Effect of Heat Stress on the Antioxidant Activity of Boar Ejaculate Revealed by Spectroscopic and Electrochemical Methods

Int. J. Electrochem. Sci., 10 (2015) 6610 - 6626 International Journal of ELECTROCHEMICAL SCIENCE www.electrochemsci.org Effect of Heat Stress on the Antioxidant Activity of Boar Ejaculate Revealed by Spectroscopic and Electrochemical Methods Pavel Horky1, Katerina Tmejova2,3, Renata Kensova2,3, Natalia Cernei2,3, Jiri Kudr2,3, Branislav Ruttkay-Nedecky2,3, Eva Sapakova4, Vojtech Adam2,3 and Rene Kizek2,3* 1 Department of Animal Nutrition and Forage Production, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, European Union 2 Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, European Union 3 Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ616 00 Brno, Czech Republic, European Union 4 Department of Languages, Faculty of Regional Development and International Studies Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic European Union *E-mail: [email protected] Received: 13 February 2015 / Accepted: 11 June 2015 / Published: 24 June 2015 An effect of heat stress on antioxidant activity and overall quality of boar ejaculates was monitored. The samples of boar ejaculates were divided into two groups according to ejaculate quality. In the group A, regarding antioxidant activity a decreased value of N,N-dimethyl -1,4-diaminobenzene method by 71 %, an increase in 2,2-diphenyl-1-picrylhydrazyl method by 104 %, and decrease in superoxide dismutase activity by 39 % was determined. Results of the metal content were as follows decrease in levels of copper by 60 % and zinc by 52 %. Also decreases in metallothionein content by 64 %, and in pH by 11 % were recorded. In the group B, in antioxidant activity again a decreased value of DMPD by 68 %, and an increase in DPPH by 207 % was recorded. In addition reduced levels of copper by 78 %, zinc by 30 %, metallothionein by 64 %, and value of pH by 11 % were determined. The results show that heat stress induces an increase in protection against oxidative stress; in addition, levels of metallothionein, zinc and copper in the boar ejaculates in both groups are decreased when compared to day 0. Keywords: Boar ejaculates; Heat stress; Antioxidant activity; Quality of ejaculates Int. J. Electrochem. Sci., Vol. 10, 2015 6611 1. INTRODUCTION The quality of boar ejaculates varies significantly during the season. The highest fluctuations are observed in all species of male livestock in the summer months at higher temperatures [1]. Also stress itself may influence the body temperature [2,3]. Quality of boar ejaculates decreases at increased heat load. A reduction of motility and concentration of sperms as well as an increase of percentage of abnormal sperm have been recorded [4]. Reduced sperm motility is caused by a lower level of ATP [57]. In the boars, the quality of ejaculate is largely influenced by season. The best quality of ejaculate is observed in spring and autumn. Conversely, in the summer, when the animals are exposed to high temperatures, significantly worse qualitative and quantitative indicators of the ejaculate are recorded. An influence of breed has also been recorded [8,9]. High temperature may also cause an excessive loading the organism with oxygen radicals and decrease in activity of antioxidant enzymes [10]. In some cases, overproduction of free radicals causes a damage of biomolecules and cancer [11] [12]. Reactive oxygen species [13] negatively affect the quality of sperms. Negative impact of these reactive oxygen species causes peroxidation of lipids that create the outer shell of sperms [14]. Reactive oxygen species may affect the metabolism of proteins in the ejaculate [15-17]. High temperature also affects the reproductive performance of breeding pigs. Selenium is part of the sperm and affects its mobility. Selenium deficiency reduces sperm motility. Zinc and copper are essential metals, which affect the concentration and motility of sperms. [18,19]. The aim of this experiment was to evaluate the effect of heat stress on biochemical parameters of ejaculate of breeding boars focusing on changes in antioxidant status (at boars with good or poor quality of ejaculates). The aim of the experiment was also to evaluate the effect of heat stress on ejaculate quality of boars. 