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Analysis of sperm chromatin structure in blue foxes (Alopex lagopus) and silver foxes (Vulpes vulpes)
Livestock Science 231 (2020) 103869
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Analysis of sperm chromatin structure in blue foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) ⁎
T
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Marta Kuchta-Gładysza, , Katarzyna Andraszekb, , Olga Szeleszczuka, Piotr Niedbałaa, Agnieszka Otwinowska-Mindurc a
Department of Animal Reproduction, Anatomy and Genomics, Faculty of Animal Sciences, University of Agriculture in Krakow, Mickiewicza 24/28 Str, 30-059 Kraków, Poland b Institute of Animal Production and Fisheries, Faculty of Agrobioengineering and Animal Husbandry, Siedlce University of Natural Sciences and Humanities, Prusa 14 Str, 08-110 Siedlce, Poland c Department of Animal Genetics, Breeding and Ethology, Faculty of Animal Sciences, University of Agriculture in Krakow, Mickiewicza 24/28 Str, 30-059 Kraków, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Foxes Sperm Comet assay Protamine Histone
The latest reports indicate that sperm function is significantly influenced by the chromatin structure of the sperm and the integrity of its DNA. The aim of the study was to determine the level of condensation in the chromatin structure of fresh semen and the degree of damage to the DNA chain in fresh and chilled semen of farmed blue and silver foxes. The sperm of 15 blue foxes and 18 silver foxes at the age of one year was used in the study. The chromatin structure was evaluated by three staining techniques: aniline blue, chromomycin A3 and acridine orange. A comet assay was used to assess sperm DNA integrity in fresh and chilled semen. The average percentage of head DNA changed over time: in blue foxes from 99.12% to 97.83%, and in silver foxes from 99.37% to 98.05%. Highly significant differences (P < 0.01) in head DNA were found between species. Within each species, highly significant differences (P < P < 0.01) were found in the chilled semen (0 h/24 h/48 h/72 h). After staining with aniline blue (AB), chromomycin A3 (CMA3) and acridine orange (AO), most sperm had normal histone retention and a stable, native DNA structure. In blue foxes, the average percentage of sperm with elevated, abnormal histone content, sperm with impaired protamination, and sperm with DNA fragmentation was 1–13%; 1–16% and 0%, respectively. The percentages for silver foxes were similar (3–17%, 5–17% and 2%).
1. Introduction Research on the semen of farmed animals has long aimed at finding the most accurate way to assess the fertilization capacity of sperm. The latest reports indicate that sperm function is significantly influenced by the chromatin structure of the sperm and the integrity (native, doublestranded structure) of its DNA. DNA integrity can be an excellent indicator of fertility (Barratt et al., 2010; Banaszewska et al., 2015a,b; Eamer et al., 2016; Jeng et al., 2016; Urbano et al., 2017). This is important in the case of animals used for insemination, as we may be dealing not only with a single incident of problematic fertilization, but also with the presence of many portions of semen with reduced chromatin quality on the market. A continuing and growing subject of interest regarding sperm chromatin condensation is how protamination and retained histones affect the epigenetic state of mature sperm (Carrell et al., 2007; Jenkins et al., 2011). For this reason, indirect methods for determining
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the number of protamines and measurements of chromatin structure (DNA or chromatin integrity), based on various staining techniques, are now widely used (Bianchi et al., 1996; Iranpour et al., 2000; Kazerooni et al., 2009). Numerous studies confirm that sperm DNA fragmentation and chromatin instability resulting from impaired histone replacement with protamines are linked to abnormal sperm parameters, fertility disorders, and embryonic mortality (Love and Kenney, 1999; Evenson et al., 2000; Aoki et al., 2005, 2006; Oliva, 2006; Oliva and Castillo, 2011). One of the tests used to assess sperm chromatin integrity is the comet assay (Single Cell Gel Electrophoresis assay – SCGE) (Singh et al., 1988; Ashby et al., 1995; Niedbala et al., 2015, Wójcik et al. 2018). Techniques used to assess sperm chromatin structure include staining with aniline blue (AB), chromomycin A3 (CMA3) and acridine orange (AO) (Andraszek et al., 2016; Banaszewska et al., 2015a,b). Morphological examination of semen is traditionally the first step in
Corresponding authors. E-mail addresses: [email protected] (M. Kuchta-Gładysz), [email protected] (K. Andraszek).
https://doi.org/10.1016/j.livsci.2019.103869 Received 9 May 2019; Received in revised form 29 October 2019; Accepted 13 November 2019 Available online 14 November 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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morphology and the structure of their genetic material. The aim of the study was to determine the chromatin structure of the spermatozoa of farmed foxes Alopex lagopus and Vulpes vulpes in fresh and chilled semen.
the assessment of male fertility. It is increasingly believed that this basic semen analysis should be expanded to include cytogenetic and molecular techniques, primarily for the determination of fertility in vivo or in vitro. Many sperm defects are not detectable at the morphological level. These are pathological changes in the sperm chromatin structure, resulting most often from impaired replacement of histone proteins with protamines, or disturbed integrity of sperm DNA or chromatin (Bianchi et al. 1996; Iranpour et al. 2000). Sperm cells with such defects may have normal morphological structure, but in terms of the quality of the genetic material they are dysfunctional (Nagvenkar et al. 2005, Kazerooni et al., 2009, Banaszewska et al., 2015a,b). The stability of the genetic material of the sperm is of great importance in assisted reproduction and semen cryopreservation (De Vos et al. 2003; Nagvenkar et al. 2005). One method of detecting unstable genetic material in sperm is the comet assay (Gedik et al., 1992; Kumaravel et al., 2009). This is a very sensitive and specific method which monitors the presence of breaks in the structure of the DNA chain of sperm and somatic cells. The SCGE technique has been used in the study of human germ cells (Baumgartner et al., 2009) as well as animal cells (Fraser and Strzeżek, 2004; Love et al., 2002; Dobrzyńska, 2005; Pronosilova et al., 2012). It is particularly important to clarify whether and in what way protamination and histone retention during condensation of sperm chromatin affect the epigenetic state of the mature spermatozoon (Carrell and Hammoud, 2010; Jenkins et al., 2011). The stability and removability of various histone modifications are important for normal spermatogenesis. Histone modifications can affect chromatin structure and gene expression in germ cells. The latest research suggests that sperm histones and specific modifications of histone methylation may play an important role after fertilization and may mark or balance genes for expression in the embryo. Stabilization of these histone modifications in ontological genes has been observed in mouse sperm, and probably occurs in all vertebrates (Chan and Trasler, 2011). Many epigenetic modifiers, including DNA methyltransferase, histone modification enzymes and their regulatory proteins, play a significant role in the development of reproductive cells (Gryzińska et al., 2013). Aberrations of DNA methylation are often found in the imprinted loci of the sperm of males with oligospermia (Dada et al., 2012). The semen of males with asthenozoospermia and teratozoospermia has a reduced level of DNA methylation (Mitchell et al., 2005; Chan and Trasler, 