Relationship between biochemical parameters and paraoxonase 1 activity of boar seminal plasma and semen quality

The aim of this study was to examine the biochemical components and the parameters of antioxidant protection in the seminal plasma (SP) of boars, as well as their relationship with semen quality parameters. Thirty-six boars were included in the study, whose ejaculates were divided into two groups: Group I (good quality semen, > 70% progressively motile sperm, < 20% spermatozoa with abnormal morphology, n = 16), and Group II (poor quality semen, < 70% progressively motile sperm, > 20% spermatozoa with abnormal morphology, n = 20). Significantly higher concentrations of ionized calcium (iCa), total cholesterol (TC), lactate-dehydrogenase (LDH) activity, as well as significantly higher values of antioxidant protection parameters: thiol groups (-SH), paraoxonase 1 (PON1), and total antioxidant capacity (TAC) ) were found in the good quality semen, while higher phosphorus (P) concentrations and increased alkaline-phosphatase (ALP) activity were found in the semen of poor quality. A negative correlation of total and progressive sperm motility with P and ALP was found in all examined semen samples, while a positive correlation was found with PON1 and TAC. The percentage of fast sperm cells positively correlated with iCa, chlorine (Cl), lactate, LDH and TAC, while a negative correlation was found with P, magnesium (Mg) and the enzyme creatine-kinase (CK). The percentage of immobile sperm positively correlated with P and ALP, and negatively correlated with TC, CK, PON1 and TAC. Elevated values of PON1 and TAC in SP and a positive correlation with sperm motility indicate the possible use of these parameters as sensitive biomarkers of boar semen quality. To the best of our knowledge there are no published data on association between PON1 activity and boar semen quality.


Introduction
The main goal of all biotechnologies in reproduction is to achieve a successful pregnancy (Parilla et al. 2019). Boar ejaculates used for artificial insemination of sows are usually selected based on motility parameters that are estimated using innovative techniques such as computer-assisted semen analysis -CASA. Although new techniques provide much more objective and detailed analyzes of sperm motility, the interrelationships of the obtained parameters in the evaluation of semen quality with in vivo fertility is still debatable (Tsakmakidis et al. 2010). Therefore, analysis of the components of seminal plasma (SP) can be of great clinical importance in the investigation of fertility or sterility of boars, as well as early detection of subfertile boars. Seminal plasma is a complex mixture of exocrine secretions of the testes, epididymis and accessory gonads of males, which contains a wide range of different organic and inorganic components (Rodríguez-Martínez et al. 2011). It plays a significant role in sperm metabolism and function (Asadpour 2012;Cannarella et al. 2020), acts as a protective factor during the passage of spermatozoa through the uterus, and is very important for preparing the uterus for embryo development and implantation . There are literature data on the influence of minerals and enzymes on semen quality (Strzezek et al. 1998;Wysocki and Strzezek 2006;Lopez-Rodriguez et al. 2013;Pipan et al. 2017), thus their investigation is of great importance because it may indicate normal or altered sperm function and / or damage (Eghbali et al. 2010; Barranco et al. 2015).
Spermatozoa are known to be very sensitive to oxidative stress (Bathgate 2011;Gosalvez et al. 2017;Lee et al. 2018). The sperm cell membrane is very sensitive to lipid peroxidation initiated by the formation and accumulation of reactive oxygen species (ROS) during sperm metabolism (Cerolini et al. 2000). Spermatozoa are sensitive to oxidative stress due to the high content of unsaturated fatty acids in their membrane (Parks and Lynch 1992;Cerolini et al. 2000), and the relatively low antioxidant capacity of SP (Brezezinska-Slebodzinska et al. 1995). Elevated ROS levels may be associated with a time-dependent decrease in sperm motility, viability, and plasma membrane integrity during storage (Am-in et al. 2011). In the protection against oxidative damage and lipid peroxidation of the sperm cell membrane, the antioxidant defense mechanisms play a significant role. The enzyme paraoxonase 1 (PON1), thiol groups (-SH) and total antioxidant capacity (TAC), which neutralize free radicals and minimize their metabolic products, oxidative stress, and protect spermatozoa from oxidative damage are just some of them. According to the available literature data, this is the first study of PON1 activity in the semen of good and poor quality, as well as on the association of this enzyme with certain parameters of boar semen quality.
Therefore, the aim of this study was to examine whether there are differences in the biochemical components and antioxidant protection parameters in the SP of boars with good and poor semen quality. Also, the aim was to examine the relationship with semen quality parameters, as well as the possibility of using some of the parameters as a possible biomarker of semen quality.