2. EXPERIMENTAL PART 2.1. Animals The experiment was performed in the sperm collection centre of boars in Velke Mezirici (Czech Republic). Twenty Duroc boars breeding stocks were included in the experiment (Sus scrofa domestica). The average age was 2 ± 0.3 years and the average weight of boars was 250 ± 20 kg. The length of the experiment was set at 135 days (May to September). Experimental animals were stalled individually (2.5 x 2.5 m) and were supplemented with water ad libitum. All animals were fed with 3.3 kg per day of basic feed mixture (Tab. 1). Table 1. The composition of the feed mixture for boars Component % in feed mixture Barley grain Wheat grain 36.00 20.36 Int. J. Electrochem. Sci., Vol. 10, 2015 6612 Oat grain SBM (soybean meal) EKPO T BergaFat Calcium carbonate Monodicalciumphosphate Mineral vitamin premix for boars 0.5% Sodium chloride Magnesium oxide L-Lysine HCl LThreonine Methionine DL 20.00 14.50 3.00 2.10 1.50 1.20 0.50 0.40 0.15 0.14 0.09 0.06 Bergafat (Berg + Schmidt, Germany) – palm oil; EKPO T (Delika – Pet, Czech Republic) – biscuit meal Figure 1. Development of temperature, which acts on both experimental groups of boars during the experimental observation (A); the sum of the temperatures during individual parts of the experiment (B), the development of the relative humidity (C) Int. J. Electrochem. Sci., Vol. 10, 2015 6613 Content of MEp (metabolizable energy of pigs) was 12.6 MJ.kg-1 of diet. Boars were divided into two groups. The first group (n = 10) of boars (group A) had values of ejaculate without pathological changes (motility of sperm cells 72 %, concentration of sperm cells 499 000.mm-3; ejaculate volume 203 ml; percentage of pathological sperm cells bellow 6 %). The second group (group B) contained boars (n = 10), which had problems with the quality of ejaculate produced (low sperm motility 67 %, and a low concentration of sperm cells 430 000.mm-3). During the whole experiment, the temperature was registered by monitoring the environment in which the boars were located. The temperature was monitored with a data logger (Voltcraft DL-121TH, Germany), which was placed at the level of the animals (1 m above the ground). The data logger recorded the actual temperature in hourly intervals; the average temperature for each day was calculated from these values. Figures 1A and 1B show average temperature and the sum of temperature values during the experiment. Figure 1C shows average and maximal relative humidity of the environment in which the experiment was carried out. Ejaculates were collected weekly, after collection they were frozen to -80 ºC and they were thawed immediately before analyses. The ejaculates, which were taken at the beginning of the experiment (day 0 -control samples) and at 45th, 90th and 135th day of the experiment, were used for analyses. The ejaculates were obtained from boars using a “jump mare” [20] Determination of the ejaculate volume, sperm concentration, its motility, and percentage of abnormal sperms were carried out according to Lovercamp et al. [20]. Ejaculate volume was determined using a measuring cylinder. Determination of sperm motility was performed within 15 minutes after collection from microscopically-mixed semen. Sperm concentration was determined photometrically. The percentage of pathological sperms was determined microscopically and they were collected prior to the start of the experiment; Photographs of sperms were taken using an inverted Olympus microscope IX 71 S8F-3 (Tokyo, Japan). Records were taken using a camera Olympus DP73 and processed with Stream Basic 1.7 software. Magnification: 600x, Resolution: 1600 × 1200 pixels. 2.2. Preparation of samples for biochemical analyses Firstly, 0.5 ml volume of native thawed ejaculate was pipetted with subsequent addition of 2 ml of liquid nitrogen and 0.5 ml of phosphate buffer. Subsequently, the sample was homogenized in an ULTRA-TURRAX T8 homogenizer (IKA, Konigswinter, Germany) at 3000 rpm for 2 minutes. After homogenization, 1 ml of phosphate buffer was added. Sample modified like this was homogenized in a vortex (Vortex-2 Genie Scientific Industries, New York, NY, USA) at 2000 rpm for 15 minutes. Subsequently, the sample was centrifuged in a Universal 32 R centrifuge (Hettich-Zentrifugen GmbH, Tuttlingen, Germany) at 16000 rpm at 4 °C for 20 minutes. Finally, supernatant was removed and used for analyses (1.5 ml). 