2011). For this reason, indirect methods are now widely used to determine the quantity of protamines and measure chromatin structure (DNA or chromatin integrity) by various staining techniques (Bianchi et al., 1996; Iranpour et al., 2000; Kazerooni et al., 2009). A number of methods have been developed to assess the integrity of sperm DNA and chromatin (Chan et al., 2001; Hekmatdoost et al., 2009; Schulte et al., 2010; Chan i Trasler, 2011). Many of these only detect damage to single- or double-stranded DNA of sperm cells. In contrast to other methods, they are based on the fact that defects of sperm chromatin structure are associated with increased DNA instability and sensitivity to endogenous and exogenous factors. Therefore, these methods involve creating conditions that cause denaturation and then assessing the ability of the sperm chromatin to maintain its integrity. Most of these methods are based on staining of sperm with fluorescent dyes (Hekmatdoost et al., 2009; Kazerooni et al., 2009, Banaszewska et al., 2015a,b). Protamination disturbances have become the subject of numerous studies on factors associated with infertility, thus showing the important role of these proteins and their impact on chromatin structure (Aoki et al., 2006; Zhang et al., 2006; Zini et al., 2007; Jenkins et al., 2011 Andraszek et al., 2016; Banaszewska et al., 2015a,b). Spermatozoa of canids, particularly farmed foxes, are not often used in andrology research. The increasingly common artificial insemination of these animals presents a challenge to precisely determine their sperm
2. Material and methods Sexually mature male blue foxes (n = 15) and silver foxes (n = 18) used for breeding were used in the study. The semen for the research was obtained manually by the breeder during an artificial insemination procedure. Directly after ejaculation, the semen was subjected to a standard analysis encompassing semen volume, sperm motility, progressive motility, and sperm concentration. Sperm concentration was measured in a Bürker chamber, and sperm motility was evaluated in a Blom chamber placed over a thermostatic hot plate (SEMIC Warsaw, Poland) to keep the sperm at 37 °C during the evaluation. Assessments were performed under a light microscope at 200–400 × magnification (Bioval, Poland). Sperm morphology (viability) was evaluated using semen smears stained with 5% eosin (Sigma Aldrich) and 10% watersoluble nigrosin (Sigma Aldrich) under a light microscope at 1500× immersion magnification (Bioval, Poland). A total of 200 sperm cells were assessed by determining the percentage of normal sperm and of sperm with the following defects: protoplasmic droplets, single bent tail, coiled tail, damaged acrosome, damaged head, and agglutinated sperm. For further analysis, as soon as possible, the semen was diluted to a concentration of 40 × 10 3 sperm/5 μl in MIII extender. 2.1. SCGE procedure The cells were briefly suspended in a gel of 0.5% LMPA (Sigma Aldrich) between two layers of 0.5% NMPA (Sigma Aldrich). To precipitate proteins, cells were lysed for 24 h in an alkaline buffer (2.5 M NaCl, 100 mM Na2EDTA, 0.4 M Tris-HCl, 1% sodium N-lauroyl sarcosinate, 10% Triton X-100, 1% DMSO, pH = 10). After 20-min incubation in a strongly alkaline TBE buffer (10 N NaOH, 200 mM EDTA, pH ≥ 13), electrophoresis was conducted (25 V, 300 mA, 20 min). Finally, the preparations were neutralized with 0.4 M Tris-HCl (Sigma Aldrich) and stained with ethidium bromide (200 µg/ml). The results of the comet assay were analysed as follows. Sperm DNA integrity was assessed on the basis of the percentage content of nuclear DNA in the comet head and the value of the Tail Moment (TM) index. Slides were examined under a Zeiss epifluorescence microscope at 400 x magnification. To assess the degree of damage to sperm DNA in the semen just after dilution (0 h) and chilling, one slide was prepared for each sample. We analysed 50 cells on each slide. Comet measurements were made in CASP 1.2.0 software. Comets observed in the chilled semen were assessed with regard to the degree of damage and then classified according to the Gedik scale (Gedik et al., 1992). 2.2. AB, CMA3, and AO staining The slides with semen were stained by three techniques: aniline blue, chromomycin A3 and acridine orange. Spermatozoa were assessed using an Olympus BX 50 fluorescence microscope with an Olympus UplanApo 100x/1.35/Oil Iris/∞/0.17 lens and a Jenoptik ProgRes camera. From each individual 500 sperm stained by each method were evaluated. AB staining was performed according to Franken et al. (2000). The slides were incubated in 3% buffered glutaraldehyde at room temperature and then stained with a 5% AB solution in 4% acetic acid (pH 3.5) for 5 min. The presence of sperm with normal histone content (light blue colour) and with high histone content (intense blue) was analysed. CMA3 staining was performed according to Lolis et al. (1996). The slides were incubated in Carnoy solution at 40 °C for 5 min and then coated with 100 μl of CMA3 solution (0.25 mg/ml in McIlvaine buffer, pH 7.0, containing 10 mM MgCl2) for 20 min. The slides were rinsed in buffer and left in buffered glycerol. 2
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intact DNA fluoresce green, while sperm with DNA fragmentation in the nucleus show fluorescence from orange to red (Fig. 3). The average percentage of head DNA in comets of blue fox and silver fox spermatozoa stored at 4 °C changed over time after ejaculation (Table 2). In blue foxes it decreased slightly from 99.12% (SEM = 0.038%) immediately after ejaculation to 97.83% (SEM = 0.062%) 72 h after ejaculation. The same downward trend in the mean percentage of head DNA was observed for silver foxes; it fell from 99.37% (SEM = 0.028%) to 98.05% (SEM = 0.045%). Our results show highly significant differences in the percentage of head DNA depending on the time after ejaculation (P < 0.01) within each foxes species. This finding may be associated in part with the small variation in the percentage of head DNA within time-after-ejaculation classes. Importantly, the differences between mean percentages of head DNA were about 1–2 pp during 72 h of storage, which confirms the protective function of the thinners used. It is worth noting that efficient storage of fox semen in a liquid state has practical significance. Additionally, analysis of the percentage of head DNA revealed significant differences (Table 2; P < 0.05) between fox species immediately after ejaculation (0 h). Highly significant differences (P < 0.01) in head DNA were found between species at 24 and 48 h after ejaculation. The mean percentage of head DNA was slightly higher for silver fox than for blue fox at each analysed time after ejaculation. Table 3 presents the percentages of sperm with abnormalities identified using the three staining techniques described above in the two species of foxes. The semen of silver foxes had a lower mean percentage of abnormal sperm when the slides were stained with AB (mean: 0.802%) or CMA3 (mean: 0.974%) than the semen of blue foxes (AB: mean 1.413%, CMA3: mean 1.747%). Analysis of the data shows that the genetic material of the sperm of silver foxes was more stable, in the case of both chromatin and DNA. No DNA fragmentation was identified by acridine orange in the sperm of silver foxes. In blue foxes, the proportion of spermatozoa with abnormal histone retention and impaired protamination was nearly twice as high as in silver foxes. Within each fox species, the mean numbers of sperm with abnormal histone retention (AB) and DNA fragmentation (AO) differed highly significantly (P < 0.01) from the mean number of sperm with impaired protamination (CMA3). There were no significant differences (P > 0.05) in the percentage of abnormal sperm between the two fox species in the case of abnormal histone retention (AB), DNA fragmentation (AO), or impaired protamination (CMA3).
