Animals and experimental study
This study was previously approved by the Bioethics Committee of the School of Veterinary Medicine and Animal Science - University of São Paulo (protocol number: 7122171213). All chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise listed.
As described in detail previously, using the same study population [27], 20 male dogs of several breeds, aging from 5 to 13 years and weighting from 10 to 30 kg were selected for this study and assigned to four experimental groups:
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(1)
BPH Group (n = 5): dogs with mean age of 10.8 years and body weight of 18.8 kg were presumptively diagnosed with BPH based on clinical signs (hematospermia, hematuria, pollakiuria, dysuria and tenesmus), marked prostatomegaly and prostatic biometry by B-mode ultrasound [28, 29].
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(2)
BPH-Finasteride Group (n = 5): dogs with mean age of 9.2 years and body weight of 23.4 kg were presumptively diagnosed with BPH (as for the BPH Group) and subjected to BPH treatment with 5 mg finasteride per animal, orally, every 24 h for 2 months [30].
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(3)
Non-BPH-Finasteride Group (n = 5): dogs with mean age of 7.4 years and body weight of 20.8 kg without BPH were subjected to finasteride treatment protocol (as for the BPH-Finasteride Group). The diagnosis of BPH was ruled out by the lack of characteristic clinical signs coupled with a normal prostate morphometry by B-mode ultrasonography [28, 29].
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(4)
Non-BPH-Untreated Group (Control Group, n = 5): dogs with mean age of 7.2 years and body weight of 22.3 kg, not presenting BPH (as for the Non-BPH-Finasteride Group) nor subjected to finasteride protocol.
To assure the appropriate sample size, an analysis was conducted with the SAS Power and Sample Size 12 (SAS Institute Inc., Cary, NC, EUA). A retrospective analysis of the data indicated there was a power of 0.99, which is considered an acceptable statistical power (at least 0.8). Hence, a minimum of 5 dogs per group were sufficient to demonstrate significant differences in the data.
Seminal collection
Semen samples were collected monthly for 60 days, i.e., Day 0, Day 30 and Day 60, considering day 0 as the first day of finasteride treatment or BPH diagnosis. Semen was collected by penile digital manipulation directly into a calibrated plastic tube connected to a funnel. The sperm-rich fraction was collected by means of visual inspection of the ejaculate, as well as part of the 3rd fraction containing prostatic fluid. While collecting the sperm-rich fraction, we also included part of the prostatic fraction, as to achieve an equal proportion 1:1 of semen:prostatic fluid (v:v).
Subsequently, conventional sperm motility (%) was assessed under light microscopy (Nikon, Eclipse E200, Japan) at 400× magnification using 5 μL of semen placed on a pre-warmed glass slide with coverslip. Only dogs that showed total motility higher than 60% were included in this study. Then, the ejaculate was processed for sperm DNA integrity analysis.
Evaluation of sperm susceptibility to DNA fragmentation
Toluidine blue (TB) staining
Semen smears were prepared and subjected to toluidine blue staining according to a protocol previously described by Rui et al. [31]. Smears were prepared with 10 μL of the sperm sample on a glass slide and subsequently fixed in 96% ethanol-acetone for 30 min at 4 °C. After drying, smears were hydrolyzed in 0.1 N HCl for 5 min at 4 °C and washed three times in distilled water for 2 min. Subsequently, smears were exposed to toluidine blue stain (0.05%) for 20 min and washed 2 times in distilled water for 2 min. Smears were evaluated under light microscopy (Leitz, Dialux 20, Germany) at 1000x magnification in a single-blind way, i.e., researchers were kept ignorant of either the group they were assessing. Sperm cells with damaged DNA were stained in blue, whereas intact DNA sperm remained unstained. For each sample, a minimum of 200 sperm cells were assessed and results were expressed in percentage (%) of DNA damaged spermatozoa.
Sperm chromatic structure assay (SCSA)
The assay was performed following the methodology adapted by Lucio et al. [32] for dogs, based on a protocol that allows the estimation of chromatin susceptibility to acid denaturation [33]. Chromatin instability after acid exposure was quantified by flow cytometer after acridine orange (AO) labeling. Based on the DNA integrity status, a metachromatic fluorescence shift is induced from green (double-strand DNA) to red (denatured single-strand DNA). We used the Guava EasyCyte™ Mini System (Guava® Technologies, Hayward, CA, USA), with a 488 nm argon laser and the following photodetector filters: PM1 (583 nm; yellow fluorescence), PM2 (680 nm; red fluorescence) and PM3 (525 nm, green fluorescence). A total of 20,000 sperm cells were considered from each sperm sample and data were analyzed using Flow Jo v8.7 Software (Flow Cytometry Analysis Software – Tree Star Inc., Ashland, Oregon, USA).
In brief, sperm samples (15 μL) were diluted in 100 μL TNE buffer (0.01 M Tris–HCl, 0.15 M NaCl, 1 mM EDTA, pH 7.4) and subsequently mixed with 400 μL of an acidified detergent solution (0.08 M HCl, 0.1% Triton X-100, 0.15 M NaCl, pH 1.2). After 30 s, sperm cells were stained by adding 600 μL of AO staining solution (0.037 M citric acid, 0.126 M Na2HPO4, 0.0011 M disodium EDTA, 0.15 M NaCl, pH 6.0). After 5 min, samples were examined by flow cytometry as described above. DNA fragmentation rates were calculated based on the percentage of spermatozoa outside the main population in an αT histogram (ratio between red fluorescence and total fluorescence) as evaluated using the Flow Jo system (Version Mac) [32, 34].