2.3. Determination of glutathione peroxidase (GPx) A Glutathione Peroxidase Cellular Activity Assay Kit (CGP1, Sigma Aldrich, USA) was used for the GPx assay, determination was done in native ejaculate. For the determination of GPx activity, a Int. J. Electrochem. Sci., Vol. 10, 2015 6614 BS 400 automated spectrophotometer (Mindray, China) was used. The experimental protocol was as it follows. A 260 µl volume of reagent R1 (0.3 mM NADPH in GPx buffer) was pipetted into a plastic cuvette with subsequent addition of 10 µl of sample and after mixing, a 30 µl volume of reagent R2 (3 mM tert-butyl hydroperoxide) was added to the cuvette which starts the reaction. The decrease in absorbance was measured at 340 nm using kinetic program for 126 seconds. 2.4. Determination of SOD Kit 19160 SOD (Sigma Aldrich, USA) was used for assay of superoxide dismutase (SOD, EC 1.15.1.1.). A 200 µL volume of reagent R1 (WTS solution diluted 20 times with buffer) was pipetted into a plastic cuvette and agent was incubated at 37 °C for 108 s. Afterwards, a 20 µL volume of sample was pipetted and in 378 s, the reaction was started by adding a 20 µL volume of reagent R2 (enzyme solution 167 times diluted with buffer). It was incubated for 72 s and then absorbance was measured at λ = 450 nm. Kinetic reaction was measured for 180 s and absorbance was read every 9 s. 2.5. Determination of antioxidant activity Solutions of 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl-1picrylhydrazyl (DPPH) and free radical (FR) assays were prepared according to Sochor et al. [21]. Brief description of the assays is below. 2.6. Determination of antioxidant activity using the ABTS test The ABTS radical method is one of the most used assays for the determination of the concentration of free radicals. It is based on the neutralization of a radical-cation arising from the oneelectron oxidation of the synthetic chromophore 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS): ABTS• – eABTS•+. This reaction is monitored spectrophotometrically by the change of the absorption value. A 150 µl volume of reagent was mixed with 3 µl of sample. Absorbance was measured at 660 nm. For calculating of the antioxidant activity, difference between absorbance at the last (12th) minute and at the second minute of the assay procedure was used. 2.7. Determination of antioxidant activity by Ferric Reducing Antioxidant Power (FRAP) method The FRAP method is based on the reduction of complexes of 2, 4, 6-tripyridyl-s-triazine (TPTZ) with ferric chloride hexahydrate (FeCl3·6H2O), which are almost colourless, and eventually slightly brownish. This chemical forms blue ferrous complexes after its reduction. Reagent preparation: Solution 1:10 mmol.L-1 solution of TPTZ in 40 mmol.L-1 of hydrochloric acid. Solution 2: 20 mmol.L-1 solution of ferric chloride hexahydrate in ACS water. Solution 3: 20 mmol.L-1 acetate buffer, pH 3.6. These three solutions (TPTZ, FeCl3, acetate buffer) are mixed in a 1:1:10 ratio. A 150 Int. J. Electrochem. Sci., Vol. 10, 2015 6615 μL volume of reagent was injected into a plastic cuvette with subsequent addition of a 3 μL sample. Absorbance was measured at 605 nm for 12 minutes. Difference between absorbance at the last (12th) minute and second minute of the assay procedure was used for calculating of the antioxidant activity. 2.8. Determination of antioxidant activity by the N,N-dimethyl -1,4-diaminobenzene (DMPD) method The compound DMPD is converted in solution to a relatively stable and coloured radical form (DMPD•+) by the action with ferric salt. After addition of a sample containing antioxidants, DMPD•+ radicals are scavenged and as a result of this scavenging, the coloured solution is decolourized [22]. Procedure for the determination was taken from Sochor et al. [23,24]. A 160 μl volume of reagent (200 mM DMPD, 0.05 M FeCl3, 0.1 M acetate buffer pH 5.25) was injected into a plastic cuvette with subsequent addition of 4 μl sample. Absorbance was measured at 505 nm. Difference between absorbance at the 10th minute and second minute of the assay procedure was used for calculating of the antioxidant activity. 