The percentages of spermatozoa with normal chromatin packaging (dull green fluorescence) and with abnormal chromatin packaging (bright green fluorescence) were determined. AO staining was performed according to Tejda et al. (1984). The staining solution was prepared by mixing 10 ml of the stock solution (1 mg AO in 1000 ml of distilled water) and 40 ml of 0.1 M citric acid with 2.5 ml of 0.3 M Na2HPO4. The slides were stained by applying 2–3 ml of the solution for 10 min and then rinsed with distilled water. The wet slides were covered with a cover slip, which was sealed with rubber cement. Microscopic analysis of the slides involved the identification of sperm cells with normal DNA structure (green fluorescence) and with damaged, single-stranded DNA (orange fluorescence). 2.3. Statistics All values were expressed as mean ± standard error of mean (SEM). Data were evaluated for normality and homogeneity of variances. The differences in sperm morphology between blue and silver foxes were analysed using a non-parametric Mann-Whitney U test. The percentages of head DNA between different times (0 h/24 h/48 h/72 h) within species were analysed using the non-parametric Friedman test and post-hoc Nemenyi test. The percentages of sperm with abnormal histone content were compared between staining methods (AB, CMA3 or AO) within species using a non-parametric Friedman test, and posthoc analyses were carried out by the Nemenyi test. All P-values less than 0.05 were considered statistically significant, and values less than 0.01 were statistically highly significant. Statistical procedures were performed using the R software package (R Core Team, 2013). 3. Results Table 1 presents detailed results of the morphological analysis of fox semen. The basic morphological analysis revealed a higher proportion of normal sperm in blue foxes. Analysis of individual sperm defects showed a higher percentage of cytoplasmic droplets, acrosome damage and agglutination in the semen of blue foxes, while the percentage of single bent tails, coiled tails, and head damage was higher in silver foxes. Highly significant differences were found (P < 0.01) between the semen of blue and silver foxes only in the case of head damage. Aniline blue selectively differentiates sperm with normal and abnormal histone retention. Sperm with abnormal, elevated histone content are identified on the slide as navy blue or bright blue, while normal sperm are much lighter than the abnormal ones (Fig. 1). Chromomycin A3 identifies sperm with normal and abnormal protamination. Sperm with abnormal protamination show bright green fluorescence on the dark background of the slide, while spermatozoa with normal chromatin structure are characterized by a light, dull fluorescence (Fig. 2). Acridine orange selectively stains sperm with normal, native DNA structure and with damaged, single-stranded DNA. Sperm with normal,
4. Discussion The comet assay is a sensitive test used to measure damage and repair at the DNA level in individual cells. This test is highly versatile, as it can be used to assess damage in a variety of types of cells and samples, such as cells of the peripheral blood, kidneys, liver, brain, gills, bone marrow, or sperm, as well as cancer cells, solid tumours, bacteria or yeasts (Collins et al., 2001; Awad et al., 2014; Wójcik et al., 2018, Gajski et al., 2019). Initially, this technique was mainly used to study human cells, but it has also been used to assess DNA damage in cells of domestic animals (horses, pigs, cattle, sheep, goats, dogs and cats) and other vertebrates: amphibians, reptiles, birds and fish, including Cyclostomata (Gajski et al., 2019). In our study, the research material was semen from two species of farmed foxes: blue and silver foxes. The SCGE method involves electrophoretic separation of nuclear DNA, which makes it possible to observe its fragmentation. The test cells are immobilized on agarose on microscope slides and then lysed to release the contents of the nucleus. The high ionic strength of the lysis buffer promotes the dissociation of proteins from DNA. Then electrophoresis is performed. The DNA is fluorescently stained with silver salts and the resulting image is analysed under a microscope. If there is damage, the image is reminiscent of a comet. The head of the comet is the cell nucleus, while the tail consists of DNA fragments that have migrated during electrophoresis. The greater the damage, the longer the tail is.
Table 1 Morphological characteristics of semen of blue foxes and silver foxes immediately after ejaculation (0 h). Sperm
Intact Cytoplasmic droplet Single bent tail Coiled tail Acrosome damage Head damage Agglutination
Blue fox Mean
SEM1)
Silver fox Mean
SEM1)
73.93A 5.13A 13.73A 0.40A 1.60A 4.53A 0.53A
2.07 0.97 1.55 0.16 0.21 0.61 0.19
69.50A 3.60A 15.90A 1.20A 1.00A 8.70B 0.10A
2.33 0.72 1.62 0.39 0.33 0.88 0.10
1) SEM – standard error of mean. A, B values within the same row designated with different letters differ highly significantly (P < 0.01). 3
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Fig. 1. Fox sperm stained with aniline blue; A – spermatozoon with elevated histone content; B - normal spermatozoon.
DNA damage can be assessed in several ways, e.g. by measuring the length of the comet tail or by visual assessment of the degree of damage on a scale of 0–4, depending on the appearance of the comet (Fraser et al., 2005; Linfor and Meyers, 2002, Gajski et al., 2019). However, the comet test also reveals other comet parameters, alternatively used to describe the result of this test, such as the percentage of DNA damage, percentage of DNA in the comet head, percentage of DNA in the tail, and tail moment (TM), which is the product of tail length and the percentage of DNA in the tail (Gajski et al., 2019). In our study, the selected parameter was head size (% head DNA), which ranged from 99.12% (SEM = 0.038%) to 97.83% (SEM = 0.062%) in blue foxes and from 99.37% (SEM = 0.028%) to 98.05% (SEM = 0.045%) in silver foxes. Niedbała et al. (2015) also chose the percentage of DNA in the comet head as a test parameter in chinchilla. They tested four different semen extenders commonly used for storing the sperm of farmed chinchilla for insemination. They showed that the best cryoprotectant, in the L4 extender, can protect the integrity of sperm DNA in chilled semen (even after 72 h), with 98.56% of DNA retained in the head of the comet. There are many papers describing the use of the comet test on the semen of farmed animals, but due to the variable factors affecting the sperm, numerical comparison with our data is difficult. The usefulness of the SCGE test for assessment of sperm should be stressed. Studies of sperm chromatin proteins in relation to fertility help to understand the importance of normal chromatin structure for sperm functions. Unfortunately, assessment of protamination to evaluate male fertility is primarily carried out in human material, while in the case of animals, this type of research is scarce and usually limited to laboratory
The measure of the level of DNA damage is the length of the tail and the amount of DNA contained in it. A reliable result is obtained by analysing 50–100 comets in gel (Końca et al., 2003; Singh et al., 2003). In our study, evaluation of the semen of farmed foxes was based on analysis of 50 cells per individual. The comet assay is most commonly carried out under alkaline conditions, but there is also a neutral form of the assay (Nandhakumar et al., 2011). The differences are fundamental, because the alkaline form of the SCGE test can detect both single- and doublestranded DNA damage. In our study, we used the alkaline method. An unquestionable advantage of the method is that several different DNA modifications can be analysed with relatively small changes in the basic procedure. The method identifies single-stranded and double-stranded DNA breaks, as well as all types of chemical modifications (e.g. apurinic sites and unstable adducts) and enzymatic modifications (oxidative damage) that can potentially become breaks. The comet assay makes it possible to detect DNA damage at the level of a single cell and to determine susceptibility to a specific genotoxic factor, as well as the efficiency of damage repair (Ashby et al., 1995; Singh et al., 1988; Slyskova et al. 2007, 2014; Azqueta et al. 2014). The comet test has mainly been used in medicine, pharmacy and biomonitoring. In domestic animals, the comet test is performed to assess the effect of feed toxins or anaesthesia on DNA integrity, or to test for complications after certain infections (Strasser et al., 2012; Hornatovich et al., 2013; Radakovic et al., 2016). In addition, the test has been used to test the semen of breeders to test the quality of sperm after cryopreservation.
