Reprod Dom Anim 49, 761–768 (2014); doi: 10.1111/rda.12364 ISSN 0936–6768

Validation of a Field Based Chromatin Dispersion Assay to Assess Sperm DNA Fragmentation in the Bottlenose Dolphin (Tursiops truncatus) M-J Sanchez-Calabuig1, C Lopez-Fernandez2, E Martınez-Nevado3, JF Perez-Gutierrez4, J de la Fuente1, SD Johnston5, D Blyde5,6, K Harrison7 and J Gosalvez2 1 Department of Animal Reproduction, Instituto Nacional de Investigaci on y Tecnologıa Agraria y Alimentaria (INIA), Madrid, Spain; 2Department of Genetics, Autonomous University of Madrid, Cantoblanco, Spain; 3Zoo-Aquarium, Madrid, Spain; 4School of Veterinary Medicine, Universidad Complutense de Madrid, Madrid, Spain; 5School of Agriculture and Food Science, University of Queensland, Gatton, Qld, Australia; 6Seaworld, Gold Coast, Qld, Australia; 7Queensland Fertility Group, Brisbane, Qld, Australia

Contents Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management of captive dolphin populations, including evaluation of sperm DNA quality. This study validated a customized sperm chromatin dispersion test (SCDt) for the bottlenose dolphin (Tursiops truncatus) as a means of assessing sperm DNA damage both in the field and in the laboratory. After performing the SCDt, two different sperm morphotypes were identified: (i) sperm with fragmented DNA showed large haloes of dispersed DNA fragments emerging from a compact sperm nucleoid core and (ii) sperm containing non-fragmented DNA displayed small compact haloes surrounded by a dense core of non-dispersed DNA and protein complex. Estimates of sperm DNA fragmentation by means of SCDt were directly comparable to results obtained following a two-tailed comet assay and showed a significant degree of correlation (r = 0.961; p < 0.001). This investigation also revealed that the SCDt, with minor modifications to the standard protocol, can be successfully conducted in the field using a LED florescence microscopy obtaining a high correlation (r = 0.993; p = 0.01) between the data obtained in the laboratory and in the field.

Introduction Over the last 20 years, there has been a concerted effort to manage the genetic diversity of the captive bottlenose dolphin (Tursiops truncatus), which is currently regarded as a protected species (Montano et al. 2012). Assisted reproductive technologies have been well established in this species, including the production of offspring following artificial insemination using both cryopreserved and sex sorted semen (O’Brien and Robeck 2006; Robeck et al. 2013). While the establishment of a genome resource bank for the dolphin and related cetacean species is now well in the realm of possibility, this objective will be greatly enhanced thorough a knowledge of how the sperm, including sperm DNA, tolerates the cryopreservation procedure. The assessment of DNA fragmentation represents an important tool for the prediction of male infertility (Evenson et al. 2002; Agarwal and Allamaneni 2005). In humans, where this aspect of sperm quality has been largely assessed, it has been shown that DNA fragmentation levels above 30–40% produce a highly significant reduction in the maintenance of pregnancy after natural mating (Larson et al. 2000). There is also a positive correlation between the proportion of spermatozoa with fragmented DNA and infertility (Agarwal and Said © 2014 Blackwell Verlag GmbH

2003). Moreover, individuals with sperm nuclear damage usually show a higher incidence of abnormal seminal parameters (Irvine et al. 2000; de la Torre et al. 2007). There are currently a range of techniques available for evaluating sperm DNA fragmentation, including variations of the comet assay (Hughes et al. 1996; Aravindan et al. 1997; Singh et al. 1998; Fraser and Strzezek 2004; Chohan et al. 2006), the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling method (TUNEL) (Lopes et al. 1998), the Sperm Chromatin Structure Assay (SCSA) (Evenson et al. 1994; Evenson 1999), the DNA breakage detection-fluorescence in situ hybridization test (DBD-FISH) (Fernandez and Gos alvez 2002) and the sperm chromatin dispersion test (SCDt) (Fernandez et al. 2005a). Sperm DNA fragmentation has recently been assessed in the bottlenose dolphin using the SCSA (Montano et al. 2012). The SCDt was firstly developed for humans (Chohan et al. 2006; Enciso et al. 2006b; Zini and Libman 2006) and later adapted in a range of mammalian species, nonhuman eutherian mammals (Fraser et al. 2010) metatheria (Johnston et al. 2007; Zee et al. 2009) and even in a prototheria species (Fernandez et al. 2003, 2005a; Enciso et al. 2006a,b; Johnston et al. 2009). The SCDt is a cost-effective procedure that can be used under field conditions (Gosalvez et al. 2008). In the specific case of the dolphin, this feature may be particularly convenient because laboratory facilities are not always close to where the animals are housed. There were two main aims to our experimental approach in this study: (i) to modify and validate the SCDt for the bottlenose dolphin spermatozoa using the comet assay as an external control and (ii) to explore the capacity and efficiency of the SCDt to be conducted under field conditions, without access to mains power and using LED fluorescence microscopy.

