Chemicals
All chemicals in this study were purchased from Sigma Chemical CO. (St. Louis, MO, USA), unless stated otherwise.
Description of Radio Electric Asymmetric Conveyer (REAC) Technology
Radio Electric Asymmetric Conveyer Technology (REAC) is a technological platform for bio and neuro modulation. A detailed description of REAC mechanism of action can be found in Maioli 2016 [2]. Briefly, REAC is an asymmetric technology, because a normal electric circuit has two physical poles: one positive and one negative (symmetrical circuit); in the REAC technology, there is only one single physical pole (asymmetrical circuit). This pole becomes the attractor (Asymmetric Conveyer) for the currents induced in the body by the radio frequency emission. This scheme has been developed to create an asymmetric circuit for better interact with the asymmetric mechanism underlying the cell polarity [24], in order to optimize its functions. In fact, REAC technology is able to modulate the current flows existing both at cellular and body level, when these are altered. Another peculiarity of REAC Technology is the low power level used in radio frequency emission. This is necessary to induce current flows of intensity comparable with those of cell polarity. Higher power levels would disturb the adjustment mechanisms of cell polarity. REAC devices use only two radio frequencies (2.4 and 5.8 GHz) because these are the most widely used and permitted at the international level. In this study, we used the biomodulation REAC-VIVSI treatment protocol. The REAC device used in this study was B.E.N.E (ASMED, Florence, Italy).
Semen source and preparation
The experimental procedures were carried out during horse breeding season (January–July). Semen collection was carried on at the Department of Stallion Reproduction of the Regional Agency for Research in Agriculture (AGRIS Sardegna, Ozieri, Sassari, Italy), while the analytical work was carried on at the laboratories of the Department of Veterinary Medicine of the University of Sassari (UNISS, Sassari, Italy). These facilities meet the requirements of the European Union for Scientific Procedure Establishments. Ejaculates were obtained by artificial vagina from eight adult stallions of different ages (from 12 to 24 years), and different breeds. In particular, stallions were identified with progressive number from 1 to 8 (stallion 1: Thoroughbred; stallions 2, 3, 4, 6: Arabian; stallions 5, 7, 8: Warmblood). The stallions were housed individually in boxes, and fed ad libitum with a diet containing adequate nutrients. At the moment of the study, they had been housed in AGRIS for over 5 years and used exclusively for breeding, and they are currently still housed at the same center. All evaluated stallions had a career in sports flat racing and show jumping.
The eight stallions enrolled in this study were used for breeding, and the frequency in semen collection during the entire breeding season was twice a week. We used for this study two ejaculates from each male, collected 15 days apart from each other. Semen was transported to the AGRIS laboratory within 5 min after collection, and it was immediately processed. Sperm concentration was evaluated by NucleocounterSp - 100 Chemometech. Thereafter, semen was diluted up to 50 ×106 spermatozoa/mL with a commercial media for liquid storage (Kenney's extender, IMV Technologies). Ejaculates from each stallion were kept separated throughout all experimental procedures and, once diluted, they were placed in two separated Falcon tubes and transported to the UNISS laboratory under controlled temperature (4 °C) within 1 h.
Upon arrival, one sample from each stallion was allocated to the treated and untreated group. The experimental groups were placed in two refrigerators at 4 °C and kept there for 72 h. The REAC device was placed in the refrigerator where treated samples were stored, and it was set at 2.4 GHz, and its conveyer electrodes were immersed into the semen liquid storage media.
Experimental design
In order to evaluate the effects of REAC technology during stallion semen liquid storage, a battery of analyses was performed. The parameters analyzed included different semen molecular and cellular features, measured before and during REAC treatment, such as viability, motility parameters, acrosome status, and DNA integrity. In addition, the oxidative status was assessed by determining lipid peroxidation, the activity of superoxide dismutase (SOD), and the total antioxidant capacity. These analyses were performed on sub-samples of cooled semen collected at 0, 24, 48, and 72 h. after the beginning of the REAC treatment. Each analysis was replicated 3 times.
