Acid–Base, Gas, Ions, and Glucose Analysis in Follicular Fluid in Holstein-Friesian Dairy Cows Is Associated with the Follicle Size in Poland

Simple Summary Reproductive failures in dairy farms are extremely costly for breeders; however, the cause of loss is often very hard to determine. The environment may reflect an influence on the quality of the oocyte follicular fluid (FF). It was suggested that FF can be used as an indicator for the functional status of the ovarian follicle in bovine species, however, the topic is still not well known. This study aimed to investigate the variations in FF parameters connected with acid–base balance, ions, and glucose analysis. It was proven that changes in the FF formula were associated with the follicle size. Abstract The lack of fertilization and early pregnancy loss is seen in the quality and maturation of the oocytes. The environment of the first divisions and maturation of the oogonia, which is also a reflection of the quality of the oocyte, is the follicular fluid (FF). Thus, the purpose of this study was to investigate the variations in parameters such as pH, pCO2, pO2, standard HCO3−, actual HCO3−, base excess (BE), base excess of extracellular fluid (BE ecf), ctCO2, ions: Na+, K+, actual ionized calcium Ca2+, adjusted ionized calcium (at pH 7.4) Ca2+ (7.4), Cl−, anion gap (AnGap), and glucose in FF collected from different size follicles in dairy cattle. The most apparent differences were related to pH, K+, and Ca2+ 7.4 concentration in comparison to changes in follicle size (p < 0.05). Several trends were also evaluated as an increase in follicular size was followed by an increase in pH, BE, and Ca2+ 7.4 value and a decrease in the K+ concentration (p < 0.05). In conclusion, there are changes in FF formularies associated with the size of follicles. However, more research is necessary to establish the reference value, which then could be one of the factors describing the quality of the follicle and the developmental potential of the associated oocyte.


Introduction
Poland, with 1.7% of global milk production (14.1 billion liters), is the second largest dairy producer in the European Union [1]. However, the increase in milk production is accompanied by a decrease in the reproductive performance of dairy cows. Decreased reproductive efficiency is mostly connected with estrus cycle disorders. Anestrus is the main cause of ineffective insemination, increased insemination index, reduced fertilization rates, and extended time of inter-pregnancy and inter-calving period, which generates huge economic losses [2]. The above circumstances encourage the search for methods of faster and more effective genetic improvement in dairy cattle breeding and the field corresponding to the emerging needs in reproductive biotechnology. The used biotechniques such as in vivo and in vitro embryo production, are showing a constant increase around the world, even observable during the COVID-19 pandemic [3]. In 2020, more than 1.5 million bovine embryos were recorded, which represents an increase of 7.0% compared with 2019 (1,518,150 vs. 1,419,336, respectively) [3]. However, also in other species, there was a substantial increase in the numbers of in vivo-derived (IVD) (in horses +13.6%; sheep +33.3%; goats +51.0%), as well as in vitro-produced (IVP) embryos (horses +37.1%; goats +204.1%) [3]. Thus, taking into account these numbers, improving the biotechniques used in reproduction is essential.
However, the biotechnology of reproduction as a very sensitive and developing field requires constant research and a better understanding of the processes such as gamete maturation and fertilization. The most common reproductive biotechnology in all mammalian species is artificial insemination (AI), which includes sperm cryopreservation. In cattle nowadays, embryo production (ET) is the fastest developing segment as mentioned earlier.
Thus, in vitro bovine embryo production is important from an economic point of view, as well as in recent years it has become a model for humans.
Follicular fluid (FF) has been examined for many years to evaluate the oocyte microenvironment. FF provides nourishment for oocyte development such as proteins, lipids, ions, carbohydrates, etc. It strongly influences the environment of oocytes. It was suggested that the main problem of inferior oocyte and embryo quality is the FF quality rather than a disruption in gonadotropin secretion in high-yielding dairy cattle [4,5]. Thus, monitoring of FF composition can be used for non-invasive diagnosis.
The material can be obtained from the ovary of slaughtered animals [4][5][6] as well as from live animals in the course of the ovum during the pick-up procedure [7][8][9]. FF has several oocyte-related functions, creating together with the cumulus cells the intrafollicular environment that supports the acquisition of developmental competence in oocytes. FF also protects the oocyte against proteolysis and extrusion during ovulation [10], thereby influencing in vitro maturation and fertilization competence of bovine oocytes [11]. As a product of the transfer of blood plasma components through the blood-follicular barrier and of the metabolism of theca and granulosa cells, FF contains several hormones such as testosterone, estradiol-17β (E2), and progesterone (P4) and some growth factors. In addition, it seems to be convenient that FF might be a perfect maturation medium as it also influences the resumption of meiosis and cytoplasmic maturation of bovine oocytes [4,5]. Thus, optimization of the in vitro maturation environment is vital for generating viable embryos.
As mentioned earlier, diluted FF is also used in media supporting the bovine oocyte maturation in vitro or for the conservation of oocytes. It was postulated that the changes in the composition of FF may be used as an evaluation parameter of oocyte quality, which can be directly related to fertility [12]. In the study performed by Matoba et al., 2014, they confirmed that analysis based on the follicular fluid metabolite profile is the best predictor of oocyte developmental competence [12]. As it was mentioned earlier FF is also used as medium enrichment. Thus, research connected with that topic is of great importance as the FF contains proteins, steroids, glycoproteins, and fatty acids-the compounds which contribute to the metabolism of cells and follicular oocytes.
However, there are only several studies connected with acid-base balance and gas analysis, and most of them are older than 15 years and/or have only evaluated a limited number of parameters [13][14][15]. Maintenance of the acid-base balance as well as equilibrium in ion concentration is fundamental for the normal functioning of biological processes, mainly due to the pH dependence of enzyme function [16]. Thus, knowledge of changes in those parameters is essential for the oocyte quality understanding. Broadening the knowledge about this topic will help with embryo transfer procedures by obtaining better quality oocytes as well as a more suitable medium for culturing them during maturation. Thus, it may be applied in the future to farm animal production.
In addition, the embryo production success rate depends also on the follicle size which is probably connected with different FF compositions [10]. In human medicine, the in vitro treatment is follicle size rather than their competence. A good example of that is that the timing of administration of hCG is mostly guided by the size of the lead follicle or lead follicular cohort. However, there is a lack of studies connected with this topic. The hypothesis is that the FF acid-base, gas, and ion composition vary in different size follicles. Thus, this study investigates the changes in the acid-base, gas, and ion concentrations in FF obtained from different size follicles in dairy cows.

