Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

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ANIREP-5207; No. of Pages 10

Animal Reproduction Science xxx (2015) xxx–xxx

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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus) Rosanna Serafini a,∗ , Juan E. Romano b , Dickson D. Varner b , Rossella Di Palo a , Charles C. Love b a b

Department of Veterinary Medicine and Animal Production, University of Naples “Federico II”, Naples, Italy Department of Large Animal Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, TX, USA

a r t i c l e

i n f o

Article history: Received 13 February 2015 Received in revised form 15 May 2015 Accepted 22 May 2015 Available online xxx Keywords: Bull sperm DNA assays Sperm motility Sperm morphology

a b s t r a c t The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than interassay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P < 0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P < 0.05). The % Ghosts and COMP-˛t were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Sperm chromatin is organized in toroids, which are stable and compact structures that are attached to the nuclear matrix by toroid linker regions. These linker regions are the most susceptible to DNA damage (Ward and Coffey, 1991; Sotolongo et al., 2003). Sperm DNA injury can result

∗ Corresponding author at: DVM, Department of Veterinary Medicine and Animal Production, University of Naples “Federico II”, via Delpino 1, Naples, 80137, Italy. Tel.: +39 3287591539. E-mail address: rosanna.serafi[email protected] (R. Serafini).

in single (ssDNA) or double strand breaks (dsDNA) due to oxidative or enzymatic damage (Aitken et al., 2013). In humans, ssDNA breaks may have a more desirable prognosis as these are easier to repair than dsDNA breaks (Sakkas and Alvarez, 2010). The ssDNA breaks may impair fertilization (Ribas-Maynou et al., 2012b; Simon and Lewis, 2011) whereas dsDNA breaks are also reported to interfere with embryonic development and implantation (Lewis and Aitken, 2005) and are associated with recurrent miscarriages (Lewis and Simon, 2010). Many techniques have been developed to examine sperm DNA integrity, such as the Sperm Chromatin Structure Assay (SCSA), the single cell gel electrophoresis

http://dx.doi.org/10.1016/j.anireprosci.2015.05.015 0378-4320/© 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Serafini, R., et al., Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus). Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.05.015

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method (Comet assay) and the Sperm Bos Halomax (SBH) assay. These tests reportedly evaluate different aspects of the sperm DNA structure. The SCSA identifies the ratio of ssDNA (abnormal) to dsDNA (native) in the exposed toroid linker regions, but not in the more compact toroids (Shaman and Ward, 2006). This assay has been widely used to evaluate sperm DNA quality in men (Evenson et al., 1980), bulls (Ballachey et al., 1987, 1988; Januskauskas et al., 2001; Januskauskas et al., 2003; Waterhouse et al., 2006; Fortes et al., 2012; D’Occhio et al., 2013), stallions (Love and Kenney, 1998) and boars (Evenson et al., 1994). In contrast, the Comet assays (i.e., neutral and alkaline) reportedly allow stain access to both the toroid and toroid linker regions (Shaman et al., 2007) for identification of ssDNA and dsDNA breaks. In the Comet assays, sperm DNA breaks migrate away from the head region to form “comets” following electrophoresis, whereas intact DNA remains in the original head position (Shaman and Ward, 2006). Computer software is available to objectively evaluate comet images. The types of DNA breaks detected by the neutral and alkaline Comet assays are unclear. Shaman and Ward (2006) suggested that the alkaline Comet assay identifies ssDNA and dsDNA breaks while the neutral Comet assay detects mainly dsDNA breaks. In contrast, Evenson et al. (2002) and Baumgartner et al. (2009) proposed that the neutral Comet assay identifies dsDNA breaks and closely associated ssDNA breaks, whereas the alkaline Comet assay identifies only ssDNA breaks. As such, it remains unclear how neutral and alkaline Comet assays differ with respect to evaluation of sperm DNA quality. Sperm from fertile men had less ssDNA and dsDNA breaks compared to subfertile men when using neutral and alkaline Comet assays (Ribas-Maynou et al., 2012b). In bulls, the neutral Comet assay detected more DNA breaks (i.e., higher tail moment) in non sex-sorted sperm, as compared to sex-sorted sperm (Boe-Hansen et al., 2005). There was no correlation between neutral Comet assay and SCSA values in this previous research. The Sperm Bos Halomax (SBH) assay has been developed for assessment of sperm DNA integrity in bulls, based on the Sperm Chromatin Dispersion Test (SCDt) for humans (Fernandez et al., 2003). The SBH assay is similar to the Comet assays with the exception that treated sperm are not exposed to an electrophoretic field. Greater DNA fragmentation produces larger halos whereas less DNA fragmentation yields smaller halos (García-Macías et al., 2007). To date no comparisons of the four assays (alkaline and neutral Comet assays, SCSA, and the SBH assay) have been reported for evaluation of bull sperm DNA. Similarly there are no reports comparing these DNA tests with conventional measures of sperm quality. The aims of the present study were to: (1) objectively measure and describe comet images and determine Comet assay repeatability; (2) evaluate and compare sperm DNA quality among the SCSA, the neutral and the alkaline Comet assays, and the SBH assay; (3) determine the relationship of sperm DNA quality tests to traditional measures of sperm quality, i.e., sperm motility and morphology.