Material and Methods Semen collection, evaluation and cryopreservation All experimental procedures were performed using five proven breeding bottlenose dolphins held at Zoo Aquarium of Madrid (male 1) and Sea World Gold Coast, Australia (males 2–5). Different frozen straws of male 1 were used to perform all the technical variations to adapt the sperm chromatin dispersion test, the comet assay and the laboratory/field experimental validation.

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M-J Sanchez-Calabuig, C L opez-Fern andez, E Martınez-Nevado, JF Perez-Gutierrez, J de la Fuente, SD Johnston, D Blyde, K Harrison and J Gos alvez

Males 2–5 were used to assess the consistency of the technique in different males. All the experiments were reviewed by the Institutional Animal Care Committee of both Zoo Aquarium of Madrid and The University of Queensland. Ejaculates were obtained by a voluntary semen collection technique (Keller 1986; Robeck and O’Brien 2004); semen was collected 1–3 times in succession until an adequate number of viable spermatozoa were obtained. Sperm concentration was determined using a Makler Counting Chamber (Sefi Medical Instruments, Haifa, Israel). Sperm total motility, progressive motility, viability and sperm morphology were assessed as previously described (Robeck and O’Brien 2004). Semen samples were adjusted to 400 9 106 spermatozoa per ml and diluted 1 : 1 (v/v) with a TRIS-egg-yolk-based buffer containing 1.5% glycerol over 5 min. The suspension was cooled to 5°C over 1 h ( 0.27°C/min) and diluted with the second fraction with glycerol to obtain a 3% final glycerol concentration and a final concentration of 200 9 106 sperm per ml over a 10-min equilibration period, loaded into 0.25 ml straws, sealed and frozen in liquid nitrogen vapour at 4.5 cm above the liquid nitrogen ( 12°C/min) for 10 min and then finally plunged into liquid nitrogen and stored until evaluation. The characteristics of the frozen-thawed bottlenose dolphin sperm samples that were used for validation from male 1 were as follows: total motility (%): 59.3  4.7, progressive motility (%): 49.3  3.7, sperm viability (%): 82.0  2.8, intact acrosomes (%): 86.8  0.7 and normal morphology (%): 80.0  6.0. Straws of the same frozen-thawed semen samples from male 1 were used for validation and also for simulating the SCDt procedure under field collection conditions. SCDt validation experiments Five different straws from the same ejaculate were used to validate the SCDt technique and produce a prototype kit specifically adapted to assess sperm DNA damage in the dolphin (Halomax, Halotech SL, Madrid, Spain). Once the methodology was established, a comet assay was simultaneously prepared using 20 different straws from two different ejaculates, and the results from both assays were directly correlated. Additionally, five straws from five different ejaculates were incubated and assessed for SDF by SCDt and comet assay at T0 (immediately after thawing) and after 0.5 h (T0.5), 1 h (T1), 4 h (T4), 24 h (T24) and 48 h (T48) of incubation at 37°C. To investigate the reproducibility of the technique when sperm samples of other dolphins were used, two different frozen-thawed semen samples obtained from males 2–5, including two replicates per male, were assessed for each sample immediately after thawing. The basic equipment used to conduct the SCDt in the laboratory (SCD-laboratory) was a standard refrigerator, a microwave oven, an incubator set up at 37°C and an epifluorescence microscope. We used a Leica DMLA model motorized fluorescence microscope (Leica Microsystems, Wetzlar, Germany); equipped with a Leica EL6000 fluorescence light source, a charge-coupled device (Leica DFC350 FX, Leica Microsystems) and Fluotar 40/609 objectives for routine scanning. Two

