it comes from uid samples instead of in vivo estimations. This study describes a non-invasive method to measure pH and % of CO 2 in the uterus of sows with cutting-edge technology and no medication. Sows were at three different reproductive conditions, estrous with no insemination E(-)AI and after insemination E(+)AI, and diestrous (non-estrous, NE). From pH and CO 2 data, HCO 3− concentration was estimated. The designed methodology allowed for in situ time-lapse recording of pH and % of CO 2 within the uterus of non-anesthetized sows. Variable oscillatory patterns of pH, CO 2 and HCO 3− were found independently of the estrous condition. Insemination did not changed the levels of uterine pH, % of CO 2 and HCO 3− concentration, -E(-)AI = E(+)AI-, but all the values were affected by the estrous cycle in a way that decreased signicantly at diestrous condition - E(-)AI and E(+)AI > NE-.

The success of fertilization starts with sperm migration within an adequate microenvironment of the female genital tract. Both, H + and HCO 3 − have a pivotal role in regulating sperm fertilization capacity, which is progressively achieved along their trajectory from the epididymis to the fertilization site in the oviduct through a gradient of increased pH and HCO 3 − concentrations [3][4][5]. Both components are directly involved in many biochemical reactions resulting in the so called sperm ∩ acitation, a process necessary for fertilization [6,7]. HCO 3 − and pH play also an important role for embryo transport, development and implantation [2,8]. The production and secretion of HCO 3 − and H + in the uterus, although not fully elucidated, includes different exchangers [9,10] which are sensitive to sex-steroids hormones[8, [10][11][12][13].
Reference in vivo levels of pH, CO 2 and HCO 3 − in the uterus and oviduct of several mammals (rabbit, monkey, human, bovine, equine or porcine) have been obtained during different stages of the estrous cycle (see Table 1), while other values were obtained from oviductal or uterine uid samples [14,15]. pH is the best characterized parameter. Uterus and oviduct pH vary between species (range from 6.83 to 7.35 and 6.7-8. 3, respectively) and is in uenced by the stage of the estrous cycle (Table 1). Contrastingly, no HCO 3 − values have been recorded directly from the uterus, so the available data were obtained from oviductal uid, either directly or from samples [14][15][16]. Likewise, very few studies and with minimal explicit information have been aimed at determining CO 2 tension (pCO 2 ) within the uterus or oviduct [14,16]. In the porcine species, pH values in the oviduct are very variable (ranged from 6.7 to 8.3) depending on the study and the stage of the cycle [15,17,18], while HCO 3 − levels of 10.0-33.1 mMol were estimated in samples of oviductal uid retrieved under surgical conditions [15]. In the uterus, pH (6.98) was only measured in a preliminary study in a series of 6 gilts [17], with no mention to CO 2 or HCO 3 -values. Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js Table 1 Summary of the studies measuring pH, CO 2 and/or HCO 3 −  has an impact on the physiological condition. Hugentobler et al. [19] compared the in uence of the anesthetic protocol on oviductal and uterine pH in cows. When intravenous anesthesia (thiopentone) was compared with inhalatory anesthesia (halothane) oviduct and blood pH was reduced, whereas uterus pH increased. For this reason, the most reliable results in this type of approaches are those with minimum or complete absence of pre-medication. This has been achieved in cows [20,21] and mares [22], where a non-invasive external approach to the uterine lumen via the cervix allowed direct pH measurements. However, until now a similar approach to the uterus has not been tried yet in pigs.
In most previous studies the in vivo measurement of uterine pH was carried out with potentiometric devices. For this, miniaturized pH glass electrodes either as a single unit or in combination with a reference electrode were extensively used [15][16][17][18][19][20][21][22][23][24][25]. Similarly, for pCO 2 measurements a pH electrode was converted to a Severinghaus type electrode covered with a Te on membrane [16]. In recent years, optical sensors have become a convenient alternative in several application areas of research, including biological systems and organisms. Different principles and working mechanisms are used depending on the analyte to be measured [26] and for optical pH and CO 2 measurements, a combination of different uorescent dyes which detect intensity changes in the time domain are used. Although optical based probes were used to assess in vivo oxygen levels within the female reproductive tract of humans [27] and pigs [28], to our knowledge, this technology has never been used in reproductive organs for pH and CO 2 measurement.