2. Materials and methods 2.1. Semen collection and general processing Thirty-three (33) beef-type (Angus or Brangus) bulls, 3–11 years old, underwent a breeding soundness examination (BSE) on a ranch in South Texas, USA. The BSE was performed in accordance with guidelines set forth by the Society for Theriogenology (Chenoweth et al., 1992). Semen was collected using an electronic ejaculator (ElectroJac 6, IDEAL Instruments, Lexington, KY, USA). Bulls (n = 20) were selected for this study, based on sperm motility and morphology values, for further analysis to compare the SCSA, the neutral and alkaline Comet assays and the SBH assay. There were ten bulls that were categorized to have the greatest sperm motility and morphology values, whereas there were 10 other bulls categorized as having the least sperm motility and morphology values. Immediately following semen collection, 10 ␮L of raw semen were diluted in 990 ␮L of Dulbecco’s phosphate-buffered saline (DPBS, Corning Cellgro, Mediatech, Inc., VA, USA) in each of four microcentrifuge tubes (1.5 mL) with snap caps (VWR International, LLC Radnor, PA, USA). In addition, 10 ␮L of raw semen were diluted in 990 ␮L of either a milk-based semen extender (INRA 96; IMV, Maple Grove, MN, USA) or a buffered formol saline solution (Kenney et al., 1983). The four tubes containing semen in DPBS were flash frozen on dry ice for later DNA evaluation (i.e., neutral and alkaline Comet assay, SCSA and SBH assays), because the farm was located several hours from the laboratory. The aliquots with semen extender were cool-stored in an Equitainer (Hamilton, Thorne Biosciences, Beverly, MA, USA) at 5–8 ◦ C to be transported to a laboratory where aliquots were analyzed for sperm concentration and sperm motility on the following day. Sperm concentration was measured with a fluorescence-based cell counter (NucleoCounterSP-100TM Chemometec, A/S, Allerød, Denmark) and sperm motility was analyzed with a computer-assisted sperm motion analysis (CASMA) system (IVOS Version 12.2.l, Hamilton, Thorne Biosciences, Beverly, MA, USA). Each sample (6 ␮L) was placed on a microscope slide (Leja Standard Count 2 Chamber slides; Leja Products, B.V., Nieuw-Vennep, The Netherlands) and slides were inserted into the CASMA. A minimum of 10 fields and 700–1000 sperm were measured for each sample. Variables of measure included: percentage of total motile sperm—TMOT; percentage of progressively motile sperm—PMOT; average path velocity—VAP (␮m/s); mean curvilinear velocity—VCL (␮m/s); straight line velocity—VSL (␮m/s) and straightness—STR (␮m/s). Preset values for the IVOS system consisted of the following: frames acquired—45/s; frame rate—60 Hz; minimum contrast—60; minimum cell size—4 pixels; straightness (STR) threshold for progressive motility—50%; average path velocity (VAP) threshold for progressive motility—30%; VAP threshold for static cells—15 ␮m/s; cell intensity—106 pixels. Sperm morphology was assessed in buffered formol saline solution and evaluated by differential-interference contrast microscopy (Olympus BX60, Olympus America, Inc., Melville, NY, USA; X 1562 magnification). A total of 100 sperm per sample were evaluated and all abnormalities


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identified on each sperm were recorded. The following sperm morphologic features were identified: normal, abnormal heads, abnormal acrosomes, detached heads, proximal or distal cytoplasmic droplets, abnormal midpieces, bent or coiled midpieces/tails, and premature germ cells (Kenney et al., 1983). 2.2. Neutral and alkaline Comet assays The protocols for the neutral and alkaline Comet assays were modified from Tice et al. (2000). Low melting point agarose (LMPA; Trevigen Inc., Gaithersburg, MD) was melted in a microwave for 10 s and then transferred to a 15 mL plastic tube and kept in a water bath at 37 ◦ C until used. Frozen semen samples were thawed in a water bath at 37 ◦ C and diluted to a concentration of 1 million/mL in DPBS, then 25 ␮L were dispensed into 250 ␮L of LMPA in an 1.5 mL microcentrifuge tubes with snap caps, floated in the water bath (∼30 s), and then vortexed for ∼5 s. Sperm/LMPA mixture (75 ␮L) were pipetted onto two Comet microscope slides (CometSlideTM 2 well/slide; Trevigen Inc. Gaithersburg, MD) for both neutral and alkaline Comet assays. Control bull sperm was included in each trial to monitor day-to-day (inter-assay) repeatability. The slides were placed in a rack (CometSlideTM Rack System Trevigen Inc., Gaithersburg, MD) and immersed in a cold (5–8 ◦ C) lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris HCl, 1% Triton X-100, 20 mM dithiothreitol-DTT, pH 10) for 30 min in the refrigerator, followed by immersion and incubation for 1 h and 15 min at 37 ◦ C in the same lysis solution with the addition of 0.1 mg/mL Proteinase K (Tritirachium album, MP Biomedicals, LLC, CA; USA). All solutions were made fresh daily and stored either in the refrigerator or incubator before adding to the Comet slides. Following lysis, slides were washed three times with deionized water at 5 min intervals and then placed in a horizontal electrophoretic unit (Fisher Scientific Electrophoresis Systems FB-SBR-2025).

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Slides for the neutral Comet assay were kept for 30 min in a chilled electrophoresis solution (500 mM NaCl, 0.1 M Tris-base, 1 mM EDTA, 0.2% DMSO, q.s. to 1.6 L deionized water, pH 9, osmolarity-mean ± SD—999.5 ± 1.34 osmol/L; range—998–1001 osmol/L). Electrophoresis was performed at 32 V (0.7 V/cm), 0.44–0.52 A, for 30 min at room temperature in the dark. Slides were subjected to drop-wise rinsing three times at 5-min intervals with a neutralization buffer (0.4 M Tris-base, pH 7.5), then dehydrated with cold 70% ethanol and left overnight, in the dark, at room temperature. –The alkaline Comet assay was performed immediately after the neutral assay. Slides were kept for 5 min in chilled electrophoresis solution (500 mM NaCl, 0.1 M Tris-base, 1 mM EDTA, 0.2% DMSO, q.s. to 1.6 L deionized water, pH 13, osmolarity-mean ± SD—1476 ± 175.05 osmol/L; range—1270–1682 osmol/L). Electrophoresis was performed at 32 V (0.7 V/cm), 0.73–0.97 A, for 15 min at room temperature in the dark. The neutralization and the dehydration steps were similar to that of the neutral Comet assay. 2.3. Slide staining, comet acquisition and scoring methods Slides were stained with 40 ␮L of diluted propidium iodide (PI; final concentration 0.38 ␮M) from a stock concentration of 9.6 ␮M PI in 1 mL Tris–EDTA buffer (10 mM Tris HCl and 1 mM EDTA) and incubated for 10 min in the dark. For each semen sample, two slides (two wells/slide) were prepared and 50 sperm/slide were counted (25 sperm/well). Samples were evaluated by fluorescent microscopy (Olympus microscope model BX60 equipped with an objective U Plan FL N, magnification 20×/0, 50 U/S2, ∞/ 0.17/ FN 26.5). Images were captured (DP Manager, Version 3.1.1.208, Olympus) with 1 s exposure time and an image size of 1360 × 1024 pixels. The imaging software (CometScore Version 1.5 TriTek Corp,