hundred spermatozoa were scored per sample, and the proportion of sperm containing a fragmented DNA molecule was calculated. As the SCDt is based on the differential production of haloes of protein-depleted chromatin, the DNA was visualized by fluorescence microscopy using GelRed (Biotium, Hayward, CA, USA). Straws of the same five frozen-thawed semen samples from male 1 were also processed under simulated field conditions (SCD-field). The aim of this experiment was to assess any possible difference between results in DNA damage of sperm processed in the laboratory and in the field and to test the capacity and reliability of the field procedure with equipment not requiring electrical supply. For this aspect of the validation procedure, the frozen-thawed semen samples were processed following incubation at 37°C at T0, T4 and T48. Equipment used in the field included a portable incubator (Minit€ ub, Tiefenbach, Germany) pre-fixed at 37°C for sperm incubation (Fig. 1a), a CO2 spray to produce a ‘cold’ surface over the slide (Fig. 1b) for microgel production and a battery-powered LED fluorescence microscope (Motic, BA310A, Barcelona, Spain; Fig. 1c) equipped with a LED-fluorescence module containing a filter block consisting in an exciter 470, dichroic 480 and barrier filter at 485. This configuration has been designed for Auramine O staining, but it is also efficient for visualization of SYBR Green DNA staining. This incubator can be operated directly off the car battery while in the field or during transport back to the laboratory (Fig. 1a). Sperm chromatin dispersion test For the SCD procedure, dolphin spermatozoa were diluted in INRA 96 (IMV Inc., L’Aigle Cedex, France) to a concentration of 10 9 106–15 9 106 sperm per ml for optimal visualization; 25 ll of this suspension was added to a vial containing low-melting-point agarose previously heated in the microwave oven (SCD-laboratory) or gas heater (SCD-field) tempered at 37°C. Spermatozoa were gently mixed with the liquefied agarose and a 10 ll aliquot of the mixture placed on a pre-treated slide and covered with a glass coverslip. The sample was solidified by chilling the slide on a refrigerated (4°C) metallic surface for 5 min (SCD-laboratory) or by using the refrigeration produced by the CO2 spray (SCD-field); it is recommended to spray the slide 10 cm away to the microgel and the use of protective gloves to avoid direct contact with CO2 (Fig. 1b). Once the microgel was formed, the coverslip was carefully removed and the slide placed horizontally in 10 ml of the lysing solution provided in the kit for 5 min for protein depletion. This can be performed at room temperature, but never at temperatures lower than 15°C. It is recommended to perform the lysis step of the procedure on top of the portable heater adjusted at 37°C as shown in Fig. 1a. The sperm-gel preparation was then rinsed in distilled water for 5 min and subsequently exposed to a sequential increasing series of ethanol (70%, 90% and 100%). At the laboratory, sperm were stained using GelRed (Biotium, Hayward, CA, DNA stained red) and © 2014 Blackwell Verlag GmbH

Dolphin Sperm Chromatin Dispersion Assay

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(a)

(c)

(b) Fig. 1. Basic equipment to perform the sperm chromatin dispersion test in the field. (a) Portable incubator pre-fixed at 37°C to incubate sperm samples; (b) CO2 spray to accelerate jellification; (c) LED-equipped fluorescence microscope.