The porcine species is being widely used as an experimental animal model in biomedicine because of its similarities with humans in many aspects (i.e. genomics and immunology system) [29,30]. Despite the differences observed in the female genital tract when compared with humans, porcine arises as a good alternative to other commonly used animal models such as rodents. In addition, to produce these porcine biomodels in a standardized scale, assistant reproductive techniques (ARTs) are necessary [31,32]. However, porcine in vitro fertilization (IVF) is far from being as e cient as in other species as rodents or bovine [32]. The main problem is polyspermy (more than one spermatozoon penetrates the oocyte), which has been related with the protocols of gamete preparation, and the medium and ambient conditions stablished during the co-incubation of gametes, which makes the zygote non-viable. Thus, when the ambient parameters mimic the physiological values, as it was recently demonstrated by adjusting the oxygen level for IVF and embryo culture [28], porcine ARTs yield better results. With regard to the pH-CO 2 -HCO 3 − levels, the sperm cells and oocytes used for IVF are directly immersed in a culture media whose values for these parameters might not be similar to the physiological. This is mainly due to the absence of appropriate knowledge about the real gures existing in the living animal. The same problem is found for the protocols of sperm preparation, since no consideration is taken towards the transitional period confronted by the sperm cells in their passage throughout the uterus. Given that there is an almost complete lack of knowledge about the regulatory mechanisms of uterine pH [19], whether the uterine ambient, and particularly its luminal pH-CO 2 -HCO 3 − levels are modulated by the presence of sperm is a topic which deserves further attention.

Animals and study design
The experiment was conducted on 12 sows [Landrace x commercial hybrids (Large White and Duroc) with 2.4 parities in average]. All animals showed normal reproductive performance previous to the experiments (total piglets born per litter 13.5 ± 1.0; live born per litter 10.9 ± 0.7). During the study, sows were housed in individual pens and fed by a commercial diet twice a day. Water was provided ad libitum. Environment was controlled by mechanical ventilation and evaporative cooling systems (temperature 22-24 ºC). Multiparous sows were weaned 28 days after farrowing. Thenceforth, sows were observed twice daily for estrous behavior. Those exhibiting vulva reddening and swelling, and a standing re ex were considered in estrous.
pH and CO 2 measurements were carried out at three different conditions (Fig. 1). The rst set of measurements was done twenty-four hours after estrous detection [E(-)AI]. Immediately after this procedure, the females were arti cially inseminated and 2 hours later pH and CO 2 measurements were repeated [E(+)AI]. For the insemination, a single dose of diluted semen was used. The inclusion criteria to use the seminal doses was total motility > 70%, viable sperm > 90% and morpho-abnomalies ≤ 15%. Postcervical AI (seminal dose of 1.5 × 10 9 sperm/45 ml) was performed with a disposable catheter (Soft&Quick®, TecnoVet S.L., Spain) [33]. Finally, 7-10 days later, when the animals were at diestrous stage (non-estrous [NE]) the same procedure as at E(-)AI condition was repeated.
Measurement of pH and CO 2 pH and CO 2 optical dipping probes − 1500 mm long and 3 mm thick-were used (DP-HP5 and DP-CD1 prototypes, respectively, PreSens Precision Sensing GmbH -Am BioPark 11-93053 Regensburg, Germany) ( Fig. 2A). Each probe was plugged into a recording unit (pH1 and PCO 2 mini, PreSens Precision Sensing GmbH), which was connected to a laptop. Probes were pre-calibrated by the manufacturer, but calibration was also checked the day before use with standard pH 7 solution (Crison Instruments S.A., Spain), or 5% CO 2 saturated saline solution (0.9%) within an embryo incubator. As pH and CO 2 values are in uenced by temperature, uterine temperature was always measured before inserting the optical probes.
This was done with a 1500 mm long and 0.5 mm thick thermistor connected to an ampli er device (ThermaDataTM Logger TCD; E.T.I. Electronic Temperature Instruments, West Sussex, UK).