Fig. 1. Images of six different bull sperm nucleoids (1, 2, 3, 4, 5, 6) using alkaline (1–3) and neutral (4–6) Comet assays. For each image (labeled on the right side): a (1–6) show microscopic views (PI staining); b (1–6) software views; c (1–6) software’s graphic representation. In b: the outer box identifies the length and width of the nucleoid and the middle vertical line identifies the center of the comet head. Different amounts of DNA migration have been selected, to indicate the most intact (1 and 4 a, b, c) and the most fragmented DNA (Images 3 and 6 a, b, c). Fig. 6 represents a Ghost image. White scale bar = 10 ␮m.


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VA) was pre-calibrated with a micrometer. Comet measurements included the following variables, grouped as dimension measures (i.e., comet length, tail length, comet height, comet area, head diameter, head area, tail area), intensity measures (comet intensity, comet mean intensity, head intensity, head mean intensity, tail intensity, tail mean intensity), % DNA in head—% H-DNA and tail—%TDNA, tail moment—TM and Olive moment—OM. Comet measures are described in the user manual as follows: a Comet is defined as all adjoining pixel in a designated shape, where each pixel intensity corresponds to the amount of DNA at that location. It must be oriented from the left (head) to the right (tail). Comet length (␮m): number of microns in horizontal direction in Comet (corresponding to total length); Tail length (␮m): Head diameter subtracted from Comet length, expressed in microns; Comet height (␮m): number of microns in vertical direction in Comet (corresponding to Total Height); Comet area (␮m2 ): number of ␮m2 in Comet; Head diameter (␮m): number of microns in horizontal direction in head; Head area (␮m2 ): number of microns in head; Tail area (␮m2 ): number of ␮m2 in tail; Total Comet intensity: sum of pixel intensity values in Comet; Mean Comet intensity: mean intensity of pixels in Comet; Total head intensity: sum of pixel intensity values in head; Mean head intensity: mean intensity of pixels in head; Total tail intensity: sum of pixel intensity values in tail; Mean tail intensity: mean intensity of pixels in tail; %DNA in head: total head intensity divided by total Comet intensity (multiplied by 100); %DNA in tail: total tail intensity divided by total Comet intensity (multiplied by 100); Tail moment: %DNA in tail multiplied by Tail length × 0.031 (constant factor related to the microscope);

Olive moment: summation of Tail intensity profile values multiplied by their relative distances to the Head center, divided by total Comet intensity. Comet images representing different patterns of DNA migration following alkaline and neutral Comet assays are presented in Fig. 1 with the corresponding Comet measurements in Table 1. When the software measures b, the image is automatically reduced in size. 2.4. Description of Ghost images in the neutral Comet assay Neutral Comet assay images in which the head is completely separated from the tail have been termed Ghosts (or Clouds or Hedgehogs, Fig. 1, Image 6a, b, c, Collins et al., 2008) and were visually identified, recorded and scored as a percentage of the total sperm counted. Previous studies suggest these comet images represent a high level of DNA damage such as apoptosis or necrosis (Kumaravel et al., 2009). 2.5. Sperm Chromatin Structure Assay (SCSA) The SCSA was performed as previously described by Love and Kenney (1998). Sperm from a control bull (i.e., good sperm quality based on a low percent of Cells Outside Main Population; COMP-˛t ) was used to standardize instrument settings daily prior to analysis of study samples. The flow cytometer was adjusted such that the mean green fluorescence was set at 500 channels (FI-1 ∼500) and mean red fluorescence at 150 channels (FI-3 @ 150). Data were stored in List-Mode and subsequently analyzed using

Table 1 Alkaline and neutral comet measures generated by the CometScore software from images c (1–6) in Fig. 1. Measure

Image number Alkaline

Comet length(␮m) Tail length (␮m) Comet height (␮m) Comet area (␮m2 ) Head diameter (␮m) Head area (␮m2 ) Tail area (␮m2 ) Comet intensity Comet mean intensity Head intensity Head mean intensity Tail intensity Tail mean intensity DNA in head (%) DNA in tail (%) Tail moment Olive moment

Neutral

1

2

3

4

5

6

54 39 45 1729 15 521 1208 251,821 15 61,088 12 190,733 16 24 76 92 52

45 38.7 30 856 6 76 780 108,661 13 8663 12 99,998 13 8 92 110 67

47 42 31 864 5 16 847 102,228 12 2174 14 100,054 12 2.1 98 128 75

36 7.4 31 771 28 615 156 179,713 24 153,145 26 26,568 18 85 15 3.4 6

79 57 32 1672 22.5 515 1157.5 283,586 18 97,932 20 185,654 17 34.5 65.5 115 64.5

66 59 42 1176 6 37 1139 280,857 25 10,159 28.5 270,698 25 4 96 177 113.5

Comet length (␮m): number of microns in horizontal direction in Comet (corresponding to total length); Tail length (␮m): Head diameter subtracted from Comet length, expressed in microns; Comet height (␮m): number of microns in vertical direction in Comet (corresponding to Total Height); Comet area (␮m2 ): number of ␮m2 in Comet; Head diameter (␮m): number of microns in horizontal direction in head; Head area (␮m2 ): number of microns in head; Tail area (␮m2 ): number of ␮m2 in tail; Total Comet intensity: sum of pixel intensity values in Comet; Mean Comet intensity: mean intensity of pixels in Comet; Total head intensity: sum of pixel intensity values in head; Mean head intensity: mean intensity of pixels in head; Total tail intensity: sum of pixel intensity values in tail; Mean tail intensity: mean intensity of pixels in tail; %DNA in head: total head intensity divided by total Comet intensity (multiplied by 100); %DNA in tail: total tail intensity divided by total Comet intensity (multiplied by 100); Tail moment: %DNA in tail multiplied by Tail length × 0.031 (constant factor related to the microscope); Olive moment: summation of Tail intensity profile values multiplied by their relative distances to the Head center, divided by total Comet intensity.