Semen sampling
Thirty-six boars, aged 1 to 3 years, Landras and Duroc breed, from 3 different AI centers were included in the study (AI Center A: n = 19, AI Center B: n = 10 and AI Center C: n = 7). In each center, the boars were kept under similar conditions, in individual boxes with straw bedding, fed commercial feed (2-3 kg of feed / day), and fed with water ad libitum. Semen samples were taken in early autumn (late September or early October). One ejaculate, no gel fraction of each boar was collected using the gloved hand technique (Shipley 1999).
Prior to dilution, 9 mL of native boar ejaculate was separated into sterile tubes to assess semen quality and to separate seminal plasma for further analysis. The ejaculate was stored at room temperature and transported in an isothermal box (15-17 °C) to the Faculty of Veterinary Medicine, University of Belgrade, Serbia.

Examination of boar semen quality
After transporting the ejaculate to the laboratory of the Department of Obstetrics, Sterility and Artificial Insemination, University of Belgrade, Serbia (2-3 h after collection), 1 mL was separated into Eppendorf microtubes, to assess semen quality.
Various motility parameters as well as sperm concentration were analyzed using the CASA system (Minitube, AndroVision, Tiefenbach, Germany). Prior to analysis, the ejaculate sample was diluted in PBS (1: 8) and warmed to 38 °C for 30 min (Laboratory heating plate with integrated controller Minitube, Tiefenbach, Germany). After heating, 2.7 µL of sample was applied to the Leja chambers (Leja, GN Nieuw Vennep, Netherlans). Ten visual fields were analyzed on a phase-contrast microscope (Motic BA310, Barcelona, Spain) with a built-in heating plate, and the analysis of mobility parameters was performed automatically using AndroVision software (Minitube Manual 12,500/0000, AndroVision, Tiefenbach, Germany). The CASA system determined the following parameters of semen quality: sperm count, total and progressive sperm motility, percentage of fast, slow and immobile sperm.
Morphology was assessed using eosin-nigrosin stained slides following standard procedures (Dott and Foster 1972). A total of 100 spermatozoa were evaluated to determine the percentage of sperm with abnormal morphology (abnormal head, abnormal tail and cytoplasmatic droplets).
After analysis, ejaculates were divided into two groups: Group I (good semen quality, > 70% progressively motile sperm, < 20% spermatozoa with abnormal morphology, n = 16), and Group II (poor semen quality, < 70% progressively motile sperm, > 20% spermatozoa with abnormal morphology, n = 20). Ejaculates with semen quality complying with the AI center´s cut-off values were included in Group I, while ejaculates with semen quality below these cut-off were considered as poor and were included in Group II.

Separation of boar seminal plasma
Seminal plasma was separated after double centrifugation at 1,500 x g for 10 min. The supernatant after the second centrifugation was examined microscopically to make sure there were no spermatozoa. After centrifugation, the seminal plasma was separated, frozen and stored at − 20 °C until analysis.

Biochemical analysis of seminal plasma
The concentration of mineral substances in the seminal plasma: ionized calcium (iCa), sodium (Na), potassium (K), chlorine (Cl), pH and lactate was determined on a gas ionic analyzer State Profile Prime + Vet (New Biomedical Corporation, Waltham, USA), using original micro sensor cards for the specified parameters from the same manufacturer.
Concentration of total proteins (TP), albumin, total cholesterol (TC), phosphorus (P), magnesium (Mg), as well as the activity of enzymes alkaline phosphatase (ALP), aspartate amino transferase (AST), creatine kinase (CK) and lactate dehydrogenase (LDH) was determined on an automatic biochemical analyzer BioSystems A15, (BioSystems, Barcelona, Spain) using original reagents from the same manufacturer.

Determination of antioxidant protection parameters
Determination of paraoxonase 1 enzyme activity (PON1) PON1 activity was measured according to the modified Alapati and Mihas method (1999). The reaction mixture (1 mL) contained 200 µL of synthetic paraoxone (diethylp-nitrophenyl) phosphate) as substrate, 50 µL CaCl 2 , 400 µL NaCl in 1 mM Tris-HCl buffer pH 8.5. After addition of the seminal plasma sample, the increase in absorbance at 412 nm, 5 min at 37 C was measured with a spectrophotometer. The absorption coefficient used to calculate the degree of hydrolysis is 18,500 M −1 cm −1 . Enzyme activity is expressed in U/mL.