Biological Samples
The samples were collected post-mortem in the slaughterhouse, which does not fall under the legislation for the protection of animals used for scientific purposes, national decree-law (Dz. U. 2015 poz. 266), and European Union law (2010-63-EU directive). Thus, no Ethical Committee's permission was needed for sample retrieval after slaughter.
FF was collected from 44 slaughtered cows (Holstein Friesian-HF breed). The inclusion criteria were good health based on veterinary clinical examination and normal reproductive tracts upon macroscopical examination after slaughter. In addition, the cows were assessed pre-slaughter by ultrasound examination of the reproductive tract. All examined cows were multiparous. Fluid from 1 to 2 follicles was collected from each ovary. The follicles were divided into 3 groups, according to their size: I: 5 to 10 mm diameter (small follicles; n = 16); II: 11 to 25 mm (dominant follicles; n = 24), III: >30 mm (cysts; n = 4). FF was obtained by aspiration directly into 1-mL gasometric and 3-mL plain tubes (Monovette 1mL LH, Sarstedt, Nümbrecht, Germany). The expulsion of the air bubbles from the syringes was performed immediately after collection, syringes were capped with a rubber stopper, and the syringes were stored on ice. FF was centrifuged at 1000× g for 1 min and the supernatant was stored at 4 • C until analysis.

Acid-Base, Gas, and Biochemical Analysis of FF
The supernatant was examined a maximum of 5 min after slaughtering. The following parameters were marked and calculated: pH; pCO 2 -partial pressure of carbon dioxide; pO 2 -partial pressure of oxygen; HCO 3 -act-actual bicarbonate concentration; HCO 3 -std-standard bicarbonate concentration; BE(B)-base excess; BE ecf-base excess of extracellular fluid; ctCO 2 -total carbon dioxide concentration; Na + , K + , Ca 2+ -actual ionized calcium; Ca++7.4-adjusted ionized calcium at pH 7.4; Cl − , AnGap-anion gap; and cAMP and glucose (Glu) FF concentration. The oximetric parameters were assessed using a critical points analyzer, RAPIDPoint 500 (Siemens, Erlangen, Germany). The biochemical parameters were assessed using the analyzer, BS-120 (Shenzhen, China).
As there is no recommendation on how to perform the FF evaluation, the measurements were carried out as recommended by the National Committee of Blood Laboratory Standards (Considerations in the Simultaneous Measurement of Blood Gases, Electrolytes and Related Analytes in Whole Blood; Proposed Guidelines). For each test, the analyzer's operating temperature was set according to the bovine rectal temperature recorded during sampling.