2,7-dibromo-4-hydroxymercurifluorescein (Sigma Aldrich, Irvine, UK, protein stained green). In the field, a LEDfluorescence module was used and the fluorochrome was adapted to the wavelength of the filter set of the microscope; in this case SYBR Green (Molecular Probes, Eurogene, OR) was the fluorochrome used for the LED-fluorescence module. Two hundred cells of each semen sample were analysed for SCDt. Comet assay The neutral comet assay reveals double-stranded DNA damage (Ds-DNAd), whereas the alkaline comet assay permits the detection of single-stranded DNA damage (Ss-DNAd) and Ds-DNAd. Both assays can also be performed simultaneously using the same slide twotailed Comet Assay to enable the dual identification of Ss-DNAd and Ds-DNAd on the same spermatozoon. A 15 ll aliquot containing a sperm concentration of 1 9 106 per ml was loaded into microgel and two different times for each run were used. For neutral comet assay, spermatozoa were treated for 5 min with the same lysis buffer as used for the SCDt and incubated in a Tris-borate-EDTA (TBE) (0.089 M Tris, 0.089 M boric acid and 0.002 M EDTA) buffer for 15 min. The first electrophoresis was performed for 12 min at 20 volts and resulted in double-stranded DNA fragments migrating to the anode. The microgel slide was then immersed in 0.9% NaCl for 2 min followed by 5 min in 0.4 M Tris-HCl buffer (pH 7.5) and 2 min in TBE. Thereafter, the slide was transferred into an alkaline solution chilled (4°C) (0.03 M NaOH and 1 M NaCl, pH 12.5) for 2.5 min and then subjected to a second electrophoresis for 4 min at 20 volts in alkaline solution at room temperature but in an orientation at 90° of its initial position in the electric field. This process causes the release of DNA fragments caused by Ss-DNAd to migrate towards the anode. To desiccate the microgels, the slides were placed in a Tris-HCl buffer for 5 min, followed by 2 min in TBE with a final series of ethanol (70%, 90% and 100%). Sperm DNA comets were stained with propidium iodide and observed under © 2014 Blackwell Verlag GmbH

fluorescent microscope (960); 200 cells were analysed for each semen sample. Image capture and processing Sperm were visualized in the laboratory using a Leica DMRB microscope and captured using a CCD Leica DFC350 FX. A merge of the resultant grey images with channel assignation for red (DNA) and green (proteins; Fig. 2) was performed for delineation of the DNA and protein domain. Images were converted to RGB files and further enhanced using grey-level images with the filtering sequence ‘find edges’ and ‘invert’, using Photoshop CS4. Statistical analysis The dynamic loss of DNA integrity and the differences between ejaculates were analysed using the nonparametric maximum-likelihood Kaplan–Meier. The logrank test, (Mantel-Cox) which estimates hazard functions between the two groups at each time interval, was utilized. Univariate analysis was performed to identify differences between the SCDt procedure and two-tailed comet assay. Differences between SCD-laboratory and SCD-field assessments were analysed using the Mann– Whitney U-test. Correlation analysis between the SCDt and the two-tailed comet assay was performed using a nonparametric Pearson test. Differences between individuals were analysed using a Kruskal–Wallis one-way analysis of variance on ranks. Statistical comparisons were performed using SPSS v.17.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results Sperm Chromatin dispersion test Frozen-thawed dolphin spermatozoa processed using the SCDt resulted in sperm DNA of two primary morphotypes: sperm nuclei which were considered as presenting fragmented DNA possessed large haloes of spotty and dispersed DNA fragments emerging from a

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M-J Sanchez-Calabuig, C L opez-Fern andez, E Martınez-Nevado, JF Perez-Gutierrez, J de la Fuente, SD Johnston, D Blyde, K Harrison and J Gos alvez

(a)

(b)

(d)

(c)

Fig. 2. Bottlenose dolphin sperm DNA fragmentation after the sperm chromatin dispersion test procedure. Sperm were stained using Gel-Red (sRed) and 2,7-dibromo-4-hydroxymercurifluorescein (Green). (a) Two selected spermatozoa presenting with fragmented (arrow) and nonfragmented DNA; (b–d) Electronic filter enhanced original micrographs to illustrate the distribution of DNA (red fluorescence) and protein (green fluorescence). Figure (d) corresponds to electronic filter enhancement on panel a