WinList software (Verity Software House, Topsham, ME, USA). Measures included Mean-˛t , Standard Deviation-˛t (SD-˛t ), COMP-˛t and Mode-˛t .

2.6. Sperm Bos Halomax (SBH) assay The SBH assay (Sperm Bos Halomax, Bos Taurus, Halotech DNA, SL, Madrid, Spain) was performed according to manufacturer’s instructions (Halotech DNA, SL, Madrid, Spain). Slides were stained with PI (final concentration 0.03 ␮M) from a stock solution, diluted (1:40×; stock solution 1.2 ␮M) in deionized water and then 1:1 mixed with antifade mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, USA). Contents of each well were stained with 2 ␮L of prepared staining solution and 600 sperm per sample were analyzed with an Olympus microscope model BX60, equipped with an objective U Plan FL N, magnification 40×/ 0, 0.75 Ph2, ∞/ 0.17. Sperm were categorized as those with presence or absence of a halo.

2.7. Statistical analysis Statistical analysis was performed using SAS, version 9.2 (SAS Institute, Inc. Corp., Cary, NC, USA). Kolmogorv–Smirnov test was used to determine data normality distribution. The coefficient of variation (CV) was calculated to measure inter- and intra-assay variability. Spearman rank correlations were used to test the relationship between non-parametric sperm parameters. A general linear model procedure (GLM) was used to determine differences between means for sperm quality values between selected groups of bulls. P-values less than 0.05 were considered statistically significant.

3. Results 3.1. Inter-assay variability (repeatability among days within control sample) For the neutral Comet assay, the lowest coefficients of variation (CV) were related to dimension measures (comet length—11% and height—11%, tail length—14%, head area—15%, head diameter—16%), %T-DNA (13%) and Olive moment (19%). The remaining variables had a CV ≥ 20%. The only alkaline Comet assay measures with CV less than 20% were comet height (11%) and comet length (18%). The CV for the intensity measures were less in the neutral Comet assay (range 21–34%) in comparison with the alkaline Comet assay (range 37–59%). Similarly, the CV for %T-DNA was less in the neutral Comet assay (13%) as compared to the alkaline Comet assay (20%). The CVs for tail moment and Olive moment were also less in the neutral Comet assay (21 and 19%, respectively) as compared to the alkaline Comet assay (47% and 35%, respectively). The CV was similar for % H-DNA with the neutral Comet assay (25%) and the alkaline Comet assay (26%). In general, CV for neutral Comet assay measures were less than that for the alkaline Comet assay measures.

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3.2. Intra-assay variability (repeatability between replicates [i.e., between slides] within bull) The CVs for intensity measures were lower in the neutral Comet assay (range 1–15%) as compared to the alkaline Comet assay (range 6–29%). The CV for the neutral and alkaline Comet assays were also less for % H-DNA (5 and 10%, respectively) but the same for % T-DNA (4%). The CV for dimension measures were similar for the neutral Comet assay (range from 3% to 14%) and the alkaline Comet assay (range 4–18%). The CVs were less for the tail and Olive moments for both neutral and alkaline Comet assays (7% and 2%, respectively). In general, intra-assay variability was less than inter-assay variability for both assays. 3.3. Comparison of neutral and alkaline Comet assay measures Comet length, tail length and head diameter were greater in the neutral Comet assay (71 ± 9, 48 ± 7 and 22 ± 4 ␮m, respectively; mean ± SD) as compared to the alkaline Comet assay (40 ± 7, 24 ± 9 and 15 ± 5 ␮m, respectively; P < 0.05). The tail and Olive moments were also greater in the neutral Comet assay (105 ± 21 and 66 ± 12 ␮m, respectively) as compared to the alkaline Comet assay (46 ± 21 and 29 ± 10 ␮m, respectively; P < 0.05), as was the % T-DNA (66 ± 9% compared with 43 ± 11%; P < 0.05). 3.4. Differences in age, sperm morphology and motility between bull groups Age was not different between high- and lowsperm-quality groups (5 ± 2, 7 ± 3; P > 0.05). The percent morphologically normal sperm was greater in the greaterthan the lesser-sperm-quality group (81 ± 6 compared with 43 ± 18%; P < 0.05). The percentages of abnormal heads, detached heads, proximal droplets, distal droplets, abnormal midpieces, bent midpieces, bent tails, coiled tails and premature germ cells were less in the greatersperm-quality group (6 ± 3, 2 ± 1, 1 ± 1, 1.5 ± 4, 1 ± 1, 5 ± 8, 0.2 ± 0.4, 4 ± 3, 0.6 ± 1, respectively) compared to the lesser-sperm-quality group (11 ± 7, 9 ± 19, 6 ± 9, 3 ± 4, 2 ± 2, 14 ± 12, 2 ± 3, 12 ± 8, 3 ± 2, respectively; P < 0.05). Sperm motility variables TMOT, PMOT, VCL, VAP and VSL were greater in the greater-sperm-quality group (90 ± 4, 65 ± 6, 207 ± 34, 107 ± 12 and 77 ± 8, respectively) than the lesser-sperm-quality group (23 ± 26, 12 ± 16, 96 ± 36, 48 ± 27 and 35 ± 22, respectively; P < 0.05). 3.5. Evaluation of DNA quality measures between bull groups Regarding SCSA measures, mean-˛t (M-˛t ) and Standard Deviation-˛t (SD-˛t ) were less in the greater-spermquality group (231 ± 10, 30 ± 9) than in the lesser-spermquality group (268 ± 48 and 69 ± 34; respectively; P < 0.05; Table 2). The COMP-˛t was less in the greater-spermquality group than the lesser-sperm-quality group (6 ± 1% compared with 39 ± 27%; respectively; P < 0.05).


