Animals and semen collection
A total of 40 Bos indicus bulls (Nellore) were selected from an artificial insemination center located in the southeast of Brazil (21°04′52″S, 48°02′24″W). The bulls were kept on their native pasture (Cynodon plectostachyus) and were given dietary supplementation to optimize their energy balance. Three ejaculates were collected from each bull using an artificial vagina according to a regular twice-a-week collection schedule between September and February of 2010. The samples were cryopreserved (TRIS-egg yolk extender, 7% glycerol in 250 μL French mini-straws) in liquid nitrogen until the evaluation. The 40 selected animals were divided into three groups according to their ages at collection time: 1.8 to 2 years (young group, 27 samples), 3.5 to 7 years (adult group, 57 samples), and 8 to 14.3 years (aged group, 36 samples).
Flow cytometry
Flow cytometry analysis was performed on an Attune® apparatus (Applied Biosystems by Life Technologies, Grand Island, NY, USA) equipped with 488 nm and 405 nm laser beams and the following emission filters: BL1 530/30 nm “band pass” (BP), BL2575/24 nm BP, BL3 640 nm “long pass” (LP), and VL1 450/40 nm BP, VL2522/30 nm BP, and VL3 603/48 nm BP.
Computer assisted semen analysis (CASA)
Frozen semen samples (250 μl straws) were thawed at 35 °C for 20 s. Microscopic evaluations were performed using an Olympus BX61 microscope (Olympus, Tokyo, Japan) equipped with phase contrast, differential interference contrast (DIC), and epifluorescence. The Computer Assisted Semen Analysis of sperm movement (CASA 12.3-Hamilton Research®, Beverly, MA, USA) used the IVOS “hardware” (Integrat Visual Optical System, Hamilton Thorne Biosciences®, Beverly, MA, USA). The following kinetic spermatic parameters were evaluated in 2 μl sample aliquots on a slide with four chambers (Leja, NL): MOT (total motility), MP (progressive motility), VAP (average path velocity), VSL (straight-line velocity), and VCL (curvilinear velocity). The fields were randomly selected for each analysis based on the absence of artifacts such as bubbles, particles, or other elements that could adversely affect the evaluation.
Sperm morphology
Sperm alterations were classified according to Blom (1973) [12] and Barth & Oko (1989) [13] into major defects (i.e., acrosome defects, proximal droplets, abnormal loose heads, abnormal contour, abnormal midpiece, vacuoles, double forms, dag defect) and minor defects (i.e., small normal heads, normal loose heads, abaxial implantation, coiled tails, distal droplets). The abnormalities were additionally grouped as head defects, tail defects (abnormal mid-piece, other tail defects), droplet defects (proximal droplet, distal droplet), and normal loose heads. A total of 200 cells (%, DIC microscopy, oil immersion, 1000×, Olympus BX61) were examined per sample, and one abnormality was recorded per cell.
Evaluation of membrane integrity - PI and FITC-PSA
One straw per batch was thawed (35 °C/20 s). The semen sample was then diluted to 2.106 sperm in 200 μl of PBS (phosphate buffer solution) and stained with 3 μL (14 μM) of propidium iodide (PI) and 50 μL (12.5 μM) of Pisum sativum agglutinin conjugated to fluorescein isothiocyanate (FITC-PSA). Samples were incubated at 37 °C for 8 min in the dark and analyzed in an Attune® flow cytometer with a 488 nm argon ion laser excitation beam and simultaneous BL1 (530/30) and BL3 (640LP) readings. The positive control semen samples were subjected to three cycles of “Flash Frozen” [14] prior to staining to promote membrane injury and positive staining in red for PI and green for FITC-PSA.
Evaluation of mitochondrial potential - JC-1
One straw with frozen semen was thawed (35 °C/20s) and diluted in PBS to obtain 2.106 sperm in 200 μl; 6 μL of JC-1 (4 μM) was added to this aliquot, incubated for 8 min at 37 °C in the dark, and analyzed using a flow cytometer with a 488 nm argon ion laser excitation beam and simultaneous BL1 (530/30 nm) and BL2 (575/24 nm) readings. The control cells (with low mitochondrial potential) were incubated with 10 μM of carbonyl cyanide-m-chlorophenylhydrazone (CCCP, Sigma, St. Louis, MO, U.S.A.) at 37 °C for 30 min and stained with JC-1, according to the protocol described previously. CCCP is an uncoupling agent that rescues protons and depolarizes the mitochondrial membrane [15].