Fig. 2. Fox sperm stained with chromomycin A3; A - spermatozoon with abnormal protamination; B - normal spermatozoon. 4
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Fig. 3. Fox sperm stained with acridine orange; A - spermatozoon with DNA fragmentation; B - normal spermatozoon.
experiments, without practical application. The role of basic proteins stabilizing the sperm DNA structure is increasingly emphasized in studies dealing with human and animal reproduction. In some cases of infertility, the direct cause is abnormalities in spermiogenesis associated with the replacement of histone proteins. Assessment of chromatin organization in sperm indicates the normality not only of spermatogenesis, but also of the paternal genome and epigenome (Martianov et al., 2005; Enciso et al., 2011). In early, round spermatids the number of histones replaced by their transitional variants exceeds 50%. Nevertheless, the classic nucleosome structure and transcriptional activity are still preserved until histone H1t and the majority of histones are dissociated and transition proteins (TP) appear (Hammoud et al., 2009). Following this chromatin reorganization, about 10–15% of histones remain in the mature sperm (Kimmins and Saccone-Corsi, 2005). The presence of histones allows the nucleosome structure to be maintained. Hammoud et al. showed that this applies to 4% of the haploid genome (Hammoud et al., 2009). The normal level of histones in chromatin is species-specific. Studies in boars and mice have found only about 1% histones in sperm chromatin, while the level in humans can be as high as 15% (Tanphaichitr et al., 1978; Gatewood et al., 1990; Bench et al., 1996). There are no data on the physiological level of histones in canine sperm. Increased histone levels have a negative effect on sperm maturation, fertilization, and early embryonic development (Jenkins et al., 2011). Elevated histone levels have been found in infertile individuals or those with reduced fertility diagnosed with oligospermia, teratozoospermia or asthenozoospermia (Zhang et al., 2006; Zini et al., 2007). Research by Kazerooni et al. (2009) on human semen has shown that in couples with recurrent miscarriages, there is an increased percentage of cells positively stained with aniline blue. Hammadeh et al. (2001) have demonstrated that positive AB staining was significantly more common in a group of patients with fertility disorders than in the healthy control
Table 2 The percentage of head DNA in comets of blue fox and silver fox spermatozoa stored at 4 °C. Time (h)
0 24 48 72
P-values2)
Blue fox Mean
SEM1)
Silver fox Mean
SEM1)
99.12A 98.84B 98.30C 97.83D
0.038 0.039 0.050 0.062
99.37A 99.12B 98.72C 98.05D
0.028 0.036 0.042 0.045
0.0324 0.0003 <0.001 0.1445
1) SEM – standard error of mean. 2) P-values indicate differences between blue foxes and silver foxes at one time (0 h/24 h/48 h/72 h). A,B,C,D values within the same column designated with different letters differ highly significantly (P < 0.01). Table 3 Effect of staining method on percentage of sperm with abnormal histone content, by species. Staining method1)
AB CMA3 AO
P-values3)
Blue fox Mean
SEM2)
Silver fox Mean
SEM2)
1.413A 1.747B 0.027A
0.322 0.370 0.027
0.802A i 0.000A
0.197 0.229 0.000
0.1762 0.1660 0.2733
1) AB – abnormal histone retention, CMA3 – impaired protamination, AO – DNA fragmentation. 2) SEM – standard error of mean. 3) P-values indicate differences between blue foxes and silver foxes within a staining method. A,B values within the same column designated with different letters differ highly significantly (P < 0.01).
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protamines, lack of protamines, maturation in the epididymis, and chromatin stability. Kazerooni et al. (2009) have observed that the number of sperm positively stained with CMA3 and AB is higher in individuals with impaired fertility. In addition, sperm viability and abnormal morphology have been found to be correlated with the percentage of spermatozoa positively stained with CMA3 and AB. This shows that abnormal chromatin packing has a destructive effect on semen parameters. This conclusion is in line with those presented by other authors (Coetzee et al., 2001; Hammadeh et al., 1998). Acridine orange staining is a simple and rapid test to assess DNA damage in sperm. When bound to double-stranded DNA it fluoresces green, while bound to RNA and single-strand DNA it fluoresces red. Evenson et al. (1980) found that spermatozoa containing denatured, single-stranded DNA reduce fertilization rates and the quality of embryos obtained in vitro. Although acridine orange has been used to study sperm of various species, the level of pathological DNA fragmentation has been determined only for humans. Up to 15% sperm with damaged DNA is considered normal, 15–25% is believed to reduce fertility, and a level above 25% indicates a high risk of infertility (Evenson et al., 1999, 2000). In bulls, a reduction in fertility has been observed when the percentage of damaged sperm exceeded 10% (Bochenek et al., 2001). Reference values have not been determined for other species Analysis of disturbances in the protamination process should complement morphological and molecular evaluation of semen. Protamination is considered one of the important parameters of male fertility (Aoki et al., 2006; Zini et al., 2007). Unfortunately, assessment of protamination is carried out primarily in human material. Research on nuclear proteins with respect to infertility demonstrates the importance of normal chromatin structure for sperm function. A more detailed understanding of the complex structure of sperm chromatin is essential for the development of new, more versatile and more accurate diagnostic tools.
population. These studies also showed that embryo mortality was correlated with increased histone content in the spermatozoa (Hammadeh et al., 2001; Kazerooni et al., 2009). Immature sperm or sperm with DNA disorders usually contain histone residues (Auger, 2010; Schulte et al., 2010; Jenkins et al., 2011). Further research is therefore necessary to determine how best to use the aniline blue staining technique. Studies on histones and their potential involvement in early embryonic development are needed to understand the epigenetic factors of male infertility. Numerous researchers are carrying out experiments to establish species reference values. Early abortion and embryonic mortality have been shown to correlate with increased histone content in spermatozoa (Hammadeh et al., 2001; Kazerooni et al., 2009). Higher histone levels have been found in infertile individuals or those with oligospermia – reduced mature sperm count, teratozoospermia – abnormal structure, and asthenozoospermia – impaired sperm motility (Zhang et al., 2006; Zini et al., 2007). In the case of animal sperm, this type of research is scarce and most often limited to laboratory experiments, without practical application. Studies of these unique proteins and their potential involvement in early embryonic development are needed to understand the epigenetic factors of male infertility. Protamines are proteins rich in arginine and cysteine. They form an extremely condensed and transcriptionally silenced conformation of sperm chromatin (Vilfan et al., 2004). In the mammalian genome, incorporation of protamines P1 and P2 is strictly regulated during spermatogenesis. Not only impaired protamination, but also the P1 to P2 ratio is a factor affecting fertility. Abnormalities of both the protamination process and the P1 to P2 ratio have been found in individuals whose semen morphology assessment shows low sperm motility or low sperm concentration. Protamination is indispensable to normal sperm chromatin condensation, which in turn affects sperm function (Aoki et al. 2006). The few published results of animal research evaluating histone retention and impaired protamination have dealt with the effect of age on the structure of sperm chromatin and its relationship to the physical characteristics of the ejaculates of breeding boars (Banaszewska et al. 2015a), and the role of sperm chromatin structure in reproduction in cocks (Banaszewska et al. 2015a). Studies in boars have found a relationship between age and sperm