Determination of the concentration of thiol (-SH) groups
The spectrophotometric method was used to determine the concentration of SH-groups Ellman (1959). The method is based on the reaction of 2.2-dinitro-5.5-dithio-benzoic acid (DTNB) with SH groups, producing p-nitrophenol with a maximum absorption at 412 nm. The concentration SHgroups in the sample was calculated using the molar absorption coefficient of p-nitrophenol (14,150 × 10 6 M − 1 cm − 1 ) and expressed in µM.

Statistical data analysis
Statistical data processing was performed using GraphPad Prism 6.00 statistical software (GraphPad Software Inc., San Diego, CA, USA). AI center and Group were included as fixed factor and their interaction was investigated using the ANOVA test. Most of the parameters except for tCa were not affected by AI center. Normal distribution was tested by the D'Agostino-Pearson omnibus statistical test. Statistical significance of differences in the tested values (P) was determined using the ANOVA test and Student's t test. Differences at the level of P < 0.05 were taken as statistically significant. However, if the distribution did not follow the Gaussian distribution, the Kruskal-Wallis test was applied. D'Agostino-Pearson omnibus statistical test has shown that only pH did not follow normal distribution. All selected parameters were above the detection limit of the test. Results are shown as mean ± standard (SE) as interquartily values. The degree of correlation of the examined parameters was determined by Spearman's correlation coefficient and by checking the statistical significance of the correlation coefficient (P < 0.05). The strength of the correlation was defined according to Evans (1996).

Semen quality and intergroup comparisons
Semen quality parameters are shown in Table 1. Boar semen of good quality had significantly higher (P < 0.001) percentage of total and progressively motile spermatozoa and significantly lower percentage of sperm with abnormal morphology (P < 0.001) compared to poor quality semen. Also, the percentage of fast spermatozoa was significantly higher in Group I (37.85%) compared to Group II (P < 0.01), while in the low quality semen the percentage of slow and immobile spermatozoa was significantly higher (P < 0.05) compared to good semen quality. I compared to Group II. Higher CK, ALT and AST enzyme activities were found in good quality semen, but without statistical significance compared to poor quality semen.

Seminal plasma parameters of antioxidant protection and comparisons between groups
Significantly higher concentrations of thiol groups (P < 0.01), higher activity of PON1 enzyme (P < 0.05) as well as significantly higher concentrations of TAC (P < 0.05) were measured in the group of boars with good quality semen (Table 3).

Seminal plasma biochemical values and intergroup comparison
The obtained values for the measured biochemical parameters of SP boars are shown in Table 2. Significantly higher (P < 0.05) iCa concentration and significantly lower P concentrations (P < 0.01) were found in the seminal plasma of boars in the group with good semen quality compared to boars' semen with poor quality semen.
The concentration of total cholesterol was 36.5 times higher in the seminal plasma of boars with good semen quality compared to poor quality semen (P < 0.05). In the seminal plasma of the semen of poor quality, a ten times higher activity of ALP (P < 0.001), and significantly lower activity of LDH (P < 0.05) of 41.70% was found, compared to the semen of better quality. The results disclosed a significantly higher lactate concentration of 32.61% in Group Table 2 Reference value (mean, median, interquartile range) biochemical values of seminal plasma parameters of boars with good (Group I, > 70% progressive sperm motility, < 20% spermatozoa with abnormal morphology, n = 16), and poor (Group II, < 70% progressive sperm motility, > 20% spermatozoa with abnormal morphology, n = 20) semen quality 18 ± 0.378 *** 26.55 ± 1.371 sp.: spermatozoa; significant differences ***P ˂ 0.001, **P ˂ 0.01, *P ˂ 0.05, t-test Table 1 Average values (mean ± SE) of semen quality parameters of boars with good (Group I, > 70% progressive sperm motility, < 20% spermatozoa with abnormal morphology, n = 16), and poor (Group II, < 70% progressive sperm motility, > 20% spermatozoa with abnormal morphology, n = 20) semen quality sperm motility (r = − 0.8170) at the level of statistical significance P < 0.001. There was a strong positive correlation with the percentage of fast spermatozoa (r = − 0.7467, P < 0.01) and a strong positive correlation with the percentage of immobile sperm (r = 0.7972, P < 0.001).
Mg concentration negatively correlated with medium strength with the percentage of fast sperm (r = − 0.5410, P < 0.05), and showed a very strong positive correlation with the percentage of slow sperm in the semen sample (r = 0.8704, P < 0.001), while the concentration of K negatively correlated with medium strength with the percentage of slow spermatozoa (r = − 0.5363).
A positive correlation of moderate strength (P < 0.05) was found between TC and total motility (r = 0.5022), progressive motility (r = 0.4959) and slow (r = 0.5884) spermatozoa, while a negative correlation of moderate strength