Statistical Analysis
Statistical analysis was performed in PQStat 1.6.4.121. (Poznan, Poland.). The numerical variables were given as the arithmetic median and standard deviation (SD), or the interquartile range (IQR), unless the variable was normally distributed according to the Shapiro-Wilk W test. The range was presented in all cases. Between-group comparisons were performed using the Kruskal-Wallis H test along with Dunn-Bonferroni's post hoc test, unless the variable was normally distributed. Additionally, a Jonckheere-Terpstra trend test was performed. The significance level was set at p < 0.05, and a test probability was considered highly significant when p < 0.01.

Results
The values for the examined FF parameters in cattle from the different groups are presented in Table 1. Table 1. Selected acid-base, gas, ions, and glucose concentrations were measured in follicular fluid in three groups depending on the size of the ovarian follicle. Data are presented as arithmetic mean and standard deviation. Different letters (a, ab, b) mean statistically significant difference (p < 0.05). Data withˆpresent a significant trend. Group I: 5 to 10 mm diameter (small follicles; n = 16); II: 11 to 25 mm (dominant follicles; n = 24), III: >30 mm (cysts; n = 4). The pH was the lowest in Group I in comparison to the other groups ( Figure 1). There was no statistically significant difference between groups, but a trend that confirmed that the decrease in follicle size was related to a lower pH value (p < 0.05) was observed. There were no significant changes between HCO 3 -std ( Figure 2), BE ecf, and BE(B) (Figure 3) values, however, there was a significant trend that confirmed an increase of these parameters connected with the increase in follicle size (p < 0.05).

Group
In ions analysis, the concentration of K + and Ca++74 in FF was significantly variable among the groups. The K + concentration was the highest in Group I (p < 0.01) and there was a trend confirming that the higher the follicular size, the lower the K + FF concentration (p < 0.01) (Figure 4). The Ca++7.4 FF concentration was the lowest in the Group I (p < 0.05) and there was a significant positive correlation found between the Ca++7.4 FF concentration and the follicular size (p < 0.05) ( Figure 5).
There were no statistically significant changes in pCO 2 , pO 2 HCO 3 -act ctCO 2 AnGap, and ions such as Na + , Ca 2+ , Cl − , and Glu FF concentration between groups.

Discussion
In the present study, follicular fluid was sampled separately for each cow and each follicle which was unique in comparison to previous studies [13,16]. There are only a limited number of articles published in the changes in acid-base, gas, and ions FF concentrations in comparison to different sized follicles in dairy cattle. Parameters such as pH, BE(B), BE ecf, pCO2, pO2, HCO3-act, HCO3-std, AnGap, and ions all together in FF have never been examined in bovine species. Recent studies focus mostly on the follicular fluid metabolome [17,18], whereas routine parameters were never evaluated which may be measured in clinical practice. Of course, pre-ovulatory follicle diameter and follicular fluid metabolome profiles are of high value but because of high cost and low availability, nowadays they cannot be used in veterinary everyday practice.
Acid-base disorders can threaten the proper process of animal reproduction. There are three main fundamental mechanisms: chemical buffering, respiratory regulation, and kidney regulation. There is only one study in which the comparison between values of the routine used acid-base balance parameters in follicular fluid and venous blood in dairy cows and heifers was performed [19]. However, only limited parameters were evaluated such as pH, pCO2 and pO2, HCO3 − , and BE and there was no evaluation of the influence of follicular size. Thus, the comparison is very hard to perform.
In our study, the FF pH had a positive correlation with follicular size, which is in line with other findings [9]. It was documented that the tendency for higher pH in FF from follicles derived from stimulated estrus exists. It was suspected that the main cause of that was a change in pCO2 in FF, which may be supported by the Henderson-Hasselbach equation [pH = 6.1 + log 10 (HCO3 − /α × pCO2)]. However, in our study, the pCO2 was constant, whereas the HCO3-std was changing as well as BE. Buffer systems carry the most of carbon dioxide (CO2), thus pCO2 correlates with HCO3 − values. In many studies, the