compact sperm nucleoid core (Fig. 2a,c arrow), whereas non-fragmented DNA spermatozoa, displayed small compact haloes surrounded by a dense core of nondispersed DNA (Fig. 2a,b). The flagellum was clearly distinguished from chromatin through double-fluorescence staining [(green emission for proteins and red emission for DNA (Fig. 2d)]. The nuclear core that resisted partially protein depletion, showed a yellow colour due to the mixture of red and green fluorescence in this region (Fig. 2a). Electronic filtering of the original images allowed enhancement of the distribution of proteins and DNA after the SCDt (Fig. 2d). Baseline and dynamic assessment of the SDF under laboratory conditions Using the SCDt under laboratory conditions, the mean level of DNA fragmentation at T0 (baseline level) in male 1 was 2.2  0.5. There was an increase (p < 0.05) in sperm DNA damage following incubation at 37°C, which is summarized in Table 1. After 1 h of incubation there was an increase in SDF equivalent to a rate of SDF (r-SDF) of 0.9 per hour. This value increased reaching a maximum after 4 h of incubation with a rSDF of 9.0 per hour. The r-SDF decreased after 24 h and 48 h of incubation to r-SDF of 2.0 per hour and 0.5 per hour, respectively (Table 1). Finally, the values for SDF were assessed at T0 in four different males and their replicates (Table 2). The consistency of the results between the different replicas was high and similar to what was observed in male 1; the baseline level of SDF observed in the different individuals was very low, and no significant differences were observed across all males (p > 0.05). Baseline and dynamic assessment of the SDF under field conditions Using male 1 under field conditions, the baseline level of SDF (T0) observed was 2.5  1.2 and no significant differences were observed when compared to the sperm examined under laboratory conditions (Table 1). The dynamic assessment showed no significant difference

between SCD-laboratory and SCD-field assays (U = 107.0; Z = 0.228) at any time point throughout incubation, and there was a high correlation between sperm DNA damage values obtained when the samples were processed under the different conditions (r = 0.993; p = 0.01; Fig. 4b). Two-tailed comet assay Following the two-tailed comet assay, three main categories of spermatozoa were observed (Fig. 3). The first group showed a small comet tail localized at the y-axis and a compacted nuclear core (Fig. 3a). The other two groups of dolphin sperm DNA comet morphologies indicated the presence of sperm DNA damage. The first of these (Fig. 3b) was characterized by DNA fragments migrating in the same direction as those described for the structural comets, but also by having a smaller nuclear core and longer comet tail. In this type of comet, there were no traces of perpendicular migration of DNA fragments (x-axis); this type corresponded to Ss-DNAd (Fig. 3b). The second type of DNA damaged spermatozoa (Fig. 3c) were characterized by DNA migration in two directions (x-and y-axis) and which depicted the presence of Ss-DNAd (y-axis) and Ds-DNAd (x-axis). The results obtained with two-tailed comet assay are shown in Table 1. The mean level of DNA breakage (%) at T 0 h was 2.8  1.1. After 0.5, 1, 4, 24 and 48 h of incubation at 37°C, the mean level (%) of DNA fragmentation was 5.4  1.5, 9.9  4.9, 47.2  4.4, 96.9  3.3 and 99.1  0.5, respectively. No differences were found between the relative proportions of SCDt and sperm presenting with single and double-stranded DNA damage (U = 309.000 and Z = 2.085) by means of the two-tailed comet assay; consequently, there was a high correlation for sperm DNA damage assessment observed between these two techniques (r = 0.961; p < 0.001; Fig. 4a).

Discussion The results of this experiment have revealed that the modified SCDt was an effective methodology for the © 2014 Blackwell Verlag GmbH

Dolphin Sperm Chromatin Dispersion Assay

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Table 1. Sperm DNA fragmentation (%) measured using the two-tailed comet assay (2T-Comet) and SCDt at different incubation times (T0, T0.5, T1, T4, T24 and T48 h) in male 1 2T-Comet (%)

T0 S-1 S-2 S-3 S-4 S-5 Mean T0.5 S-1 S-2 S-3 S-4 S-5 Mean T1 S-1 S-2 S-3 S-4 S-5 Mean T4 S-1 S-2 S-3 S-4 S-5 Mean T24 S-1 S-2 S-3 S-4 S-5 Mean T48 S-1 S-2 S-3 S-4 S-5 Mean

(SD)

(SD)

(SD)

SCDt (%)