2.9. Determination of antioxidant activity using the DPPH• test The DPPH test is based on the ability of the stable 2,2-diphenyl-1-picrylhydrazyl free radical to react with hydrogen donors. The DPPH• radical displays an intense UV-VIS absorption spectrum. In this test, a solution of radical is decolourized after reduction with antioxidant (AH) or a radical (R•) in accordance with the following scheme: DPPH• + AH → DPPH-H + A•, DPPH• + R• → DPPH-R. A 150 µl volume of reagent was incubated with 15 µl of sample. Absorbance was measured at 505 nm for 12 minutes. Output ratio was achieved by the calculation of difference of absorbance at the last (12th) minute and at the second minute of the assay procedure. 2.10. Determination of antioxidant activity by the FR method This method is based on ability of chlorophyllin (the sodium-copper salt of chlorophyl) to accept and donate electrons with a stable with a stable change of maximum absorption. This effect is conditioned by an alkaline environment and the addition of catalyst. A 150 μl volume of reagent was injected into a plastic cuvette with subsequent addition of a 6 μl sample. Absorbance was measured at 450 nm at the second minute of assay and the last (12th) minute. Difference between these two absorbancies was considered as an outputting value and served for the calculation of antioxidant activity. 2.11. Determination of zinc Determination of zinc by differential pulse voltammetry was performed using a 797 VA Computrace instrument connected to 813 Compact Autosampler (Metrohm, Switzerland), using a standard cell with three electrodes. A hanging mercury drop electrode (HMDE) with a drop area of 0.4 Int. J. Electrochem. Sci., Vol. 10, 2015 6616 mm2 was the working electrode. An Ag/AgCl/3M KCl electrode was the reference and platinum electrode was the auxiliary. For data processing, 797 VA Computrace software by Metrohm CH was employed. The analysed samples were deoxygenated prior to measurements by purging with argon (99.9%). Acetate buffer (0.2M CH3COONa + CH3COOH, pH 5) as a supporting electrolyte was used. The supporting electrolyte was exchanged after each analysis. The parameters of the measurement were as follows: initial potential of -1.2 V, end potential -0.7 V, deoxygenating with argon 90 s, deposition 420 s, time interval 0.04 s, step potential 5 mV, modulation amplitude 25 mV, adsorption potential -1.2 V, volume of injected sample: 10 µl, volume of measurement cell 2 ml (10 μl of sample, 1990 μl of acetate buffer) [25,26]. 2.12. Preparation of samples for electrochemical determination of copper – microwave digestion To 10 µl ejaculate 500 µl of digestion mixture (350 µl HNO3 + 150 µl H2O2) was added. Samples were digested in MW Anton Paar, rotor MG-65. The program (SUP 6) begins and ends with the same ten-minute-long-step, beginning with the power of 50 W and ending with the power of 0 W. Microwave power was 100 W in the main part of the program (30 min.). 2.13. Determination of metallothionein Differential pulse voltammetric measurements were performed with a 747 VA Stand instrument connected to 693 VA Processor and an 695 Autosampler (Metrohm, Switzerland), using a standard cell with three electrodes and cooled sample holder and measurement cell to 4 °C (Julabo F25, JulaboDE). A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm2 was the working electrode. An Ag/AgCl/3M KCl electrode was the reference and glassy carbon electrode was the auxiliary. For data processing VA Database 2.2 by Metrohm CH was employed. The analysed samples were deoxygenated prior to measurements by purging with argon (99.999 %) saturated with water for 120 s. Brdicka supporting electrolyte containing 1 mM Co(NH3)6Cl3 and 1 M ammonia buffer (NH3(aq) + NH4Cl, pH = 9.6) was used. The supporting electrolyte was exchanged after each analysis. The parameters of the measurement were as follows: initial potential of -0.7 V, end potential of -1.75 V, modulation time 0.057 s, time interval 0.2 s, step potential 2 mV, modulation amplitude - 250 mV, Eads = 0 V, volume of injected sample: 10 µl, volume of measurement cell 2 ml (10 μl of sample, 1990 μl of Brdicka solution) [27-31]. 