Viability and motility parameters assessment
In vitro viability was assessed by eosin-nigrosin stain. Briefly, the eosin–nigrosin solution was prepared as described by Pintado et al. [25]. Briefly, 10 g nigrosin was dissolved in distilled water by boiling, and filtered into a cylinder containing 0.7 g eosin, 7.5 ml of 50 mmol glucose l–1, and 7.5 ml tartrate phosphate buffer (TPB) (50 mM Na2HPO4 l–1, 25 mM KH2PO4 l–1, 77 mM potassium sodium tartrate l–1), and the volume made up to 100 mL. The solution was kept at 5 °C. Staining was carried out by mixing an aliquot of spermatozoa suspended in saline medium with eosin–nigrosin solution (1:3 dilution) for 30 s before preparing a smear and drying on a warm plate at 37 °C. At least 200 cells were counted for each slide. Sperm motility parameters were assessed using a computer-assisted sperm analysis (CASA) system (Sperm Class Analyser, S.C.A. v 3.2.0, Microptic S.L., Barcelona, Spain) with setting of 25 frames acquired to avoid sperm track overlapping, minimum contrast 10, minimum velocity of average path 30 μm/s, progressive motility > 80% straightness. This system has a specific set-up for stallion sperm evaluation. In particular, it was set up as follows: minimum contrast – 70; low and high static size gates – 0.6–4.32; low and high intensity gates – 0.20–1.92; low and high elongation gates 7–91; default cell size – 10 pixels; default cell intensity −80. For each sample, 5 μL subsample of sperm suspension was loaded into a pre-warmed analysis chamber with a depth of 10 μm (Makler Counting chamber, Sefi-Medical Instruments ltd., Biosigma S.r.l., Italy) and a minimum of 500 sperms per subsample were analyzed in at least four different microscopic fields. Sperm motility was assessed at 37 °C at 40 × using a phase contrast microscope. The parameters evaluated included: percentage of progressive motile spermatozoa (PM); percentage of rapid spermatozoa (rapid); average path velocity (VAP, mm/s; the average velocity of the smoothed cell path); curvilinear velocity (VCL, mm/s; the average velocity measured over the actual point to point track followed by the cell); straight-line velocity (VSL, mm/s; the average velocity measured in a straight line from the beginning to the end of the track); linearity index (LIN, %; the average value of the ratio VSL/VCL); straightness index (STR, %; the average value of the ratio VSL/VAP); amplitude of lateral head displacement (ALH, mm; the mean width of the head oscillation as the sperm swim); beat cross-frequency (BCF, Hz; the frequency of sperm head crossing the average path in either direction); wobble (WOB; VAP/VCL × 100, %; a measure of the oscillation of the actual trajectory about its spatial average path).
Acrosome integrity
Acrosome integrity was evaluated by incubating spermatozoa with fluorescein isothiocynatelabeled PisumSativum agglutinin (FITC-PSA). The aliquots of sperm suspension were incubated for 15 min at 39 °C with FITC-PSA (5 μg/mL in phosphate buffered saline [PBS], pH 7.4), and propidium iodide (PI; 14 μg/mL in phosphate buffered saline [PBS], pH 7.4). In order to reduce background fluorescence, unbound PSA and PI were removed by adding 200 μL of PBS and spermatozoa were washed by centrifugation in a micro centrifuge at 800 g for 2 min. The supernatant was aspirated and the pellet re-suspended in 100 μL of PBS. After washing, a 10 μL sample was put on a slide and cover slipped. The slide was immediately dried by leaving at 37 °C for 10 min for immobilization of sperm cells. To evaluate the stained sperm cells, at least 200 cells were counted in duplicate for each sample, using a Diaphot (Nikon, Japan) epifluorescence microscope. Spermatozoa with intact plasma membrane and intact acrosome were PI and FITC-PSA negative (no fluorescent staining), those with intact plasma membrane and damaged acrosome were PI negative and FITC-PSA positive (emitting green fluorescence), those with damaged plasma membrane and intact acrosome were PI positive and FITC-PSA negative (emitting red fluorescence), and finally those with damaged plasma membrane and damaged acrosome were PI and FITC-PSA positive (emitting both green and red fluorescence).