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Table 2 The mean ± SD for neutral Comet assay, % Ghosts, SCSA and SBH measures between bull groups. Comet measures are grouped as Comet (length, area, height, mean intensity, intensity), Head (diameter, area, mean intensity, % DNA), Tail (length, area, height, mean intensity, intensity, % DNA), Moment (tail and Olive). % Ghosts corresponds to neutral Ghosts. SCSA measures are abbreviated as M-˛t -Mean-˛t , SD-˛t -Standard Deviation-˛t , COMP-˛t -Cell outside main population and Mo-˛t -Mode-˛t . SBH-Sperm Bos Halomax is indicated as % halos. For each group n = 10. Neutral cometa

Measure

Group greater-sperm-quality (n = 10)

Group lesser-sperm-quality (n = 10)

Comet

Length Area* Height Mean intensity Intensity

72 ± 14 1865 ± 578 36 ± 5 17 ± 4 311,977 ± 159,869

66 ± 11 1400 ± 331 34 ± 4 17 ± 4 227,741 ± 86,252

Head

Diameter* Area* Mean intensity Intensity* % DNA

24 ± 3 703 ± 95 19 ± 4 125,608 ± 40,760 45 ± 14

20 ± 5 466 ± 164 19 ± 4 85,249 ± 39,639 40 ± 16

Tail

Length Area Mean intensity Intensity % DNA

48 ± 15 1162 ± 535 16 ± 4 186,370 ± 129,133 54 ± 14

46 ± 14 934 ± 330 15 ± 4 142,492 ± 74,296 60 ± 16

Moment

Tail Olive % Ghosts*

91 ± 37 59 ± 23 3±2

104 ± 46 72 ± 33 17 ± 12

SCSAb

M-˛t * SD-˛t * COMP-˛t * Mo-˛t

231 ± 10 30 ± 9 6±1 228 ± 11

268 ± 48 69 ± 34 39 ± 27 240 ± 42

SBHc

% halos

12 ± 4

20 ± 13

* a b c

Within rows, means are different (P < 0.05). n = 100 sperm. n = 5000 sperm. n = 600 sperm.

Neutral Comet assay mean intensity measures (for comet, head and tail) were not different between greaterand lesser-sperm-quality groups (P > 0.05; Table 2). Head intensity, head diameter, head area and comet area were greater in the greater-sperm-quality group (125,608 ± 40,760 pixels, 24 ± 3 ␮m, 703 ± 95 ␮m2 and 1865 ± 578 ␮m2 ) than in the lesser-sperm-quality group (85,249 ± 39,639 pixels, 20 ± 5 ␮m, 466 ± 164 ␮m2 and 1400 ± 331 ␮m2 ; respectively; P < 0.05). The % Ghosts were less in the greater-sperm-quality group than lessersperm-quality group (3 ± 2% compared with 17 ± 12%; respectively; P < 0.05). Alkaline Comet assay measures were similar between sperm-quality groups (P > 0.05). Similarly, the % halos, as measured by SBH assay, were not different between groups (12 ± 4, 20 ± 13; respectively; P > 0.05; Table 2).

measures and alkaline Comet assay measures (P > 0.05). Neither COMP-˛t nor % Ghosts was correlated with % halos (P > 0.05). However, Mean-˛t and SD-˛t were correlated to % halos (r = 0.58; P < 0.05) and % Ghosts (r = 0.60 and 0.79, respectively; P < 0.05). Many positive correlations were detected between neutral and alkaline Comet assay measures. Values are reported in Table 3. The % Ghosts were correlated to neutral head area, head diameter and head intensity (r = −0.83; −0.59; −0.54, respectively; P < 0.05). The % Ghosts were not correlated to any of the alkaline Comet assay measures (P > 0.05). The % halos was not correlated to any of the neutral Comet assay measures (P > 0.05), but was correlated to alkaline % H-DNA and % T-DNA (r = 0.51 and −0.51, respectively; P < 0.05).

3.6. Correlations among DNA sperm quality assays

3.7. Relationship between DNA quality and sperm motility measures

The Mean-˛t was inversely correlated to neutral head area (r = −0.67; P < 0.05). The COMP-˛t and SD-˛t were inversely correlated to neutral head intensity (r = −0.58 and −0.49, respectively; P < 0.05). SD-˛t was also inversely correlated with neutral head area and head diameter (r = −0.63 and −0.46, respectively; P < 0.05). COMP-˛t was inversely correlated with neutral head area, head intensity and head diameter (r = −0.73, −0.52 and −0.58, respectively; P < 0.05). The COMP-˛t was correlated to % Ghosts (r = 0.83; P < 0.05). No correlations were detected between SCSA

The Mean-˛t was inversely correlated to TMOT and PMOT (r = −0.45 and −0.50, respectively; P < 0.05). The COMP-˛t and SD-˛t were inversely correlated to TMOT, PMOT, VAP, VCL and VSL (r = −0.81, −0.65; −0.78, −0.71; −0.81, −0.77; −0.81, −0.73; −0.69 and −0.70, respectively; P < 0.05). The % Ghosts (inversely) and neutral head area (positively) were correlated to TMOT, PMOT, VAP, VCL and VSL (r = −0.77, 0.68; −0.76, 0.68; −0.80, 0.65; −0.76, 0.64; −0.75 and 0.59, respectively; P < 0.05). No correlations were