At the three sampling conditions the following routine was followed. Brie y, a disposable post-cervical catheter (Soft&Quick®, TecnoVet S.L., Spain) was used to facilitate the approach to the uterine lumen (body and horns) with the probes (Fig. 2B). When the catheter was positioned in the cervix (Fig. 2C), the inner cannula was gently pushed cranially to "help to open" the cervical lumen (Fig. 2D). The inner cannula was then removed and replaced by an endovascular catheter − 900 mm long and 2.92 mm inner diameter-(reference 54-89001, Terumo Europe), which allowed the temperature probe to reach the uterine cavity. Temperature was recorded for 10 min, and the stable temperature value used as a reference for pH and CO 2 measurement in each animal. The endovascular catheter was then replaced by the pH probe, which was pushed cranially through the insemination catheter until no further progression was possible (Fig. 2E). When maximum insertion was reached, estimation of the insertion length within the reproductive tract was calculated by subtracting the total length of the probe to the distance from the rima vulvae to the recording unit. After a period of stabilization (2-5 min), and an indication to measure at the precise de ned temperature, pH data were recorded every 5 seconds for a total of 10 min (Fig. 2F).
The pH probe was then replaced by the CO 2 probe, and the same procedure was followed for the recording of CO 2 values. It is worth of mention that every time a probe was replaced the external opening of the insemination catheter was kept sealed to avoid the penetration of air within the cervix. All the protocol was done without any medication. In general terms the technique used to approach the uterine lumen with the probes was successful. After implanting the insemination catheter in the cervix, the progress of the inner cannula up to 16 cm towards the uterine cavity helped to widen the distal portion of the cervical canal. Then, the successive approaches with the endovascular catheter -with temperature probe inside-, the pH and CO 2 probes were successful in nearly all the animals. One subjective but unequivocal sign of having reached the uterine cavity was the hand-feeling of a smooth progression of the probe after having overpassed the inner most cervical cushion. However, in two sows at NE condition, the deepest part of the cervical canal did not allow for further progress of the pH and CO 2 probes. On the other hand, CO 2 data from one pig at the three experimental situations were dismissed because inaccurate readings of the sensor, and from another pig at E(+)AI because of some bleeding was observed. Hence, the approach was successful in 68 out of 72 attempts (94.4%, pH and CO 2 probes in 12 animals at three different conditions) and the recording of data in 64 occasions (88.9%). The average insertion length of the pH and CO 2 probes was 64.2 ± 12.6 cm and 62.0 ± 11.5 cm, respectively. The average temperature of the uterus was 38.2 ± 0.3 ºC.
pH measurements within the uterus during estrous (before and after insemination) and diestrous stages Individual plots of the timeline progress of all recorded pH values were represented ( Fig. 3A-C). In some cases, the timeline depicted waved pattern, with several variations higher than 0.2 pH units (Fig. 3A), while other displayed a quite a at pattern with few and smooth waves (Fig. 3C). Characterization of at and waved patterns was attempted depending on the number of waves higher than an established criterion, i.e. "a waved pattern displays > 2 waves of > 0.2 pH units". However, as at and waved patterns were found in animals of the three experimental conditions, and even within the same animal (i.e. Figure 3A, red and blue lines looked like a waved pattern while green line was rather at) no statistical association between the de ned pattern and the experimental conditions was found (Chi-squared test P > 0.05).
A comparison of the uterine pH with statistical signi cance between the three experimental conditions is shown in Fig. 4. An immediate effect of AI on the uterus pH was dismissed. Although the average pH in E(-)AI sows was a bit lower than in the E(+)AI group (0.06 units), such a small difference was not found to be signi cant (7.05 ± 0.13 and 7.11 ± 0.16, respectively, P > 0.05). However, uterine pH in sows of the NE group (6.93 ± 0.16) was signi cantly lower than in groups E(-)AI and E(+)AI (P = 0.017 and P = 0.007, respectively), revealing a signi cant increase of the uterus pH at estrous. CO 2 (%) levels within the uterus during estrous (before and after insemination) and diestrous stages Plots of the timeline for the recorded % of CO 2 were obtained for each pig (Fig. 3D-F). As for the pH, the timeline variation of CO 2 % described arbitrary curves with a variable number of undulations, indistinctly of the experimental group and individual.