DNA integrity assessment
DNA damage was assessed by single-cell gel electrophoresis (comet assay). Analysis of the shape and length of "comet" tail, just like the DNA content in the tail, gives an assessment of DNA damage. The neutral comet assay allows the detection of double-strand breaks by subjecting lysed cell nuclei to an electrophoretic field at neutral pH [26], here performed according to the method described by Sakkas et al.[27], with slight modifications. Briefly, sperm suspension (30 μL) was diluted in low-melting-point agarose at 37 °C (80 μL; 1% w/v). A 100-μL mixture of sperm-agarose was immediately pipetted onto 1% w/v normal-melting-point agarose-coated slides. Slides were immersed in ice-cold lysing solution (2.5 M NaCl, 100 mM EDTA, 10 mMTris, 1% Triton X, and 10 mMdithiothreitol [DTT]; pH = 10) for 1 h at 4 °C. Slides were then immersed in lysing solution supplemented with proteinase K (10 μg/mL). Incubation was performed during 1 h at 37 °C. After this step, slides were rinsed in PBS and then placed in a horizontal electrophoresis tank filled with freshly prepared electrophoresis neutral buffer (Tris-acetate-EDTA [TAE], pH 7.3). Electrophoresis was performed at 10 V and 6 mA for 20 min. Following electrophoresis, the slides were neutralized with Tris–HCl buffer (pH 7.5) for 5 min and then fixed in methanol.
Slides were stained with propidium iodide (PI), mounted with a coverslip and analyzed under an epifluorescence microscope. Digital comet images were captured with an Olympus microscope equipped with a CCD camera and Olympus CellF software. Fifty comets were measured per replicate sample (i.e., slide circle) using Comet Score software (TriTek Corp., Sumerduck, Virginia, USA). Scored parameters included percentages of head and tail DNA (a measurement of the proportion of total DNA that is present in the comet head and tail).
Sample preparation for the oxidative parameters analysis
Five mL of semen samples (50×106 spermatozoa/mL) was centrifuged at 1500 g for 10 min. The obtained pellets were treated for cellular extraction with PBS containing 0,1% Triton X-100 (500 μL of PBS-Triton X-100 0,1% every 250×106 total spermatozoa). Malondialdehyde concentration (MDA) and superoxide dismutase (SOD) activity were assayed in cellular extracts, while trolox equivalent antioxidant capacity (TEAC) was determined in both cellular extracts and extracellular supernatants.
Superoxide dismutase (SOD) activity
SOD activity was measured enzymatically as a decrease of the XTT (3'-(1-[(Phenylamino)-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzenesulphonic acid hydrate) reduction by superoxide anion generated by xanthine oxidase [28].