Identification of DNA protamination by chromomycin A3 (CMA3)
In order to identify defects on sperm protamination process, the chromomycin A3 technique was performed based on protocols previously described by Rahman et al. [35] and Simões et al. [36]. A positive control sample of deprotaminated dog sperm was prepared by exposing spermatozoa to a solution of 0.001% Triton X-100 and 5 mM DTT in 200 μL phosphate buffered saline (PBS) for 15 min, followed by incubation in a solution of 1 M NaCl and 5 mM DTT in H2O for 2 h. Subsequently, the positive control and all other tested sperm samples were washed in PBS and fixed in Carnoy’s solution (3:1 methanol:acetic acid; Merck, Darmstadt, Germany) at 4 °C for 10 min. Then, smears were prepared and subsequently treated with 12.5 μL CMA3 solution [0.25 mg/ml in 1000 μL of McIlvaine buffer (7 ml of 0.1 M citric acid + 32.9 ml of Na2 HPO4 .7H2O, 2 M, pH 7.0, containing 10 mM MgCl2)] for 20 min. The slides were additionally stained for 2 to 5 min with Hoechst 33342 (5 mg/ml) in order to determine the total number of sperm cells. Then, slides were washed in PBS. Microscopic images were captured using fluorescence microscopy (Leica DMR, 400x magnification) with appropriate filters (460–470 nM) in a single-blind manner. A minimum of 200 cells were counted and results were expressed in percentage of sperm showing protamine deficiency (%).
Direct verification of DNA fragmentation by the modified sperm chromatin dispersion assay (SCDA)
The assay was performed according to the procedure of Shanmugam et al. [37] and Fernández et al. [38]. Initially, the protocol was validated with the use of the sperm-rich fraction collected by digital manipulation from six sexually mature dogs of several breeds. All dogs had proven semen quality, confirmed by previous breeding soundness examination. Only sperm-rich fractions with a minimum of 60% total sperm motility were used. After sperm collection, one aliquot of each sample was kept at 5 °C and the remaining sample was exposed to ultraviolet light (Fluxo Veco VLFS-12 M, Campinas, São Paulo) for 4 h at 25 °C in order to artificially induce DNA fragmentation. Both aliquots were then mixed to obtain different known proportions of fragmented DNA sperm (0, 25, 50, 75 and 100%). Sperm smears were subsequently prepared on a glass slide with 10 μL of each mixture of damaged/intact DNA sperm. Evaluation of DNA fragmentation was performed using the modified chromatin dispersion assay (SCDA). A minimum of 200 sperm cells were counted and results were expressed as percentage (%) of DNA fragmented spermatozoa in a single-blind way, i.e., researchers were kept ignorant of either the group they were assessing. A high linear regression coefficient (R2 = 0.97, p = 0.001) between the observed and the expected percentages of spermatozoa exhibiting DNA damage attested the validation of the SCD assay for dogs (Fig. 1).
For each sperm sample of the present experiment, equal volumes (1:1) of diluted semen (1 × 106 sperm/mL) and 1% low-melting agarose were mixed at 37 °C. An aliquot of 10 μL of this mixture was pipetted on a glass slide coated with 0.65% normal melting point agarose and subsequently covered with a cover slip and kept for 10 min at 4 °C in order to solidify. Immediately after careful removal of the cover slip, the slides were immersed horizontally for 3 min at 22 °C in the dark in a tray containing acid denaturation solution (0.08 N HCl). This condition generates restricted single-stranded DNA (ssDNA) motifs from DNA breaks. Subsequently, the denaturation was stopped and proteins were removed by transferring the slides for 2 h at 4 °C to a tray with neutralizing and lysis solution (10 mM Tris, 4 mM DTT, 2% Triton X-100, 100 mM Na2 - EDTA, 2.5 M NaCl, pH 11). The slides were washed in Tris-borate-EDTA buffer (0.09 M Tris-borate and 0.002 M EDTA, pH 7.5) for 2 min, dehydrated in sequential 70, 90 and 100% ethanol (2 min each) and, then, air dried. Prepared slides were horizontally covered with a mix of Wright’s stain and buffer solution (380 mg Na2 PO4, 547 mg KH2 PO4 in 100 mL distilled water) for 10 min with continuous air flow. The stain was poured off and slides were briefly washed in tap water and dried.
The stained slides were evaluated under a light microscope and 200 sperm were evaluated per slide for halo size and dispersion pattern at 1000× magnification in a single-blind manner. The nuclei with large to medium halo size were considered sperms with non-fragmented DNA, while nuclei with small halo size or without halo were considered as sperm cells with fragmented DNA.
Statistical analysis
All data were evaluated using SAS System for Windows (SAS Institute Inc., Cary, NC, USA). Effects of BPH, finasteride, moment of evaluation (days 0, 30 and 60) and interactions between these factors, were estimated by repeated measures analysis of variance (Mixed Procedure of SAS). If no triple interactions (BPH X finasteride X Timing) existed, the following interactions were considered: Timing x finasteride, Timing x BPH and finasteride x BPH. If no dual significant interactions were observed, then effects of groups were analyzed by merging all time points and conversely, time points were compared by combining all groups; otherwise, comparisons were performed taking both effects into account. Differences between BPH and finasteride treatment were analyzed using parametric and non-parametric tests, according to the residual normality (Gaussian distribution) and variance homogeneity. Data were transformed if one of these assumptions was not respected. When transformations were not successful, non-parametric tests were used. Moreover, differences between BPH and finasteride treatment were analyzed using Student t-test (parametric variables) and Wilcoxon test (nonparametric variables). Results were described as untransformed means ± SE. The significance level was P < 0.05.