G Model

ARTICLE IN PRESS



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Table 3 Correlation coefficients between neutral and alkaline Comet measures. Comet measures are grouped as Comet (length, area, height, mean intensity, intensity), Head (diameter, area, mean intensity, % DNA), Tail (length, area, height, mean intensity, intensity, % DNA), Moment (tail and Olive). Alkaline

Neutral Comet

Comet

Head

Tail

Moment

Head

Tail

area

height

mean intensity

intensity

diameter

area

0.69 0.65 0.61 0.58 0.65

0.72 0.74 0.71 0.67 0.74

0.65 0.68 0.68 0.65 0.69

0.78 0.74 0.69 0.72 0.78

0.59 0.67 0.44 0.49

0.45 0.50

Diameter Area Mean intensity Intensity

0.57 0.45

0.45 0.57 0.65 0.61

0.57 0.61 0.57

0.50 0.72 0.62

Length Area Mean intensity Intensity

0.63 0.67 0.56 0.66

0.56 0.66 0.66 0.71

0.49 0.60 0.66 0.67

0.67 0.70 0.69 0.76

Tail Olive

0.52 0.53

Length Area Height Mean intensity Intensity

0.57 0.55 0.55

0.46

mean intensity

intensity

area

mean intensity

intensity

0.69 0.75 0.76 0.69 0.73

0.67 0.78 0.82 0.65 0.72

0.65 0.57 0.50 0.55 0.60

0.59 0.56 0.53 0.60 0.62

0.70 0.60 0.52 0.63 0.68

0.57 0.61 0.54

0.38 0.53 0.53 0.46

0.58 0.43

0.49 0.62 0.56

0.68 0.58

0.54 0.68 0.71 0.73

0.54 0.73 0.72 0.75

0.59 0.58 0.49 0.59

0.46 0.48 0.58 0.57

0.61 0.58 0.57 0.64

0.45

0.47 0.48

0.49 0.51

0.45

r-values differ P < 0.05 or greater.

detected between either the alkaline Comet assay measures or % halos and any of the sperm motility measures (P > 0.05).

3.8. Relationship between DNA quality and sperm morphology measures Regarding SCSA measures, Mean-˛t and SD-˛t were inversely correlated to % normal sperm (r = −0.62 and −0.74, respectively; P < 0.05) and positively correlated to premature germ cells and coiled tails (r = 0.53, 0.48; 0.76 and 0.55, respectively; P < 0.05). Similarly, the COMP-˛t was inversely correlated to % normal sperm (r = −0.85) and positively correlated to premature germ cells, coiled tails and bent tails (r = 0.82, 0.63, 0.46, respectively; P < 0.05). Mode-˛t was the only SCSA measure correlated to detached heads (r = −0.45; P < 0.05). Similarly, SD-˛t was the only SCSA measure correlated to abnormal midpieces (r = 0.45; P < 0.05). Among neutral Comet assay measures, head area was correlated with % normal sperm, coiled tails and detached heads (r = 0.68, −0.57 and −0.56, respectively; P < 0.05). Neutral mean intensity measures (i.e., comet, head and tail) were also correlated with detached heads (r = 0.62, −0.56 and 0.56, respectively; P < 0.05). The % Ghosts were inversely correlated to % normal sperm (r = −0.75; P < 0.05) and positively correlated to premature germ cells, coiled tails and detached heads (r = 0.62, 0.78, 0.53, respectively; P < 0.05). Among the alkaline Comet assay measures, comet length and tail mean intensity were positively correlated to detached heads (r = 0.47 and 0.44; respectively; P < 0.05). The head area, % H-DNA and % T-DNA were correlated to abnormal midpieces (r = −0.51; −0.46; 0.46; respectively; P < 0.05). Regarding the SBH assay, % halos was not correlated to any sperm morphology measure (P > 0.05).

4. Discussion The present study used a software program to objectively describe and measure comet sizes thereby eliminating the need for subjective visual estimation (e.g., the presence or absence of a comet shape). Previous studies have not described the dayto-day (inter-assay) repeatability of Comet assays. In the present study, to measure inter-assay variability, a control sperm sample was included with each electrophoretic run (i.e., only 10 samples could be placed in the electrophoresis chamber for each run). In general, for both Comet assays, dimension measures (comet and tail length, comet height, head diameter and area) had the least variability, while intensity measures were greatest. The % T-DNA was the most repeatable measure which is consistent with a previous study (Collins et al., 2008). The tail and Olive moments had less variability in the neutral Comet assay, as compared to the alkaline Comet assay. In general, the neutral Comet assay had less variability than the alkaline Comet assay. Intra-assay repeatability for this study was greater than inter-assay repeatability and similar to that reported by Hughes et al. (1997). Based on results of the present study, one slide (i.e., 50 sperm nucleoids) rather than two may be satisfactory to evaluate the DNA quality using the Comet assay. Data from the present study suggest that more variability occurs among days due to reagent preparation rather than slide preparation. Further, it appears that the neutral Comet assay dimension measures are more repeatable than intensity and moment (i.e., tail/Olive) measures. The moment measures likely exhibit more variability because they incorporate intensity, a highly variable measure, into the value. It is unclear which Comet assay (neutral or alkaline) may be more appropriate for evaluation of sperm DNA