Correlations between the biochemical variables and parameters of antioxidant protection in the seminal plasma and sperm quality variables in boars
The correlations between biochemical and antioxidant protection parameters and boar semen quality are displayed in Table 4. The percentage of fast spermatozoa positively correlated with moderate strength with the concentration of iCa (r = 0.5386), Cl (r = 0.4784), lactate (r = 0.5114), and LDH activity (r = 0.5924), while a negative correlation was found between the above parameters and the percentage of slow sperm: iCa (r = − 0.4953), Cl (r = − 0.4700), lactate (r = − 0.5058) and LDH activity (r = − 0.4715) at the level of statistical significance P < 0.05.
Phosphorus concentration showed a strong negative correlation with spermatozoa motility (r = − 0.7972), progressive Table 3 Reference value (mean, median, interquartile range) of parameters of antioxidative protection seminal plasma parameters of boars with good (Group I, > 70% progressive sperm motility, < 20% spermatozoa with abnormal morphology, n = 16), and poor (Group II, < 70% progressive sperm motility, > 20% spermatozoa with abnormal morphology, n = 20) semen quality .5777 -0.5987 ** sp. spermatozoa; tCa: total calcium; iCa: ionized calcium; P: phosphorus; Mg: magnesium; Na: sodium; K: potassium; Cl: chloride; TC: total cholesterol: TP: total protein; ALP: alkaline phosohatase; CK: creatine kinase; ALT: alanine aminotransferease; AST: aspartate amino transferase; LDH: lactate dehydrogenase; -SH: thyol (sulfhydryl) groups: PON1: paraoxonase 1; TAC: total antioxidant capacity Significant differences: *P ˂ 0.05; **P ˂ 0.01; ***P ˂ 0.001, Spearman Rank correlation recorded for ion concentrations in SP boars were similar to previously published results (Massanyi et al. 2003;Lopez-Rodriguez et al. 2013;Pipan et al. 2017). In order to clearly determine the influence of calcium on boar semen quality parameters, in addition to the concentration of tCa, we also determined the concentration of iCa in the SP. In our work, no clear association of Ca with sperm motility parameters was found, as well as differences in concentration between semen of good and poor quality, which is in line with the findings of Lopez- Rodriguez et al. (2013).
It is a known fact that the binding of Ca ions to the sperm cell membrane can lead to differences in the concentrations obtained by its measurement in the ejaculate in relation to seminal plasma (Owen and Katz 2005). However, it is interesting that in our work, a positive effect of iCa on sperm velocity was determined, which was confirmed by a positive correlation with fast and a negative correlation with slow sperm. Our research is consistent with previous studies in men with sperm hypomotility in whom significantly lower iCa concentrations were found although there were no differences in tCa concentration in SP compared to semen with normal sperm motility (Prien et al. 1990). During capacitation, spermatozoa undergo various functional changes, which include changes in the composition of the cell membrane, remodeling of acrosomes, increased sperm motility and mitochondrial activity. During capacitation, there is a significant increase in intracellular iCa and ROS, as well as the phosphorylation of the amino acid tyrosine present in certain sperm proteins (Yeste et al. 2015;Betarelli et al. 2018;Rocco et al. 2018). It is generally accepted that the medium for sperm capacitation should be a source of various ions, including bicarbonate and iCa (Visconti 2009;Stival et al. 2016;Chaves et al. 2021).
In boars, Na and Cl ions are the most abundant ions in the seminal plasma , which have a significant impact on the metabolism, function, motility and integrity of the sperm membrane (Lopez-Rodriguez et al. 2013). Significantly higher concentrations of Cl and Na ions in the seminal plasma of boars with poor semen quality compared to good quality semen, as well as a negative correlation of Cl ions with normal sperm morphology were found by Lopez- Rodriguez et al. (2013). Although no differences in Na and Cl ion concentrations between groups were found in our work, the positive effect of Cl ions on sperm velocity was confirmed by a positive correlation of this ion with the percentage of fast spermatozoa and a negative correlation with the percentage of slow spermatozoa. Cl ion efflux and increased cAMP induce sperm capacitation, increased motility, and activation of proteolytic enzymes necessary for the penetration through the egg membrane (Luconi et al. 2004;Modi et al. 2007). Although previous studies have described that extracellular Na ions improve motility (Kong was found with the percentage of immobile spermatozoa (r = − 0.5022). Spermatozoa concentrations were strongly positively correlated with TC (r = 0.6217, P < 0.05).
ALP enzyme activity strongly (P < 0.001) negatively correlated with total spermatozoa motility (r = − 0.9122), progressive motility (r = − 0.9032), percentage of fast spermatozoa (r = − 0.8853). It showed also a very strong positive correlation with the percentage of immobile spermatozoa (r = 0.9122), and a moderate positive (P < 0.05) correlation with the percentage of slow spermatozoa (r = 0.5376) and a strong correlation with spermatozoa concentration (r = 0.6201).
A moderate (P < 0.05) positive correlation was found between CK enzyme activity and total motility (r = 0.5592) and progressive motility (r = 0.5438). A negative correlation with the percent of immobile spermatozoa was recorded also (r = − 0.5592).
PON1 activity positively (P < 0.05) correlated with total motility (r = 0.5315), progressive motility (r = 0.4969). A negative correlation with the percentage of immobile sperm (r = − 0.5315) and percentage of spermatozoa with abnormal morphology (r = − 0.4891) was present. A strong positive correlation (P < 0.05) was found between total motility (r = 0.6693) and progressive motility (r = 0.6682) with TAC, while the correlation between fast spermatozoa and TAC was very strong (r = 0.9711, P < 0.01). Also, a strong negative correlation was found between TAC and the percentage of immobile spermatozoa (r = − 0.6693, P < 0.05), and moderate correlation (r = − 0.5987, P < 0.01) between TAC and percentage of spermatozoa with abnormal morphology.