Evaluation of protamination – CMA3 assay
Sperm samples were diluted in 200 μl of McIlvaine’s buffer (2 × 106 sperm) and centrifuged at 300 g for 5 min; the supernatant was discarded, and the sample was resuspended in 200 μl of Carnoy solution (3:1 methanol: acetic acid) and incubated at 4 °C for 5 min. Samples were washed twice with PBS and incubated for 60 min at room temperature with 200 μL chromomycin A3 (CMA3) staining solution (0.4 μM). Samples were washed, resuspended in 1 ml of PBS, and read in the flow cytometer at 488 nm and BL2 filter (575/24 nm); 10,000 events corresponding to sperm were assessed. Semen samples used as positive controls were thawed and centrifuged in McIlvaine’s buffer at 300 g for 5 min, resuspended in PBS containing 5 mM dithiothreitol (DTT) and triton X-100 0,1%, and incubated for 15 min at 37 °C to break disulfide bridges between protamine chains and permeate the cell membrane. Samples were washed twice to remove the detergent solution and DTT, fixed in a Carnoy solution for 45 min [16], stained as described above and read using a 488 nm argon ion laser excitation beam and BL1 filter (530/30 nm); 10,000 events were assessed.
DNA oxidative – 8-OHdG assay
Guanine oxidation was evaluated using the protocol described by DeIuliis et al. (2009) [17]: thawed semen samples were centrifuged in PBS (300 g), resuspended in 100 μL of DTT (2 mM), and incubated at 37 °C for 45 min. Samples were subsequently washed with PBS, with the addition of 100 μL of 4% paraformaldehyde per 100 μL of PBS, and incubated at 4 °C for 15 min. Cells were washed in PBS, and incubated in 100 μL Triton ×100 0,1% at room temperature for 15 min; cells were then prepared according to the OXYDNA® kit (Biotrin, Ireland) procedure, diluted in distilled water at 1:25, and incubated in 1:9 anti-8-OHdG antibody labeled with FITC solution at room temperature for 60 min. Stained samples were washed, resuspended in 1 mL of PBS, and read with a 488 nm argon ion laser excitation beam and BL1 filter (530/30 nm); 10,000 events were assessed. Positive control samples were incubated in DDT (2 mM) for 45 min and were further incubated for 60 min, with the addition of hydrogen peroxide (2 mM) and ferrous sulfate (1 mM).
Chromatin integrity - SCSA
Sperm samples were adjusted to 2 × 106 spermatozoa/mL in PBS to a final volume of 200 μL, to which 400 μl of acid detergent solution (0.08 M HCl, 15 M NaCl, and 0.1% Triton ×100, pH 1.2) was subsequently added. After 30 s of incubation, 1.2 mL of Acridine Orange (AO) solution was added (6 μg of AO per mL of buffer – 0.037 M citric acid, 0.126 M Na2HPO4, 1.1 M EDTA, and 0.15 M NaCl, pH 6.0). The samples were read 3 min after the addition of acid detergent, and 10,000 events were assessed [18]. The assessments were analyzed using the Flowjo® program (Tree Star, Inc., San Carlos, CA, USA) to obtain the total percentage of sperm with damaged chromatin (% DFI). Positive control samples were incubated in DDT (2 mM) for 45 min. Hydrogen peroxide (2 mM) and ferrous sulfate (1 mM) were then added, and samples were incubated for 60 min.
Statistical analysis
Our null hypothesis (H0) was that there were no differences in DNA integrity and types of chromatin damage in cryopreserved sperm from bulls at different ages. The statistical analyses were performed using the SAS software, “Statistical Analysis System” software (release 9.2. SAS Institute Inc., Cary, NC, USA, 2008). The percentage data were transformed into arcsine to obtain a normal distribution. The results were examined using the “two-way” ANOVA (bulls × group). The averages were compared by the Duncan test and were considered significant when p < 0.05. Correlation (r) and determination (r2) coefficients and the regression equation were calculated for selected variables [19].