2.14. Determination of concentration of selenium in ejaculate The concentration of selenium was determined using differential pulse voltammetry by a 797 VA Computrace and an 889 IC Sample Center (Metrohm, Switzerland) using a standard cell with three electrodes. A hanging mercury drop electrode (HMDE) with a drop area of 0.4 mm2 was the working electrode. An Ag/AgCl/3M KCl electrode was the reference and glassy carbon electrode was the auxiliary. The analysed samples were deoxygenated prior to measurements by purging with argon Int. J. Electrochem. Sci., Vol. 10, 2015 6617 (99.999 %) saturated with water for 120 s. The parameters of the measurement were as follows: deposition potential -0.6 V, accumulation time 200 s, pulse amplitude 0.03 V, pulse time 0.05 s, voltage step 0.006 V, voltage step time 0.1 s, sweep rate 0.06 V/s, equilibration time 30 s. Total volume of the measuring vessel was 2 ml (1980 µl of electrolyte and 20 µl of sample). The electrolyte for selenium was prepared by using 0.015 mM ammonium sulphate with addition of copper sulphate; the final concentration of CuSO4 in solution was 0.05 mM. pH of this electrolyte was adjusted to 2.2 using sulphuric acid. The scan was in the range of potentials -0.4 V to -0.9 V and the characteristic peak of selenium was recorded at potential of -0.7 V. 2.15. Photometric determination of malondialdehyde The principle of malondialdehyde determination is reaction between malondialdehyde (MDA) with thiobarbituric acid (TBA) under formation of TBA-MDA-TBA adduct that absorbs strongly at 535 nm. Trichloroacetic acid (TCA) is added to the sample because of its ability to precipitate proteins, bilirubin, unsaturated fatty acids and lipoproteins. A 300 µL sample of blood plasma was mixed with 10 µL 0.5 M solution of butylated hydroxytoluene (BHT) in 96 % ethanol (v/v) and 310 µL 20 % TCA (v/v) prepared in 0.6 M HCl. After 20 min incubation on ice mixture was centrifuged at 11,000 rpm for 15 min. Subsequently, 400 µL of supernatant was mixed with 800 µL of 30 mM TBA and mixture was incubated in a thermomixer Comfort (Eppendorf, Germany) at 90 °C for 30 min. After cooling in ice MDA absorbance was measured using a spectrophotometer at 535 nm and the concentration was subtracted from the calibration curve [32]. 2.16. Determination of glucose Glucose is oxidized by glucose oxidase to gluconolactone in the presence of oxygen. The resulting hydrogen peroxide is oxidized by 4-aminoantipyrine and phenol to quinone red in the presence of peroxidase (POD). The intensity of generated red colour is directly proportional to the concentration of glucose and can be determined photometrically. Into plastic cuvettes, 200 µl reagent (Greiner, Germany, 0.1 M phosphate buffer pH 7.5, 0.75 mM phenol, 0.25 mM 4-aminoantipyrin, glucose oxidase ≥ 15kU / l peroxidase ≥ 1.5 U / l) was added followed by 20 µl of the sample. The absorbance was measured at λ = 505 after an incubation lasting 10 minutes. The absorbance values of reagents and absorbance values after 10 minutes of incubation with the sample were used to calculate glucose content in the sample. 2.17. Determination of pH pH in the ejaculate was determined using a pH meter inoLab level 3 (WTW GmbH, Weilheim, Germany). Int. J. Electrochem. Sci., Vol. 10, 2015 6618 2.18. Statistic The data were processed statistically using STATISTICA.CZ, version 10.0 (Czech Republic). Results are expressed as mean from 3 measurements ± standard deviation. Statistical significance was determined by examining the basic differences among individual samples using ANOVA and Scheffé’s method (two-factor analysis) for the parameters GPx, SOD; DMPD; DPPH; FRAP; ABTS; FR; MDA; glucose; pH; metallothionein; levels selenium; zinc and copper; quality of ejaculate volume of ejaculate; motility of sperms; concentration of sperms and abnormal sperms. Differences with P<0.05 were considered significant. 3. RESULTS Figure 2. Antioxidant activity measured by methods FRAP - Ferric Reducing Antioxidant Power and FR - Free Radicals (A); DMPD - N-dimethyl -1,4-diaminobenzene and DPPH-2,2-diphenyl-1picrylhydrazyl (B); ABTS-2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid and lipid peroxidation measures as a content of MDA - malondialdehyde (C); pH and glucose (D). Results are expressed as mean from 3 measurements ± standard deviation; P<0.05. Experiment lasted 135 days and no health problems were observed in experimental animals. 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