Discussion
Early identification of boars with reduced fertility is one of the priorities of the AI program . It is generally accepted that conventional semen quality assessment methods provide only a rough estimate of the potential fertility of boars (Dyck et al. 2011). Therefore, it is very important to examine the composition of SP as a potential source of biomarkers that would help to identify subfertile boars that remain undiagnosed when testing the semen by conventional methods (Perez-Patino et al. 2019).
In our study, the biochemical composition, as well as the parameters of antioxidant protection in the SP of boars with good and poor semen quality were examined. The values aforementioned authors added exogenous BSA to the extendor which directly influences boar sperm motility. Roca et al. (2020) found that more than 3% of boar SP proteomes are enzymes. Significantly higher activity of ALP enzymes of 10 times was found in SP of semen of poor quality in our work, while Clements et al. (2010) found lower activity of this enzyme in boars with azoospermia. However, Lopez-Rodriguez et al. (2013) did not find differences in ALP activity between good and poor quality boar semen. Although previous work indicates a positive correlation between ALP activity and the percentage of progressively motile sperm (Pesch et al. 2006;Lopez Rodriguez et al. 2013), our study found a very strong negative correlation of this enzyme with the percentage of total and progressively motile spermatozoa and the percentage of fast spermatozoa, while a strong positive correlation was found with the percentage of immobile spermatozoa indicating the negative impact of increased ALP activity in SP on semen quality parameters. ALP is known to catalyze the hydrolysis of phosphate groups of many substrates, but the role of this enzyme in the reproductive tract has not been fully elucidated (Price et al. 2009). Given the insufficient data, as well as differences in literature citations, further research ALP activity in SP boars is needed. Elevated ALP values in the semen of poorer quality, as well as a negative impact on semen quality parameters were accompanied by elevated P concentrations in SP.
It is known that the activity of CK in the SP in men is related to the quality of semen, the functionality of the sperm cell membrane, and is involved in various metabolic processes during maturation (Collodel et al. 2019). However, in our work, no significant differences were found between the groups in the activity of CK, ALT and AST enzymes. However, a positive effect of CK on sperm motility was found, which was confirmed by a positive correlation of this enzyme with the total and progressive sperm motility. This enzyme converts ADP to ATP with concurrent hydrolysis of phosphocreatinine to creatinine (Guo et al. 2019), and is necessary for sperm function (Banihani and Abu-Alhayjaa 2016). Because sperm cells are energy-dependent, low levels and decreased creatine metabolism may be associated with low sperm concentration and reduced motility (Ostojic et al. 2022).
Mitochondrial substrates, i.e. pyruvate and lactate have a significant effect on sperm motility and mitochondrial function, compared to glycolytic substrates (glucose) due to direct use in the mitochondria (Dar et al. 2018;Guo et al. 2019), which is confirmed by a positive correlation of lactate and LDH with fast spermatozoa and a negative correlation with slow spermatozoa. Lactate dehydrogenase plays a significant role in the metabolic processes that provide energy for the survival, motility and fertilization potential Holt and Harrison 2002) and correlate with normal sperm morphology (Lopez-Rodriguez et al. 2013), in our study Na concentration had no effect on semen quality. The negative correlation of K ions with the percentage of slow spermatozoa found in our work confirms the fact that K helps to preserve sperm motility, which is in line with the findings of Johnson et al. (2000).