Discussion
In the present study, follicular fluid was sampled separately for each cow and each follicle which was unique in comparison to previous studies [13,16]. There are only a limited number of articles published in the changes in acid-base, gas, and ions FF concentrations in comparison to different sized follicles in dairy cattle. Parameters such as pH, BE(B), BE ecf, pCO 2 , pO 2 , HCO 3 -act, HCO 3 -std, AnGap, and ions all together in FF have never been examined in bovine species. Recent studies focus mostly on the follicular fluid metabolome [17,18], whereas routine parameters were never evaluated which may be measured in clinical practice. Of course, pre-ovulatory follicle diameter and follicular fluid metabolome profiles are of high value but because of high cost and low availability, nowadays they cannot be used in veterinary everyday practice.
Acid-base disorders can threaten the proper process of animal reproduction. There are three main fundamental mechanisms: chemical buffering, respiratory regulation, and kidney regulation. There is only one study in which the comparison between values of the routine used acid-base balance parameters in follicular fluid and venous blood in dairy cows and heifers was performed [19]. However, only limited parameters were evaluated such as pH, pCO 2 and pO 2 , HCO 3 − , and BE and there was no evaluation of the influence of follicular size. Thus, the comparison is very hard to perform.
In our study, the FF pH had a positive correlation with follicular size, which is in line with other findings [9]. It was documented that the tendency for higher pH in FF from follicles derived from stimulated estrus exists. It was suspected that the main cause of that was a change in pCO 2 in FF, which may be supported by the Henderson-Hasselbach equation [pH = 6.1 + log 10 (HCO 3 − /α × pCO 2 )]. However, in our study, the pCO 2 was constant, whereas the HCO 3 -std was changing as well as BE. Buffer systems carry the most of carbon dioxide (CO 2 ), thus pCO 2 correlates with HCO 3 − values. In many studies, the monitoring of blood pH is suggested as useful for adjusting health, we also found FF pH value as important [20,21].
The HCO 3 − production is related to the ions concentration. Ions such as Na + , K + , Ca 2+ , and Cl − play major roles in determining the acid-base balance in biological fluids [22]. They have an impact on the acid-base and water balance, the cell membranes pumping systems, and the energy balance influencing the synthesis of milk ingredients such as lactose, etc. [22].
One of the most important ions in reproduction is Ca 2+ which influences estrogen synthesis. Its concentration increases during follicle development [23], which was also confirmed in our study. In addition, the importance of calcium signaling was confirmed in research connected with various artificial oocyte activation (AOA) methods [24], suggesting a higher fertilization rate when the Ca 2+ concentration increases. The increased fertilization rate (approximately 29% ICSI vs. approximately 50% ICSI-Ca) was confirmed and it was obtained by ICSI media calcium supplementation (Intracytoplasmic Sperm Injection) compared with the control group in humans [25]. On the other hand, the implantation capacity may be decreased by low calcium concentrations, while impaired development after implantation may be caused by too high a Ca 2+ level [25]. Increased Ca 2+ concentration may lead to the atypical expression of calcium-related protein genes or the production of reactive oxygen species (ROS). The dynamics of the Ca 2+ demand vary significantly from immature to mature oocytes and the cumulus complex. The decrease in Ca 2+ stores at the end of maturation reflects the activity of Ca 2+ in the plasma membrane and the decrease in intercellular communication by gap junctions between cumulus cells observed during meiosis. However, the study on low calcium level oocyte in vitro maturation revealed the delayed extrusion of the first polar body (PB1), and the impaired oocyte cytoplasmic maturation, including mitochondrial and endoplasmic reticulum distribution in cattle [26]. This finding suggests that bovine oocytes that are ready for fertilization have a lower requirement for Ca 2+ stores, but the process of achieving full readiness for fertilization, metaphase II, requires an adequate concentration of calcium. This observation may confirm the difference between bovine and mouse oocytes as previously described by Boni (2002) [4]. However, the exact calcium-dependent mechanism is unknown and studies describing the bovine FF Ca 2+ concentration are unique.
In our study, only the Ca++7.4 FF concentration was correlated with the follicular size. This is probably connected with the sample handling. In our study, we also decided to perform Ca++7.4 evaluation to avoid the potential confounding effects of ex vivo changes to serum pH. As a means to correct pH change and avoid the sample handling requirements, laboratorians have developed pH adjustment equations [27,28]. Proteins (particularly albumin) may bind additional hydrogen ions. So, when the pH decreases, hydrogen ions effectively compete with free calcium for available negative charges on proteins, and the protein-bound calcium level decreases whereas the circulating free calcium blood content increases. Thus, measuring the adjusted ionized calcium at pH 7.4 is beneficial for this study.