DS

DS + SS

SS

1.3 1.6 1.3 1.6 1.0 1.4  0.3

0.3 0.0 0.3 1.6 0.0 0.44  0.7

0.6 0.0 0.6 1.0 2.6 1.0  1.0

2.2 1.6 2.2 4.2 3.6 2.8  1.1

1.0 0.8

1.0 2.3

1.7 3.2

3.7 6.3

0.7 0.8  0.2

2.3 1.9  0.8

3.3 2.7  0.9

6.3 5.4  1.5

1.4 1.0 6.7

1.0 1.0 5.7

3.0 4.0 1.7

5.4 6.0 14.1

6.7 4.0  3.2

5.5 3.3  2.7

1.7 2.6  1.1

13.9 9.9  4.8

SDF-Comet

SDF-lab

SDF-field

2.0 2.2 1.7 3.0 2.3 2.2  0.5

1.7 2.3 1.7 4.5 2.3 2.5  1.2

r-SDF-lab

1.1 2.7 1.3 3.0 1.3 2.1  0.9 3.0 1.2 3.7 4.0 3.7 3.1  1.1

14.7 14.7 33.7

4.0 4.0 9.3

32.3 32.3 0.3

51.0 51.0 43.3

(SD)

33.7 24.2  11.0

9.3 6.7  3.1

0.3 16.3  18.5

43.3 47.2  4.4

22.0 21.0 38.0 25.0 37.0 30.0  8.3

(SD)

76.7 29.1 44.0 93.0 44.0 57.4  26.5

10.3 39.8 34.0 2.3 34.0 24.1  16.6

11.0 22.3 21.0 2.0 21.0 15.5  8.8

98.0 91.2 99.0 97.3 99.0 96.9  3.3

61.0 60.0 76.3 77.0 75.0 69.9  8.6

(SD)

85.0 47.6 56.2 95.9 96.8 76.3  23.0

8.0 43.8 40.0 3.1 1.2 19.2  20.9

6.0 7.4 2.6 0.0 2.0 3.6  3.0

99.0 98.8 98.8 99.0 100 99.1  0.5

73.0 80.0 88.0 85.0 79.0 81.0  5.8

0.9 18.0 22.0 41.0 23.0 37.0 28.2  10.1

9.0

2.0 82.0 84.0 85.0 90.0 85.0 85.2  2.9

0.5

S, structural comet; DS, double strand break comet; SS, single strand break comet; S, sample; rSDF, rate of increase of Sperm DNA fragmentation; SDF-lab, Sperm DNA fragmentation at laboratory conditions. SDF-field, Sperm DNA fragmentation at field condition; SD, standard deviation.

Table 2. Sperm DNA fragmentation (%) measured using the SCDt in males 2–5 immediately after thawing

S-1 S-1 S-2 S-2 Mean (SD)

Replicate Replicate Replicate Replicate

1 2 1 2

Male 2

Male 3

Male 4

Male 5

2 1 1 1 1.3  0.5

0 1 0 0 0.3  0.5

1 0 0 1 0.5  0.6

1 1 2 1 1.3  0.5

detection of bottlenose dolphin sperm DNA fragmentation and that it could be performed under field conditions using LED fluorescence microscopy, producing equivalent results to those obtained under laboratory conditions. Indeed, we demonstrated the reproducibility of the technique as males 2–5 showed © 2014 Blackwell Verlag GmbH

the same morphotypes when compared to male 1. A strong correlation was also observed between the twotailed comet assay and the SCDt, so that it can be concluded that the large haloes observed after the SCDt in all bottlenose dolphins in this study were representative of the presence of single and/or double-stranded breaks in dolphin sperm DNA. The small comet tail spermatozoa found in the comet assay corresponded to a structural feature of bottlenose dolphin sperm DNA, which is likely to be primarily related to the presence of alkali labile sites in the mature sperm of all mammalian species (Singh et al. 1998), and hence not related with DNA damage. The dynamic assessment of sperm DNA damage following incubation confirms that the dolphin sperm cell is subjected to iatrogenic damage resulting in increased presence of single and double strand DNA breaks. It is interesting to note the low fragmentation

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M-J Sanchez-Calabuig, C L opez-Fern andez, E Martınez-Nevado, JF Perez-Gutierrez, J de la Fuente, SD Johnston, D Blyde, K Harrison and J Gos alvez

(a)

(a)

(b)

(b)

(c)

Fig. 4. (a) Correlation between frequency of the sperm presenting DNA fragmentation (SDF) assessed with the chromatin dispersion test (SCDt) and two-tailed comet assay (2T-COMET) under laboratory conditions (r = 0.961; p < 0.01); (b) Correlation between frequency of the sperm presenting DNA fragmentation after the SCDt procedure in the laboratory and in the field (r = 0.993; p < 0.01)