count, with increased histone content and impaired protamination in younger individuals. The percentage of spermatozoa with disturbed chromatin structure was highest in boars under two years old. Unfortunately, the effect of ageing on sperm chromatin quality has only been carried out in humans (Sharma et al., 2015). Animals used for insemination are often young individuals whose sperm production capacity is an indicator for reproduction. In such animals, including boars, the spermiogenesis process may not be fully functional, which results in the presence of spermatozoa with an unsatisfactory chromatin structure in their semen. This leads to an increased presence of spermatozoa with histones that have not been fully replaced and abnormally incorporated protamines, and thus to reduced reproduction (Banaszewska and Kondracki, 2012; Banaszewska et al., 2015a). Abnormal protamination in breeding cocks did not exceed a few percent of the reproductive cells analysed. However, comparison of the two commercial lines showed that the F15 line had better semen quality, motility, and reproductive parameters than the Flex line (Banaszewska et al., 2015b). Sperm staining with aniline blue (AB) and chromomycin A3 (CMA3) is a rapid test of chromatin quality in sperm cells. Aniline blue is a specific dye for proteins with high lysine content. This includes histones, which are replaced by protamines during spermiogenesis. Aniline staining reveals abnormal, excessive histone content in the sperm cell. Such sperm are more susceptible to DNA damage and impaired chromatin stability. Studies have shown early abortion and foetal mortality to be correlated with elevated histone content in sperm (Hammadeh et al., 2001; Kazerooni et al., 2009). Abnormalities in sperm chromatin can occur on several levels: histone replacement with
Declaration of Competing Interest The authors hereby confirms that the manuscript has not been published in whole or in part, nor is being considered for publication elsewhere. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.livsci.2019.103869. References Andraszek, K., Szeleszczuk, O., Niedbała, P., Kuchta-Gładysz, M., 2016. Preliminary research on evaluation of sperm morphometry and chromatin structure in the semen of silver fox (Vulpes vulpes). Folia Pomer. Univ. Technol. Stetin. Agric. Aliment. Pisc Zootech 326 (38), 5–16 2. Aoki, V.W., Liu, L., Carrell, D.T., 2005. Identification and evaluation of a novel sperm protamine abnormality in a population of infertile males. Hum. Reprod. 20, 1298–1306. Aoki, V.W., Liu, L., Jones, K.P., 2006. Sperm protamine 1/protamine 2 ratios are related to in vitro fertilization pregnancy rates and predictive of fertilization ability. Fertil. Steril. 86, 1408–1415. Ashby, J., Tinwell, H., Lefevre, P.A., Browne, M.A., 1995. The single cell gel electrophoresis assay for induced DNA damage (comet assay): measurement of tail length and moment. Mutagenesis 10, 85–90. Auger, J., 2010. Assessing human sperm morphology: top models, underdogs or biometrics? Asian. J. Androl. 12, 36–46. Awad, W.A., Ghareeb, K., Dadak, A., Hess, M., Boehm, J., 2014. Single and combined effects of deoxynivalenol mycotoxin and a microbial feed additive on lymphocyte dna damage and oxidative stress in broiler chickens. Plos One 9, 1–6. Azqueta, A., Slyskova, J., Langie, S.A.S., Gaivao, O'Neill, I., Collins A., 2014. Comet assay to measure DNA repair: approach and applications. Front. Genet. 5, 288. Banaszewska, D., Andraszek, K., Biesiada-Drzazga, B., 2015a. Evaluation of sperm chromatin structure in the semen of insemination boars. Bull. Vet. Inst. Pulawy 59, 271–277. Banaszewska, D, Andraszek, K, Biesiada-Drzazga, B, Przyborski, M., 2015b. Identification
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Analysis of sperm chromatin structure in blue foxes (Alopex lagopus) and silver foxes (Vulpes vulpes) ⁎
T
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Marta Kuchta-Gładysza, , Katarzyna Andraszekb, , Olga Szeleszczuka, Piotr Niedbałaa, Agnieszka Otwinowska-Mindurc a
Department of Animal Reproduction, Anatomy and Genomics, Faculty of Animal Sciences, University of Agriculture in Krakow, Mickiewicza 24/28 Str, 30-059 Kraków, Poland b Institute of Animal Production and Fisheries, Faculty of Agrobioengineering and Animal Husbandry, Siedlce University of Natural Sciences and Humanities, Prusa 14 Str, 08-110 Siedlce, Poland c Department of Animal Genetics, Breeding and Ethology, Faculty of Animal Sciences, University of Agriculture in Krakow, Mickiewicza 24/28 Str, 30-059 Kraków, Poland
A R T I C LE I N FO
A B S T R A C T
Keywords: Foxes Sperm Comet assay Protamine Histone
The latest reports indicate that sperm function is significantly influenced by the chromatin structure of the sperm and the integrity of its DNA. The aim of the study was to determine the level of condensation in the chromatin structure of fresh semen and the degree of damage to the DNA chain in fresh and chilled semen of farmed blue and silver foxes. The sperm of 15 blue foxes and 18 silver foxes at the age of one year was used in the study. The chromatin structure was evaluated by three staining techniques: aniline blue, chromomycin A3 and acridine orange. A comet assay was used to assess sperm DNA integrity in fresh and chilled semen. The average percentage of head DNA changed over time: in blue foxes from 99.12% to 97.83%, and in silver foxes from 99.37% to 98.05%. Highly significant differences (P < 0.01) in head DNA were found between species. Within each species, highly significant differences (P < P < 0.01) were found in the chilled semen (0 h/24 h/48 h/72 h). After staining with aniline blue (AB), chromomycin A3 (CMA3) and acridine orange (AO), most sperm had normal histone retention and a stable, native DNA structure. In blue foxes, the average percentage of sperm with elevated, abnormal histone content, sperm with impaired protamination, and sperm with DNA fragmentation was 1–13%; 1–16% and 0%, respectively. The percentages for silver foxes were similar (3–17%, 5–17% and 2%).
1. Introduction Research on the semen of farmed animals has long aimed at finding the most accurate way to assess the fertilization capacity of sperm. The latest reports indicate that sperm function is significantly influenced by the chromatin structure of the sperm and the integrity (native, doublestranded structure) of its DNA. DNA integrity can be an excellent indicator of fertility (Barratt et al., 2010; Banaszewska et al., 2015a,b; Eamer et al., 2016; Jeng et al., 2016; Urbano et al., 2017). This is important in the case of animals used for insemination, as we may be dealing not only with a single incident of problematic fertilization, but also with the presence of many portions of semen with reduced chromatin quality on the market. A continuing and growing subject of interest regarding sperm chromatin condensation is how protamination and retained histones affect the epigenetic state of mature sperm (Carrell et al., 2007; Jenkins et al., 2011). For this reason, indirect methods for determining
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the number of protamines and measurements of chromatin structure (DNA or chromatin integrity), based on various staining techniques, are now widely used (Bianchi et al., 1996; Iranpour et al., 2000; Kazerooni et al., 2009). Numerous studies confirm that sperm DNA fragmentation and chromatin instability resulting from impaired histone replacement with protamines are linked to abnormal sperm parameters, fertility disorders, and embryonic mortality (Love and Kenney, 1999; Evenson et al., 2000; Aoki et al., 2005, 2006; Oliva, 2006; Oliva and Castillo, 2011). One of the tests used to assess sperm chromatin integrity is the comet assay (Single Cell Gel Electrophoresis assay – SCGE) (Singh et al., 1988; Ashby et al., 1995; Niedbala et al., 2015, Wójcik et al. 2018). Techniques used to assess sperm chromatin structure include staining with aniline blue (AB), chromomycin A3 (CMA3) and acridine orange (AO) (Andraszek et al., 2016; Banaszewska et al., 2015a,b). Morphological examination of semen is traditionally the first step in
Corresponding authors. E-mail addresses: [email protected] (M. Kuchta-Gładysz), [email protected] (K. Andraszek).