8


quality (Singh et al., 1989; Haines et al., 1998; Zee et al., 2009; Enciso et al., 2011; Ribas-Maynou et al., 2012a). In the present study, the visual appearance and the length of the neutral and alkaline comet tails showed marked differences. The neutral comets were elongated as compared to the rounder-shaped alkaline comets (Fig. 1), suggesting that sperm DNA remains in a more compact form in the alkaline comet, resulting in fewer actual comet shapes. Interestingly, the head diameter and area were smaller and less intense in the alkaline Comet assay as compared to the neutral Comet assay, suggesting that less DNA remained in the alkaline processed nucleoid. Therefore, sperm DNA that is more “fragmented” prior to alkaline processing may result in the production of smaller fragments that may disappear visually (Simon and Carrell, 2013). Therefore, the visualized alkaline comet may not represent 100% of the sperm DNA, but rather only the remaining DNA that is more “resistant” to the alkaline conditions. The concept of “lost” DNA is also supported by the lack of “Ghost” images in the alkaline Comet assay which further suggests that such conditions may “dissolve” fragmented DNA and prevent imaging. The finding in the present study is similar to that reported previously in which it was suggested that “highly fragmented” DNA, caused by apoptosis from irradiated human lymphocytes, may not be identified under alkaline conditions (Czene et al., 2002). It is unclear whether the Ghosts identified in the present study resulted from apoptosis or necrosis (Fairbairn et al., 1995; Kumaravel et al., 2009; Ersson and Moller, 2011), or other factors. Comet measures in the present study associated with Ghost images (Fig. 1, Image 6b; Table 1) included a small head diameter and area, less head intensity, lesser % H-DNA and a greater % T-DNA, as well as greater tail and Olive moments. There were more Ghosts in the group with lesser, as compared to greater, sperm quality (i.e., 17% compared with 3%). The values for % Ghosts were positively correlated with values for detached heads, coiled tails and premature germ cells. These sperm abnormalities represent severe sperm abnormalities (coiled tails), immature sperm forms (premature germ cells) or sperm associated with prolonged sperm storage in the reproductive tract (detached heads). Values for Ghosts were also correlated with the values for SCSA measures, Mean-˛t , SD-˛t , and COMP-˛t . Interestingly, in the lesser sperm quality group, the % COMP-˛t (39%) was twice the % Ghosts (17%) suggesting that Ghosts may reflect a more “damaged” DNA, while the SCSA identifies a less “damaged” DNA construct as well as the more “damaged” sperm. These combined results suggest that Ghosts represent a highly fragmented DNA form (i.e., immature or highly damaged) that is highly susceptible to the alkaline de-condensing conditions but not to the milder conditions of the neutral Comet assay and the SCSA and, therefore, cannot be identified in the alkaline Comet assay because of the complete dissolution. In addition, under strong alkaline conditions (i.e., pH 13) sperm DNA undergoes to spontaneous base loss forming alkaline labile sites (Singh et al., 1989; Haines et al., 1998), which are converted to single-stranded breaks at high pH and detected by the alkaline Comet assay. The alkaline Comet assay may, therefore, induce more DNA breaks than the neutral Comet assay and also result

in the loss of more DNA. As previously reported by Singh et al. (1989), the greater amount of ssDNA breaks in human and mouse sperm under alkaline conditions may be due to those normally occurring alkaline labile sites, which may have a protective role before and during fertilization, and may be involved in the initiation of DNA de-condensation in the embryo. In the present study, % Ghosts were negatively correlated with some neutral Comet assay measures (i.e., head diameter, area, intensity); however, %Ghosts were not correlated with any of the alkaline Comet assay measures; therefore, the neutral Comet assay may be more appropriate for the evaluation of sperm DNA as it can identify these ghost forms and does not affect alkaline labile sites. In the present study, four DNA quality assays were compared in two groups of bulls selected based on features of sperm morphology and motility (greater compared to lesser). Most SCSA measures (Mean-˛t , SD-˛t , COMP-˛t ), and % Ghosts identified differences in DNA quality between the two groups; however, the SBH assay values were not different between groups. In addition, neutral Comet assay measures of comet area, head diameter, head area, and head intensity were greater in the group with greater sperm quality. No alkaline comet measures were different between the two groups. The SCSA measures, Mean-˛t , SD˛t , COMP-˛t , were inversely correlated with neutral comet head measures (diameter, area, and intensity) suggesting that a decrease in head dimension is associated with a decrease in DNA quality. Interestingly, there was no association between SCSA measures and alkaline Comet assay measures. The values for the SBH assay were correlated to those for the Mean-˛t and SD-˛t , but not the COMP˛t or the % Ghosts. This is in contrast to a previous study with human sperm (Chohan et al., 2006), in which the SCDt (Halotest in humans) was correlated (r = 0.86; P < 0.001) with the DNA fragmentation index (% DFI; i.e., synonymous with COMP-˛t ). Another study with bulls (García-Macías et al., 2007) indicated that the SCSA and SBH assays predicted small changes in fertility (i.e., non-return rate-NNR) when comparing the DNA quality of 60 bulls divided into three fertility groups, even though there was no correlation in values between the assays. Differences among studies may be due to variation in operator and test subjectivity, especially for the SBH and the Comet assays. In addition, these assays involve evaluation of less than 100 sperm. In comparison, the flow cytometer analyzes thousands of sperm in a few minutes. Sperm motility and morphology are classic methods for assessing sperm quality. The two groups of bulls in the present study were selected based on these features to allow comparison of DNA quality assays with these traditional tests. In the present study, SCSA assay was reliable for distinguishing different qualities of DNA in the sperm cells. This assay has long been considered valuable for measuring DNA quality and its relationship to fertility has been reported for many species (Evenson et al., 1980; Ballachey et al., 1987; Evenson et al., 1994; Sailer et al., 1996; Love and Kenney, 1998; Larson et al., 2000; Januskauskas et al., 2001; Januskauskas et al., 2003; Waterhouse et al., 2006; Morrell et al., 2008; Didion et al., 2009; Nordstoga et al., 2013; Oleszczuk et al., 2013). Therefore, it was used in the present study as a reference assay to which the other