Mg is known to be one of the key macronutrients in many enzymatic reactions, and consequently associated with sperm motility and membrane preservation (Juyena and Stelletta 2012;Lopez-Rodriguez et al. 2013). Although in our work the concentrations of Mg did not differ between the groups of semen of good and poor quality, the negative impact of this macroelement on sperm velocity was confirmed by a strong positive correlation with the percentage of slow sperm and a negative correlation with the percentage of fast spermatozoa. Kasperczyk et al. (2015) believe that Mg indirectly promotes the development of oxidative stress, which in turn leads to reduced sperm motility. Pipan et al. (2017) found a negative correlation between Mg and progressive spermatozoa motility in chilled semen after three days of storage. However, there are data in the literature that indicate that the addition of Mg diluents for boar semen improves sperm viability (Szczesniak-Fabianczyk et al. 2003). Szczesniak-Fabianczik et al. (2003), in their research, added magnesium fumarate to the extender, which resulted in better semen quality and prolonged sperm survival. The addition of external Mg could lead to increased activity of the Ca 2+ Mg 2+ -ATPase enzyme, which is involved in maintaining the motility of spermatozoa (Kumosani et al. 2008).
A number of seminal plasma proteins bind to membrane receptors on the spermatozoa, thus having a significant impact on their function (Parilla et al. 2019). There are numerous papers in the literature that indicate a positive effect of protein concentration (Caballero eta al. 2008;Novak et al. 2010) especially albumin (Zhang et al. 2015;Fu et al. 2017) on semen quality. Exogenous bovine serum albumin (BSA) has been shown to have an antioxidant role in protecting the spermatozoa from cold shock by inhibiting the initiation of lipid peroxidation (Gadea 2003;Funahashi and Sano 2005;González-Cadavid et al. 2014), and that BSA is crucial in vitro to initiate boar sperm capacitation (Chaves et al. 2021). Albumin together with superoxide dismutase and glutathione peroxidase plays a significant role in neutralizing ROS generated during cellular metabolism (González-Cadavid et al. 2014). However, in our work, no differences were found in the concentration of protein or albumin between semen of good and poor quality, as well as their association with semen quality parameters. The discrepancy most likely occurred because we examined the SP components of fresh boar ejaculates, while the 2006). Thiol (sulfhydryl, -SH) groups, which are among the most important endogenous non-enzymatic antioxidants, play an important role in protecting proteins from oxidative damage, as they are the first to oxidize in cells, thus preventing oxidation of other functional groups of enzymes and proteins (Dalle-Donne et al. 2006). Although significantly higher concentrations of thiol groups in good quality semen were found, in our paper no association of thiol group concentrations with sperm motility parameters was found, but a negative correlation was established with the number of spermatozoa and percentage spermatozoa with abnormal morphology. Our research is consistent with literature data in the field of human medicine suggest a reduction in thiol groups in SP as a biomarker of oxidative stress (Piomboni et al. 2012) and altered sperm function (Aljabari et al. 2007).
Increased sperm metabolism amplifies the production of free radicals in the SP, thus creating a condition known as oxidative stress, which depletes antioxidant defense mechanisms. Barranco et al. (2021) found a negative association between oxidative stress index (OSI) and sperm motility. Decreased TAC values in SP are associated with increased lipid peroxidation of sperm cell membranes, which may result in their reduced ability to fertilize (Žura Žaja et al. 2016). The direct correlation between TAC in the SP and semen quality in our work was confirmed by significantly higher concentrations of TAC in good quality semen, as well as a positive correlation with total, progressive motility and the percentage of fast spermatozoa and negative correlation with percentage spermatozoa with abnormal morphology. The interrelationship of the examined parameters indicates the importance of the antioxidant protection mechanisms in the defense of spermatozoa from ROS. Similar to our research Am-in et al. (2011) found a positive correlation between TAC and motility, viability, normal morphology and integrity of the sperm cell membrane. Barranco et al. (2015) found that reducing TAC in the SP increases the sensitivity of boar sperm to cryopreservation.