A by-group comparison of the data with statistical signi cance is displayed in Fig. 4. The average % of uterine CO 2 in E(-)AI group was 14.45 ± 3.58, and in E(+)AI group 13.12 ± 5.09. No effect of AI on the uterine % CO 2 was observed (P > 0.05), so the presence of sperm in the uterus for 2 hours hardly changed the % CO 2 . Conversely, the average uterine % CO 2 at NE condition was 8.44 ± 3.71, representing a very strong decrease between estrous and diestrous stages (P NE vs E(−)AI = 0.0002 and P NE vs E(+)AI = 0.0019).

Discussion
The in vivo characterization of the physiological ambient within the female reproductive organs of mammals is important for both basic studies in the eld of reproductive biology and ARTs. For this reason, the knowledge of the physical and chemical properties of the reproductive milieu by in situ estimation or after uid collection from the oviduct, uterus and vagina has been largely attempted in humans, livestock and animal models [27,34]. With regards to the pH-CO 2 -HCO 3 − biological regulator, very few studies have been addressed to the uterus (Table 1), which has only been approached in a few livestock species, but mainly cows. In pigs, only one work measured the uterine pH in vivo to a reduced number of animals via laparotomy [17]. In the present work, a more extensive study has been carried out and reference values of pH-CO 2 -HCO 3 − levels in the porcine uterus have been obtained at two different stages of the estrous cycle and after insemination. Besides, to minimize iatrogenesis, all measurements were performed via a completely non-invasive cervical approach and without any medication. The combined use of a post-cervical AI catheter with miniaturized and semirigid pH and CO 2 dipping probes allowed successful approaches to the uterine cavity (body and horns) in more than 90% of the attempts. Although the method did not give a visual evidence of the position of the sensors inside the uterine cavity, it was validated by both the perception of a smooth progression of the probes after having crossed the cervical canal (subjective), and the gures of insertion length of the probes (objective). The latest criterium was veri ed by comparing our results with reported gures of the length of the reproductive tract -from the rima vulvae to the uterine cavity-measured in ex-vivo organs of multiparous sows [35]. Our estimated insertion length for the pH and CO 2 probes − 64.2 ± 12.6 cm and 62 ± 11.5 cm, respectivelywas higher than the reported post-mortem length of those organs (56.25 ± 6.01 cm). On the other hand, the total absence of medication or animal distress during the procedure guaranteed that the measurements were the closest to the physiological values, as it was also the case when a similar postcervical approach was carried out for estimation of uterine pH in cows [20,21,25].
Another novelty of the present work is the use of optical probes for the estimation of pH and CO 2 . Up to now, virtually all in vivo measurements of pH in the female reproductive organs were based on a miniaturized pH glass electrode combined with a reference electrode. While this technology proved useful and accurate for pH estimation, that was not the situation for CO 2 . Miniaturization of CO 2 potentiometric sensors is technically more limited because a Severinghaus-type electrode is built by covering the pH microelectrode with a cap and a Te on membrane in the tip. In addition to size restrictions for in vivo usage, the potentiometric CO 2 sensors have scattered readings when immersed in complex uid matrix such as the oviductal or uterine uids ([16]; our experience with unpublished results). This most probably explains the lack of previous reports for CO 2 estimation in vivo (Table 1). Unlike the potentiometric sensors, luminescent (optical) probes can be miniaturized to a few microns and display stable and accurate readings when immersed in complex biological matrix such as the reproductive uids. This has already been proved for the determination of oxygen levels in vivo in reproductive organs of women and pigs [27,28]. For the rst time, this work describes how pH and CO 2 levels can be accurately measured in pigs with optical dipping probes directly inserted in the uterus. This is more relevant because from pH and CO 2 levels, HCO 3 − concentration was estimated, hence allowing a complete in vivo characterization of the bicarbonate buffer system. The relevance of characterizing this system for the optimization of ARTs protocols and the fact that the physical characteristics of the sensors (length, thickness, exibility) can be customized under the manufacturer´s advice envisages an increasing usage of this technology in other organs and/or species.