https://doi.org/10.1016/j.livsci.2019.103869 Received 9 May 2019; Received in revised form 29 October 2019; Accepted 13 November 2019 Available online 14 November 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
Livestock Science 231 (2020) 103869
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morphology and the structure of their genetic material. The aim of the study was to determine the chromatin structure of the spermatozoa of farmed foxes Alopex lagopus and Vulpes vulpes in fresh and chilled semen.
the assessment of male fertility. It is increasingly believed that this basic semen analysis should be expanded to include cytogenetic and molecular techniques, primarily for the determination of fertility in vivo or in vitro. Many sperm defects are not detectable at the morphological level. These are pathological changes in the sperm chromatin structure, resulting most often from impaired replacement of histone proteins with protamines, or disturbed integrity of sperm DNA or chromatin (Bianchi et al. 1996; Iranpour et al. 2000). Sperm cells with such defects may have normal morphological structure, but in terms of the quality of the genetic material they are dysfunctional (Nagvenkar et al. 2005, Kazerooni et al., 2009, Banaszewska et al., 2015a,b). The stability of the genetic material of the sperm is of great importance in assisted reproduction and semen cryopreservation (De Vos et al. 2003; Nagvenkar et al. 2005). One method of detecting unstable genetic material in sperm is the comet assay (Gedik et al., 1992; Kumaravel et al., 2009). This is a very sensitive and specific method which monitors the presence of breaks in the structure of the DNA chain of sperm and somatic cells. The SCGE technique has been used in the study of human germ cells (Baumgartner et al., 2009) as well as animal cells (Fraser and Strzeżek, 2004; Love et al., 2002; Dobrzyńska, 2005; Pronosilova et al., 2012). It is particularly important to clarify whether and in what way protamination and histone retention during condensation of sperm chromatin affect the epigenetic state of the mature spermatozoon (Carrell and Hammoud, 2010; Jenkins et al., 2011). The stability and removability of various histone modifications are important for normal spermatogenesis. Histone modifications can affect chromatin structure and gene expression in germ cells. The latest research suggests that sperm histones and specific modifications of histone methylation may play an important role after fertilization and may mark or balance genes for expression in the embryo. Stabilization of these histone modifications in ontological genes has been observed in mouse sperm, and probably occurs in all vertebrates (Chan and Trasler, 2011). Many epigenetic modifiers, including DNA methyltransferase, histone modification enzymes and their regulatory proteins, play a significant role in the development of reproductive cells (Gryzińska et al., 2013). Aberrations of DNA methylation are often found in the imprinted loci of the sperm of males with oligospermia (Dada et al., 2012). The semen of males with asthenozoospermia and teratozoospermia has a reduced level of DNA methylation (Mitchell et al., 2005; Chan and Trasler, 2011). For this reason, indirect methods are now widely used to determine the quantity of protamines and measure chromatin structure (DNA or chromatin integrity) by various staining techniques (Bianchi et al., 1996; Iranpour et al., 2000; Kazerooni et al., 2009). A number of methods have been developed to assess the integrity of sperm DNA and chromatin (Chan et al., 2001; Hekmatdoost et al., 2009; Schulte et al., 2010; Chan i Trasler, 2011). Many of these only detect damage to single- or double-stranded DNA of sperm cells. In contrast to other methods, they are based on the fact that defects of sperm chromatin structure are associated with increased DNA instability and sensitivity to endogenous and exogenous factors. Therefore, these methods involve creating conditions that cause denaturation and then assessing the ability of the sperm chromatin to maintain its integrity. Most of these methods are based on staining of sperm with fluorescent dyes (Hekmatdoost et al., 2009; Kazerooni et al., 2009, Banaszewska et al., 2015a,b). Protamination disturbances have become the subject of numerous studies on factors associated with infertility, thus showing the important role of these proteins and their impact on chromatin structure (Aoki et al., 2006; Zhang et al., 2006; Zini et al., 2007; Jenkins et al., 2011 Andraszek et al., 2016; Banaszewska et al., 2015a,b). Spermatozoa of canids, particularly farmed foxes, are not often used in andrology research. The increasingly common artificial insemination of these animals presents a challenge to precisely determine their sperm
2. Material and methods Sexually mature male blue foxes (n = 15) and silver foxes (n = 18) used for breeding were used in the study. The semen for the research was obtained manually by the breeder during an artificial insemination procedure. Directly after ejaculation, the semen was subjected to a standard analysis encompassing semen volume, sperm motility, progressive motility, and sperm concentration. Sperm concentration was measured in a Bürker chamber, and sperm motility was evaluated in a Blom chamber placed over a thermostatic hot plate (SEMIC Warsaw, Poland) to keep the sperm at 37 °C during the evaluation. Assessments were performed under a light microscope at 200–400 × magnification (Bioval, Poland). Sperm morphology (viability) was evaluated using semen smears stained with 5% eosin (Sigma Aldrich) and 10% watersoluble nigrosin (Sigma Aldrich) under a light microscope at 1500× immersion magnification (Bioval, Poland). A total of 200 sperm cells were assessed by determining the percentage of normal sperm and of sperm with the following defects: protoplasmic droplets, single bent tail, coiled tail, damaged acrosome, damaged head, and agglutinated sperm. For further analysis, as soon as possible, the semen was diluted to a concentration of 40 × 10 3 sperm/5 μl in MIII extender. 2.1. SCGE procedure The cells were briefly suspended in a gel of 0.5% LMPA (Sigma Aldrich) between two layers of 0.5% NMPA (Sigma Aldrich). To precipitate proteins, cells were lysed for 24 h in an alkaline buffer (2.5 M NaCl, 100 mM Na2EDTA, 0.4 M Tris-HCl, 1% sodium N-lauroyl sarcosinate, 10% Triton X-100, 1% DMSO, pH = 10). After 20-min incubation in a strongly alkaline TBE buffer (10 N NaOH, 200 mM EDTA, pH ≥ 13), electrophoresis was conducted (25 V, 300 mA, 20 min). Finally, the preparations were neutralized with 0.4 M Tris-HCl (Sigma Aldrich) and stained with ethidium bromide (200 µg/ml). The results of the comet assay were analysed as follows. Sperm DNA integrity was assessed on the basis of the percentage content of nuclear DNA in the comet head and the value of the Tail Moment (TM) index. Slides were examined under a Zeiss epifluorescence microscope at 400 x magnification. To assess the degree of damage to sperm DNA in the semen just after dilution (0 h) and chilling, one slide was prepared for each sample. We analysed 50 cells on each slide. Comet measurements were made in CASP 1.2.0 software. Comets observed in the chilled semen were assessed with regard to the degree of damage and then classified according to the Gedik scale (Gedik et al., 1992). 2.2. AB, CMA3, and AO staining The slides with semen were stained by three techniques: aniline blue, chromomycin A3 and acridine orange. Spermatozoa were assessed using an Olympus BX 50 fluorescence microscope with an Olympus UplanApo 100x/1.35/Oil Iris/∞/0.17 lens and a Jenoptik ProgRes camera. From each individual 500 sperm stained by each method were evaluated. AB staining was performed according to Franken et al. (2000). The slides were incubated in 3% buffered glutaraldehyde at room temperature and then stained with a 5% AB solution in 4% acetic acid (pH 3.5) for 5 min. The presence of sperm with normal histone content (light blue colour) and with high histone content (intense blue) was analysed. CMA3 staining was performed according to Lolis et al. (1996). The slides were incubated in Carnoy solution at 40 °C for 5 min and then coated with 100 μl of CMA3 solution (0.25 mg/ml in McIlvaine buffer, pH 7.0, containing 10 mM MgCl2) for 20 min. The slides were rinsed in buffer and left in buffered glycerol. 2