Higher values of antioxidants, as well as the association with the parameters of semen quality, indicate their significant role in the protection of spermatozoa from ROS. In our study, significantly higher activities of PON1 and TAC in good quality seminal plasma were found, as well as their positive correlation with sperm motility parameters and negative correlation with percentage spermatozoa with abnormal morphology, which is why these parameters could be used as biomarkers of boar semen quality. To the best of our knowledge there are no published data on association between PON1 activity and boar semen quality.
Future work should focus on examining the biochemical and redox status of individual ejaculate fractions as their composition may differ. Microelements such as Se, Zn and Cu could be investigated in future studies as they are known of sperm cells (Diaconescu et al. 2014). This enzyme is a very important indicator of semen fertility, with higher activities in the semen of boars with a high percentage of total motile spermatozoa (Sopkova et al. 2015). The positive influence of LDH on the quality of boar semen in our work was confirmed by the higher activity of this enzyme in the semen of good quality, but also by the positive correlation with fast spermatozoa and the negative correlation with slow spermatozoa.
Spermatozoa are very sensitive to oxidative stress (Aitken et al. 1996;Radomil et al. 2011;Li et al. 2018) and the formation and accumulation of ROS during sperm metabolism (Cerolini et al. 2000). Decreased antioxidant defense mechanisms in the middle portion and the tail of spermatozoa, as well as increased lipid peroxidation of polyunsaturated fatty acids in their cytoplasmic membrane (Cerolini et al. 2000) adversely affect sperm function and survival (Hernandez et al. Bila, 2007Bans et al. 2010Bathgate 2011;Gosalvez et al. 2017).
The enzyme PON1 plays a significant role in the protection against oxidative damage as part of the enzymatic antioxidant system, which neutralizes free radicals and their metabolic products (Gugliucci et al. 2013). Li et al. (2018) found the highest activity of PON1 in the SP fraction rich in spermatozoa, and also that PON1 is positively correlated with the response of sperm cells to cryopreservation, because this enzyme minimizes oxidative stress during this procedure (Li et al. 2018). Significantly higher PON1 activity found in our work in good quality semen, as well as its positive correlation with total and progressive sperm motility and negative correlation with percentage spermatozoa with abnormal morphology indicates a significant role of this enzyme in defending sperm cells from oxidative stress, and thus a positive impact on semen quality. This claim is supported by the negative correlation of PON1 with the percentage of immobile sperm. PON1 binds to the cholesterol membrane of spermatozoa, protects and delays the oxidation of lipoproteins, thus protecting sperm from oxidative stress, and consequently positively affecting the motility and integrity of the membrane of these cells (Barranco et al. 2015. In our work, the increased activity of PON1 is accompanied by a higher concentration of TC in boar SP with good semen quality, as well as a positive correlation between TC and sperm motility. Our research is consistent with Am-in et al. (2011) finding that boar semen with poorer motility have a lower total cholesterol content, as well as being positively correlated with reduced sperm motility. This claim is supported by the negative correlation of PON1 with the percentage of immobile sperm. Proteins are also important target molecules for ROS, due to their high presence in the structures of biological systems and involvement in most functional processes within cells (Dalle-Donne et al.
to be important as antioxidants and enter into the composition of antioxidant enzymes Se-dependent gluthathione peroxidase and Cu, Zn superoxide dismutase.

Conclusion
The results of this paper indicate the association of the biochemical parameters of SP with boar semen quality parameters such as iCa, LDH activity and ALP could be included insemen quality assessment. Analyses activity of PON1 and TAC could provide extra information about reproductive health of boars and could therefore add valuable information to the clinical examination of boars with poor semen quality problems and could be used as biomarkers of boar semen quality.