One remarkable result obtained from the experiments was that pH, CO 2 and HCO 3 − levels were higher during the estrous stage independently of the insemination condition. It is worth noting that the levels of reproductive hormones are quite different at estrous and diestrous stages. While the estrous is mainly dominated by estrogens, the situation changes during the rst days of diestrous, when progesterone levels start to raise (reviewed by [36]). There are some evidences pointing that such hormonal changes have an impact on the pH-CO 2 -HCO 3 − system. In rats, the expression of the different isoforms of carbonic anhydrase in the uterine tissue is directly regulated by the amount of estrogen and progesterone [37], which suggests that the physiological uterine levels of pH-CO 2 -HCO 3 − are dynamic and likely modi ed by  [34].
Also, CFTR (cystic brosis transmembrane conductance regulator), which is responsible of numerous secretory responses, is an active regulator of the uterine secretion of HCO 3 − [9,39]. CFTR expression is enhanced in the uterus of females in early estrous but not in other stages [11] where progesterone is enhanced [40,41], suggesting a higher secretion of HCO 3 − during estrous which is in accordance with our observations. Hydration of CO 2 via carbonic anhydrase is another likely mechanism by which HCO 3 − accumulates in the uterine uid [14]. Likewise, in the case of pH our ndings are in agreement with the literature, where a more alkaline uterine environment is necessary for sperm transport, capacitation and fertilization [39] as occurs during the estrous stage, while an acidic pH, as observed in diestrous, is essential for embryo post-implantation [42].
Therefore, during estrous the uterine ambient is prepared to receive the spermatozoa, whose high levels of HCO 3 − and alkaline environment, among other components, are necessary to initiate the sperm capacitation, a necessary process for fertilization. It has been previously demonstrated that semen deposition causes a local shift in the gene expression of the female genital tract [43,44], including some genes involved in pH regulation [45]. However, our results showed that the pH-CO 2 -HCO 3 − system did not change after semen deposition within the uterus. Nevertheless, several factors could be involved in this fact. First, it has been shown that seminal plasma proteins and other components activated changes in the gene expression of the endometrium [44,46,47]; but in our study, the level of seminal plasma in the insemination dose was low because the ejaculate was diluted in commercial extender, so its impact on the genomic expression of the endometrium could be limited. Second, although uterus is colonized by the sperm within minutes after deposition [48] the differences detected in gene expression were evaluated 24 h after semen deposition [43][44][45], while our measurements were carried out only 2 hours after insemination. Thus, further studies with different time-points after insemination are necessary for a better understanding of the interactions between the uterine ambient and the sperm.
In vivo approaches to characterize the reproductive milieu such as the one in this study may have important bene ts for the e ciency of ARTs. It has been already mentioned that when the physiological O 2 tension found in the oviduct of sows was mimicked in the laboratory, both the nal e ciency of  [49,50]. Also, an in uence of the uterine uid composition on sperm selection and consequent embryo culture was observed in vitro in several species including porcine [51][52][53]. But, in spite of these ndings, the current laboratory conditions of boar sperm preparation prior to IVF are yet quite standard in the literature, using a temperature of 38.5 ºC, 5% CO 2 , 15-25 mMol of HCO 3 − and ~ 7.2-7.4 pH. This is relevant because such conditions are far from those observed in our study at the estrous stage, not only in their particular gures (except the temperature) but also in the fact that they are not steady but rather dynamic or even oscillatory. Therefore, as sperm preparation methods should be as physiological as possible [32]

Conclusions
This study shows for the rst time the combined values of pH, CO 2 , and HCO 3 − in the uterus of the sow.
By using a non-invasive approach and no medication during the procedure, precise reference values were obtained at different stages of the estrous cycle, and after insemination. The presence of sperm in the uterus hardly affected these parameters but were highly in uenced by the estrous cycle, so that higher gures of pH, CO 2 , and HCO 3 − were found at estrous than at non-estrous stage (diestrous). The study contributes to a better understanding of the in vivo regulation of the pH-CO 2 -HCO 3 − system in the uterus.
Based on our ndings, and trying to mimic the physiological ambient, a new set of parameters may be established for the in vitro media where sperm are treated prior to IVF.    Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js