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intact DNA fluoresce green, while sperm with DNA fragmentation in the nucleus show fluorescence from orange to red (Fig. 3). The average percentage of head DNA in comets of blue fox and silver fox spermatozoa stored at 4 °C changed over time after ejaculation (Table 2). In blue foxes it decreased slightly from 99.12% (SEM = 0.038%) immediately after ejaculation to 97.83% (SEM = 0.062%) 72 h after ejaculation. The same downward trend in the mean percentage of head DNA was observed for silver foxes; it fell from 99.37% (SEM = 0.028%) to 98.05% (SEM = 0.045%). Our results show highly significant differences in the percentage of head DNA depending on the time after ejaculation (P < 0.01) within each foxes species. This finding may be associated in part with the small variation in the percentage of head DNA within time-after-ejaculation classes. Importantly, the differences between mean percentages of head DNA were about 1–2 pp during 72 h of storage, which confirms the protective function of the thinners used. It is worth noting that efficient storage of fox semen in a liquid state has practical significance. Additionally, analysis of the percentage of head DNA revealed significant differences (Table 2; P < 0.05) between fox species immediately after ejaculation (0 h). Highly significant differences (P < 0.01) in head DNA were found between species at 24 and 48 h after ejaculation. The mean percentage of head DNA was slightly higher for silver fox than for blue fox at each analysed time after ejaculation. Table 3 presents the percentages of sperm with abnormalities identified using the three staining techniques described above in the two species of foxes. The semen of silver foxes had a lower mean percentage of abnormal sperm when the slides were stained with AB (mean: 0.802%) or CMA3 (mean: 0.974%) than the semen of blue foxes (AB: mean 1.413%, CMA3: mean 1.747%). Analysis of the data shows that the genetic material of the sperm of silver foxes was more stable, in the case of both chromatin and DNA. No DNA fragmentation was identified by acridine orange in the sperm of silver foxes. In blue foxes, the proportion of spermatozoa with abnormal histone retention and impaired protamination was nearly twice as high as in silver foxes. Within each fox species, the mean numbers of sperm with abnormal histone retention (AB) and DNA fragmentation (AO) differed highly significantly (P < 0.01) from the mean number of sperm with impaired protamination (CMA3). There were no significant differences (P > 0.05) in the percentage of abnormal sperm between the two fox species in the case of abnormal histone retention (AB), DNA fragmentation (AO), or impaired protamination (CMA3).
The percentages of spermatozoa with normal chromatin packaging (dull green fluorescence) and with abnormal chromatin packaging (bright green fluorescence) were determined. AO staining was performed according to Tejda et al. (1984). The staining solution was prepared by mixing 10 ml of the stock solution (1 mg AO in 1000 ml of distilled water) and 40 ml of 0.1 M citric acid with 2.5 ml of 0.3 M Na2HPO4. The slides were stained by applying 2–3 ml of the solution for 10 min and then rinsed with distilled water. The wet slides were covered with a cover slip, which was sealed with rubber cement. Microscopic analysis of the slides involved the identification of sperm cells with normal DNA structure (green fluorescence) and with damaged, single-stranded DNA (orange fluorescence). 2.3. Statistics All values were expressed as mean ± standard error of mean (SEM). Data were evaluated for normality and homogeneity of variances. The differences in sperm morphology between blue and silver foxes were analysed using a non-parametric Mann-Whitney U test. The percentages of head DNA between different times (0 h/24 h/48 h/72 h) within species were analysed using the non-parametric Friedman test and post-hoc Nemenyi test. The percentages of sperm with abnormal histone content were compared between staining methods (AB, CMA3 or AO) within species using a non-parametric Friedman test, and posthoc analyses were carried out by the Nemenyi test. All P-values less than 0.05 were considered statistically significant, and values less than 0.01 were statistically highly significant. Statistical procedures were performed using the R software package (R Core Team, 2013). 3. Results Table 1 presents detailed results of the morphological analysis of fox semen. The basic morphological analysis revealed a higher proportion of normal sperm in blue foxes. Analysis of individual sperm defects showed a higher percentage of cytoplasmic droplets, acrosome damage and agglutination in the semen of blue foxes, while the percentage of single bent tails, coiled tails, and head damage was higher in silver foxes. Highly significant differences were found (P < 0.01) between the semen of blue and silver foxes only in the case of head damage. Aniline blue selectively differentiates sperm with normal and abnormal histone retention. Sperm with abnormal, elevated histone content are identified on the slide as navy blue or bright blue, while normal sperm are much lighter than the abnormal ones (Fig. 1). Chromomycin A3 identifies sperm with normal and abnormal protamination. Sperm with abnormal protamination show bright green fluorescence on the dark background of the slide, while spermatozoa with normal chromatin structure are characterized by a light, dull fluorescence (Fig. 2). Acridine orange selectively stains sperm with normal, native DNA structure and with damaged, single-stranded DNA. Sperm with normal,
4. Discussion The comet assay is a sensitive test used to measure damage and repair at the DNA level in individual cells. This test is highly versatile, as it can be used to assess damage in a variety of types of cells and samples, such as cells of the peripheral blood, kidneys, liver, brain, gills, bone marrow, or sperm, as well as cancer cells, solid tumours, bacteria or yeasts (Collins et al., 2001; Awad et al., 2014; Wójcik et al., 2018, Gajski et al., 2019). Initially, this technique was mainly used to study human cells, but it has also been used to assess DNA damage in cells of domestic animals (horses, pigs, cattle, sheep, goats, dogs and cats) and other vertebrates: amphibians, reptiles, birds and fish, including Cyclostomata (Gajski et al., 2019). In our study, the research material was semen from two species of farmed foxes: blue and silver foxes. The SCGE method involves electrophoretic separation of nuclear DNA, which makes it possible to observe its fragmentation. The test cells are immobilized on agarose on microscope slides and then lysed to release the contents of the nucleus. The high ionic strength of the lysis buffer promotes the dissociation of proteins from DNA. Then electrophoresis is performed. The DNA is fluorescently stained with silver salts and the resulting image is analysed under a microscope. If there is damage, the image is reminiscent of a comet. The head of the comet is the cell nucleus, while the tail consists of DNA fragments that have migrated during electrophoresis. The greater the damage, the longer the tail is.
Table 1 Morphological characteristics of semen of blue foxes and silver foxes immediately after ejaculation (0 h). Sperm
Intact Cytoplasmic droplet Single bent tail Coiled tail Acrosome damage Head damage Agglutination
Blue fox Mean
SEM1)
Silver fox Mean
SEM1)
73.93A 5.13A 13.73A 0.40A 1.60A 4.53A 0.53A
2.07 0.97 1.55 0.16 0.21 0.61 0.19
69.50A 3.60A 15.90A 1.20A 1.00A 8.70B 0.10A
2.33 0.72 1.62 0.39 0.33 0.88 0.10
1) SEM – standard error of mean. A, B values within the same row designated with different letters differ highly significantly (P < 0.01). 3
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Fig. 1. Fox sperm stained with aniline blue; A – spermatozoon with elevated histone content; B - normal spermatozoon.