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a)
$$\mathrm{Xanthine}+{\mathrm{O}}_2\overset{\mathrm{XO}}{\to}\mathrm{uric}\;\mathrm{acid}+{{\mathrm{O}}_2}^{-}$$
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b)
$${{\mathrm{O}}_2}^{\hbox{-} }+\mathrm{X}\mathrm{T}\mathrm{T}\;\left(\mathrm{detector}\right)\to \mathrm{reduced}\;\mathrm{X}\mathrm{T}\mathrm{T}$$
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c)
$${{2\mathrm{O}}_2}^{\hbox{-} }+2{\mathrm{H}}^{+}\overset{\mathrm{SOD}}{\to }{\mathrm{H}}_2{\mathrm{O}}_2+{\mathrm{O}}_2$$
SOD activity was assessed as the competition between reaction c and b which is measured as a decrease of the rate of XTT reduced. The reaction mixture contained 40.5 mM sodium phosphate buffer pH 7.8; 15 mM xanthine; EDTA 12,5 mM; XTT 30 mM and 50 μL of sample to complete a final volume of 500 μL. The reaction was initiated by the addition of xantine oxidase (XO) (0.15 mUI) and the absorbance change at 470 nm was monitored each minute for 3 min total with a Hitachi spectrophotometer (U-2000). The values of SOD in the samples were expressed in U/mL and calculated using a standard curve (0,065-0,8 U/ml). One enzyme unit (IU) is defined as the amount of SOD capable of transforming 1.0 mmole/min of O2•¬
Quantification of lipid peroxidation end products: malondialdehyde (MDA)
MDA, one of the several low-molecular-weight end-products of LPO, was evaluated by the TBARS assay using thiobarbituric acid and a spectrophotometric method according to the TBA test described by Spanier and Traylor [29], with some modifications. 100 μL of each sample (cell extract and extracellular supernatant) were added to 100 μL glacial acetic acid 33%, 75 μL SDS 10%, 100 μL Tris–HCl 50 mM pH 7,4 and 250 μL TBA 0,75%. The mixture was then incubated for 1 h at 100 °C and immediately cooled on ice. After 10 min 200 μL of acetic acid 33% were added and samples were centrifuged for 20 min at 7000 g. The supernatant absorbance was then read with Thermo Electron Corporation Genesys 10UV spectrophotometer (Thermo Fisher Scientific, Rodano, Milano, Italy), at 535 nm. The values of MDA in the samples were expressed in μM units and calculated using a standard curve.
Trolox equivalent antioxidant capacity (TEAC)
Cell extract and extracellular supernatant antioxidant capacity was determined using the method described by Re et al.[30], and modified by Lewinska et al.[31]. Briefly, a fresh solution was prepared by dissolving 19.5 mg 2,20-azinobis (3- ethylbenzthiazoline −6-sulphonic acid [ABTS]) and 3.3 mg potassium persulphate in 7 mL of 0.1 M phosphate buffer, pH 7.4. This solution was stored in the dark for 12 h for completion of the reaction. ABTS solution was diluted (usually approximately 1:80) in 0.1 mol/L phosphate buffer, pH 7.4, to give an absorbance reading at 734 nm of 1.0. The absorbance of the mixture was measured twice in a spectrophotometer (ThermoElecrom Corporation Genesys 10 UV, Madison, Wisconsin, USA), at 734 nm, 3 min after mixing a sample with the ABTS•¬˙ solution. The extent of ABTS•¬˙ bleaching is proportional to the activity of antioxidants in a given sample. The antioxidant capacity was expressed as TEAC, the concentration of trolox producing the same effect as the sample studied.
The values of TEAC in the samples were calculated using a standard curve (5–20 mMtrolox in a total volume of 550 mL) and were expressed as mMTrolox equivalent for extracellular supernatant and nmoli of trolox equivalent/109 spermatozoa for cell extract.
Statistical analyses
Statistical analyses were performed using the statistical software program Statgraphic Centurion XV (version15.2.06 for Windows; Stat Point Technologies Inc., Warrenton, VA, USA), and a probability of p < 0.05 was considered to be the minimum level of significance. Data are expressed as mean ± S.E. Differences in sperm parameters between the experimental groups at the different time points (hours of treatment) were assessed by general lineal model where: Y = μ + hours of treatment + group + hours of treatment x group + stallion. Hours of treatment and group were considered fixed factors and stallion a random factor. The method used to discriminate between the means was Fisher’s least-significant-difference (l.s.d.) procedure. The probabilities obtained by the l.s.d. test were corrected by Bonferroni’s correction for multiple comparisons. Data were normally distributed (Shapiro Wilk W test: P >0.05).