Fig. 3. Two-tailed comet assay of bottlenose dolphin sperm DNA: (a) non-fragmented DNA represented as a sperm nuclei with a small migrating comet tails; (b) Single strand breakage DNA present in one spermatozoa (Ss-DNAd); (c) Ss-DNAd and double strand breakage DNA (Ds-DNAd) seen as two large DNA comets with migrating in perpendicular position

level of the five bottlenose dolphin sperm DNA when the sperm samples were assessed immediately after thawing. This indicates that cryopreservation by itself has very little effect on sperm DNA breakage and that the damage was only detectable after sperm incubation. This is a controversial issue in the literature because some studies claim that cryopreservation affects the level of sperm DNA damage immediately after thawing (Zribi et al. 2012), yet in this study we were unable to see this effect using either comet assay or the SCDt. We suggest that the DNA per se, is not affected by the cryopreservation procedure, but the cryodamage caused to the cell during the freeze-thaw process ultimately

contributes to oxidative damage and the increase in sperm DNA damage with incubation. While the stability of sperm DNA following cryopreservation has been reported in other species including humans (Duru et al. 2001), boar (Evenson et al. 1994) and bull (van der Schans et al. 2000), sperm DNA fragmentation in the bottlenose dolphin based on observations from the present study showed a detectable increase in the level of sperm DNA damage after 1 h incubation at 37°C. In the present study, a similar increase in sperm DNA fragmentation was observed in both the SCDt and comet assays. A positive correlation between these two techniques has also been reported in frozen-thawed spermatozoa from different species including humans (Fernandez et al. 2005b; Portas et al. 2009; Zee et al. 2009; Fraser et al. 2010). Other studies have also found valuable correlation between SCDt and other DNA integrity tests such as the chromatin structure assay (SCSA) and the Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) (Evenson and Wixon 2005; Fernandez et al. 2005b; Chohan et al. © 2014 Blackwell Verlag GmbH

Dolphin Sperm Chromatin Dispersion Assay

2006; Zhang et al. 2010) confirming the validity of this technique. Our observations of SDF performed with the five males examined in this study are in accordance with a previous study of frozen-thawed dolphin spermatozoa using the SCSA technique (Montano et al. 2012) where the level of DNA fragmentation immediately after thawing was low (6.6  4.1%). When the sperm DNA fragmentation was evaluated in male 1 after incubation at 37°C, there was a higher increase compared to the results in the cited study where sperm DNA fragmentation remained unchanged after 24 h of incubation. One interpretation as the relative differences in these results may be related to the age of sperm from male 1 in the present study, so it cannot be discounted that the DNA fragmentation dynamics of this specific animal may be related to his age. Moreover, it has been shown in humans and other mammals, that differences in DNA fragmentation dynamics exist between individuals and even between different ejaculates of the same individual (Fraser and Strzezek 2004). Further dynamic studies with sexually mature and proven males of different ages bottlenose dolphins are required to evaluate difference in sperm DNA fragmentation dynamics between individuals in this species. A high correlation was found between SCD-laboratory and SCD-field processed samples as previously described (Gos alvez et al. 2008). In this respect, SCDt assay has significant advantages over other methods for determining SDF including its rapid application, lack of requirement for expensive equipment and simplicity of use. It is not possible to perform these alternative sperm DNA damage assessment techniques in the field as flow cytometry (Evenson et al. 2002), electrophoresis (Aravindan et al. 1997) or DNA labelling using enzymatic reactions (Gorczyca et al. 1993) are required. Hence, one of the most important uses of the SCDt assay is its ability to determine DNA quality at the time of sperm collection and immediately prior to cryopreservation of artificial insemination with both fresh and frozen-thawed semen samples. Indeed, as a CO2 spray is used to accelerate solidification of the microgel, the SCDt can be performed in

Validation of a field based chromatin dispersion assay to assess sperm DNA fragmentation in the bottlenose dolphin (Tursiops truncatus).

Over the last two decades, there have been significant advances in the use of assisted reproductive technology for genetic and reproductive management...
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