Furthermore, calcium-activated potassium channels activation is connected with the concentration of Ca 2+ [29]. Thus, when the K + concentration decreases, the Ca 2+ concentration increases [30], which is in line with our results. The FF K + concentration was negatively correlated with the size of the follicle. It was documented that the highest FF K + concentration occurs just before ovulation in sheep, mares, and women [23]. This may relate to the permeability of the follicle membrane to K + and Na + . It was postulated that during the activation of murine oocytes, the membrane potential changes [31]. The decrease in K + concentration is associated with the development of the follicle which was also confirmed in our study. K + ions are transferred from the extracellular to the intracellular space. There are a limited number of such studies performed on cattle. A recent study showed that the average concentrations of sodium and potassium in FF in heifers (Na: 139.87 ± 1.83 mEq/L; K: 3.96 ± 0.12 mEq/L) were lower than in cows in lactation (Na: 138.60 ± 1.75 mEq/L; K: 4.00 ± 2.06 mEq/L), however, were still very similar to our results [31].
Other ions also participate in oocyte activation, such as Na + and Cl − . In our study, the Na + FF concentration was not dependent on the follicular size as well as Cl − . It was documented that Cl − ions activate the steroidogenesis connected with luteinizing hormone stimulation in chicken granular cells and the adrenal glands of rats [29]. In cattle, the Cl − FF concentration in lactating cows was higher (120.80 ± 3.40 mEq/L) than in heifers (97.90 ± 3.73 mEq/L) [30] which was probably connected with the decreased steroidogenesis in lactating cows compared to dairy heifers during the estrus. The Na + , Cl − , and K + concentrations in the FF were similar to those given in other studies [13,14,19,32,33].
In one study, the glucose level decreased with follicle size, however, the distinction between follicles was different than in our study (small < 4, medium = 6-8, large > 10 [mm]) [9]. The authors summarized that glucose metabolism is less intensive in large follicles compared with small ones or it relates to an increasing amount of follicular fluid. However, a more recent metabolomic study confirmed that altered glucose likely contributes to reduced developmental competence of oocytes [17]. Thus, the glucose concentration should be constant, which is in line with our findings as well as others [33,34].
The authors are aware that a higher number of cows would be beneficial for the strength of obtained results. Thus, the main limitation of our study is limited number of animals. Due to that, we were unable to establish reference values for gas and ion concentrations in FF. The American Society for Veterinary Clinical Pathology guidelines are very strict in determining the reference values in animals [34]. Thus, further analysis connected with the larger dataset would be beneficial in defining acid-base, gas, and ion FF ranges in dairy cattle.
In addition, the preanalytical changes have been reported during the acid-base and gas analysis. However, the sampling procedure was carried out anaerobically and the special glass syringes dedicated to this purpose were used. In addition, the analysis was performed as fast as possible in the slaughterhouse. Thus, we have made every effort to minimize the preanalytical changes in FF parameter values. In addition, we are aware that methodology or laboratory equipment may influence the differences obtained for the parameters. However, we evaluated the measurements in the routinely used equipment. Nutrition may also influence the ion concentration which is called the dietary cation-anion difference (DCAD) [35,36]. However, it was documented that most of the ions in FF are a result of local metabolism, not connected with blood concentration [9,19].

Conclusions
In conclusion, our study proved there are differences in pH and K + and Ca ++ FF concentration an association with the size of follicles. These findings may expand knowledge connected with oocyte health evaluation as well as medium composition for in vitro fertilization. Electrolyte as well as acid-base disturbances are a threat to the proper animal reproduction process. However, still, in cattle, there are a lack of studies related to abnormalities in oocyte health which is crucial to prevent the emergence of further breeding problems more effectively.  Institutional Review Board Statement: Biological sample collection was performed post-mortem, which does not fall under the legislation for the protection of animals used for scientific purposes, national decree-law (Dz. U. 2015 poz. 266), and European Union law (2010-63-EU directive). Thus, no Ethical Committee's permission was needed for sample retrieval after slaughter.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.