assays could be compared. Several neutral Comet assay measures compared favorably to SCSA for assessment of DNA quality and for differentiating sperm of greater or less quality. The relationship between sperm morphology and DNA integrity highlighted that abnormalities in sperm development (i.e., bent tails, bent midpieces and coiled tails) may also include abnormal DNA development. A unique feature of the neutral Comet assay was the presence of Ghosts which appeared to be related to sperm quality. In contrast to the neutral Comet assay, the alkaline Comet assay and SBH assay did not distinguish between the two categories of sperm quality set in the present study. In a previous study, the alkaline Comet assay was unable to distinguish differences in sperm DNA quality between fertile and unfertile men unless the sperm were subjected to irradiation (Hughes et al., 1996). In contrast, Irvine et al. (2000) reported that the alkaline Comet assay could be used to distinguish between fertile and infertile men. Similarly, Ribas-Maynou et al. (2012b) reported less ssDNA breaks in fertile men as compared to infertile men when using the alkaline Comet assay. However, these investigators also found less dsDNA breaks in fertile men as compared with infertile men when using the neutral Comet assay. Data from the present study suggest that the neutral Comet assay is more valuable than the alkaline Comet assay for assessing sperm DNA quality in bulls because it readily identifies a decrease in comet area, head diameter, head area, and head intensity as well as an increase in % Ghosts in association with less sperm DNA quality. These endpoints are in agreement with SCSA measures. The lack of standardized protocols, species differences and variations in evaluation methods make study results difficult to compare. The SBH assay, similar to the alkaline Comet assay, may dissolve DNA or the assay conditions may not be sufficient to relax the DNA and allow discrimination between sperm of greater and lesser DNA quality.

5. Conclusion The present study is the first to compare neutral and alkaline Comet assays, the SCSA and the SBH assay, in sperm of bulls selected on classical features of sperm motility and morphology. Among these sperm DNA quality tests, the SCSA and the neutral Comet assay were able to distinguish between the greater- and lessersperm quality groups. The alkaline Comet assay and the SBH assay were not able to differentiate between these two groups, nor were they correlated with sperm motility and morphology. Standardization of methods for the neutral Comet assay would aid the ability to compare results among laboratories. Future studies are needed to assess the relationship of neutral Comet assay results and fertility and, thereby, its utility in clinical practice.

Conflict of interest The authors declare no conflict of interest.

9

Authors’ contributions R.-S. contributed in samples analysis and in writing the manuscript. J.E.-R. contributed in samples collection and animal recruitment. D.D.-V. contributed in manuscript revision and experimental design. R.-D.P. contributed in manuscript revision. C.C.-L. contributed in experimental design, direction of the work and statistical analysis. Acknowledgements This work was supported by Legends Premier Stallion Season Auction, Texas A&M University. The authors would thank Dr. Fulgione Domenico and Dr Maselli Valeria at the Department of Structural and Functional Biology – University of Naples Federico II for the use of the Comet software. References Aitken, R.J., Bronson, R., Smith, T.B., De Iuliis, G.N., 2013. The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies. Mol. Hum. Reprod. 19, 475–485. Ballachey, B.E., Hohenboken, W.D., Evenson, D.P., 1987. Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility. Biol. Reprod. 36, 915–925. Ballachey, B.E., Evenson, D.P., Saacke, R.G., 1988. The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls. J. Androl. 9, 109–115. Baumgartner, A., Cemeli, E., Anderson, D., 2009. The comet assay in male reproductive toxicology. Cell. Biol. Toxicol. 25, 81–98. Boe-Hansen, G.B., Morris, I.D., Ersboll, A.K., Greve, T., Christensen, P., 2005. DNA integrity in sexed bull sperm assessed by neutral Comet assay and sperm chromatin structure assay. Theriogenology 63, 1789–1802. Chenoweth, P.J., Spitzer, J.C., Hopkins, F.M., 1992. A new bull breeding soundness evaluation form. Proc. Soc. Theriogenol., 63–70. Chohan, K.R., Griffin, J.T., Lafromboise, M., De Jonge, C.J., Carrell, D.T., 2006. Comparison of chromatin assays for DNA fragmentation evaluation in human sperm. J. Androl. 27, 53–59. Collins, A.R., Oscoz, A.A., Brunborg, G., Gaivao, I., Giovannelli, L., Kruszewski, M., Smith, C.C., Stetina, R., 2008. The comet assay: topical issues. Mutagenesis 23, 143–151. Czene, S., Testa, E., Nygren, J., Belyaev, I., Harms-Ringdahl, M., 2002. DNA fragmentation and morphological changes in apoptotic human lymphocytes. Biochem. Biophys. Res. Commun. 294, 872–878. Didion, B.A., Kasperson, K.M., Wixon, R.L., Evenson, D.P., 2009. Boar fertility and sperm chromatin structure status: a retrospective report. J. Androl. 30, 655–660. D’Occhio, M.J., Hengstberger, K.J., Tutt, D., Holroyd, R.G., Fordyce, G., Boe-Hansen, G.B., Johnston, S.D., 2013. Sperm chromatin in beef bulls in tropical environments. Theriogenology 79, 946–952. Enciso, M., Iglesias, M., Galan, I., Sarasa, J., Gosalvez, A., Gosalvez, J., 2011. The ability of sperm selection techniques to remove single- or double-strand DNA damage. Asian J. Androl. 13, 764–768. Ersson, C., Moller, L., 2011. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments. Mutagenesis 26, 689–695. Evenson, D.P., Darzynkiewicz, Z., Melamed, M.R., 1980. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 210, 1131–1133. Evenson, D.P., Thompson, L., Jost, L., 1994. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 41, 637–651. Evenson, D.P., Larson, K.L., Jost, L.K., 2002. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. J. Androl. 23, 25–43. Fairbairn, D.W., Olive, P.L., O’Neill, K.L., 1995. The comet assay: a comprehensive review. Mutat. Res. 339, 37–59. Fernandez, J.L., Muriel, L., Rivero, M.T., Goyanes, V., Vazquez, R., Alvarez, J.G., 2003. The sperm chromatin dispersion test: a simple method for the determination of sperm DNA fragmentation. J. Androl. 24, 59–66.



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