DNA damage can be assessed in several ways, e.g. by measuring the length of the comet tail or by visual assessment of the degree of damage on a scale of 0–4, depending on the appearance of the comet (Fraser et al., 2005; Linfor and Meyers, 2002, Gajski et al., 2019). However, the comet test also reveals other comet parameters, alternatively used to describe the result of this test, such as the percentage of DNA damage, percentage of DNA in the comet head, percentage of DNA in the tail, and tail moment (TM), which is the product of tail length and the percentage of DNA in the tail (Gajski et al., 2019). In our study, the selected parameter was head size (% head DNA), which ranged from 99.12% (SEM = 0.038%) to 97.83% (SEM = 0.062%) in blue foxes and from 99.37% (SEM = 0.028%) to 98.05% (SEM = 0.045%) in silver foxes. Niedbała et al. (2015) also chose the percentage of DNA in the comet head as a test parameter in chinchilla. They tested four different semen extenders commonly used for storing the sperm of farmed chinchilla for insemination. They showed that the best cryoprotectant, in the L4 extender, can protect the integrity of sperm DNA in chilled semen (even after 72 h), with 98.56% of DNA retained in the head of the comet. There are many papers describing the use of the comet test on the semen of farmed animals, but due to the variable factors affecting the sperm, numerical comparison with our data is difficult. The usefulness of the SCGE test for assessment of sperm should be stressed. Studies of sperm chromatin proteins in relation to fertility help to understand the importance of normal chromatin structure for sperm functions. Unfortunately, assessment of protamination to evaluate male fertility is primarily carried out in human material, while in the case of animals, this type of research is scarce and usually limited to laboratory
The measure of the level of DNA damage is the length of the tail and the amount of DNA contained in it. A reliable result is obtained by analysing 50–100 comets in gel (Końca et al., 2003; Singh et al., 2003). In our study, evaluation of the semen of farmed foxes was based on analysis of 50 cells per individual. The comet assay is most commonly carried out under alkaline conditions, but there is also a neutral form of the assay (Nandhakumar et al., 2011). The differences are fundamental, because the alkaline form of the SCGE test can detect both single- and doublestranded DNA damage. In our study, we used the alkaline method. An unquestionable advantage of the method is that several different DNA modifications can be analysed with relatively small changes in the basic procedure. The method identifies single-stranded and double-stranded DNA breaks, as well as all types of chemical modifications (e.g. apurinic sites and unstable adducts) and enzymatic modifications (oxidative damage) that can potentially become breaks. The comet assay makes it possible to detect DNA damage at the level of a single cell and to determine susceptibility to a specific genotoxic factor, as well as the efficiency of damage repair (Ashby et al., 1995; Singh et al., 1988; Slyskova et al. 2007, 2014; Azqueta et al. 2014). The comet test has mainly been used in medicine, pharmacy and biomonitoring. In domestic animals, the comet test is performed to assess the effect of feed toxins or anaesthesia on DNA integrity, or to test for complications after certain infections (Strasser et al., 2012; Hornatovich et al., 2013; Radakovic et al., 2016). In addition, the test has been used to test the semen of breeders to test the quality of sperm after cryopreservation.
Fig. 2. Fox sperm stained with chromomycin A3; A - spermatozoon with abnormal protamination; B - normal spermatozoon. 4
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Fig. 3. Fox sperm stained with acridine orange; A - spermatozoon with DNA fragmentation; B - normal spermatozoon.
experiments, without practical application. The role of basic proteins stabilizing the sperm DNA structure is increasingly emphasized in studies dealing with human and animal reproduction. In some cases of infertility, the direct cause is abnormalities in spermiogenesis associated with the replacement of histone proteins. Assessment of chromatin organization in sperm indicates the normality not only of spermatogenesis, but also of the paternal genome and epigenome (Martianov et al., 2005; Enciso et al., 2011). In early, round spermatids the number of histones replaced by their transitional variants exceeds 50%. Nevertheless, the classic nucleosome structure and transcriptional activity are still preserved until histone H1t and the majority of histones are dissociated and transition proteins (TP) appear (Hammoud et al., 2009). Following this chromatin reorganization, about 10–15% of histones remain in the mature sperm (Kimmins and Saccone-Corsi, 2005). The presence of histones allows the nucleosome structure to be maintained. Hammoud et al. showed that this applies to 4% of the haploid genome (Hammoud et al., 2009). The normal level of histones in chromatin is species-specific. Studies in boars and mice have found only about 1% histones in sperm chromatin, while the level in humans can be as high as 15% (Tanphaichitr et al., 1978; Gatewood et al., 1990; Bench et al., 1996). There are no data on the physiological level of histones in canine sperm. Increased histone levels have a negative effect on sperm maturation, fertilization, and early embryonic development (Jenkins et al., 2011). Elevated histone levels have been found in infertile individuals or those with reduced fertility diagnosed with oligospermia, teratozoospermia or asthenozoospermia (Zhang et al., 2006; Zini et al., 2007). Research by Kazerooni et al. (2009) on human semen has shown that in couples with recurrent miscarriages, there is an increased percentage of cells positively stained with aniline blue. Hammadeh et al. (2001) have demonstrated that positive AB staining was significantly more common in a group of patients with fertility disorders than in the healthy control
Table 2 The percentage of head DNA in comets of blue fox and silver fox spermatozoa stored at 4 °C. Time (h)
0 24 48 72
P-values2)
Blue fox Mean
SEM1)
Silver fox Mean
SEM1)
99.12A 98.84B 98.30C 97.83D
0.038 0.039 0.050 0.062
99.37A 99.12B 98.72C 98.05D
0.028 0.036 0.042 0.045
0.0324 0.0003 <0.001 0.1445
1) SEM – standard error of mean. 2) P-values indicate differences between blue foxes and silver foxes at one time (0 h/24 h/48 h/72 h). A,B,C,D values within the same column designated with different letters differ highly significantly (P < 0.01). Table 3 Effect of staining method on percentage of sperm with abnormal histone content, by species. Staining method1)
AB CMA3 AO
P-values3)
Blue fox Mean
SEM2)
Silver fox Mean
SEM2)
1.413A 1.747B 0.027A
0.322 0.370 0.027
0.802A i 0.000A
0.197 0.229 0.000
0.1762 0.1660 0.2733
1) AB – abnormal histone retention, CMA3 – impaired protamination, AO – DNA fragmentation. 2) SEM – standard error of mean. 3) P-values indicate differences between blue foxes and silver foxes within a staining method. A,B values within the same column designated with different letters differ highly significantly (P < 0.01).
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protamines, lack of protamines, maturation in the epididymis, and chromatin stability. Kazerooni et al. (2009) have observed that the number of sperm positively stained with CMA3 and AB is higher in individuals with impaired fertility. In addition, sperm viability and abnormal morphology have been found to be correlated with the percentage of spermatozoa positively stained with CMA3 and AB. This shows that abnormal chromatin packing has a destructive effect on semen parameters. This conclusion is in line with those presented by other authors (Coetzee et al., 2001; Hammadeh et al., 1998). Acridine orange staining is a simple and rapid test to assess DNA damage in sperm. When bound to double-stranded DNA it fluoresces green, while bound to RNA and single-strand DNA it fluoresces red. Evenson et al. (1980) found that spermatozoa containing denatured, single-stranded DNA reduce fertilization rates and the quality of embryos obtained in vitro. Although acridine orange has been used to study sperm of various species, the level of pathological DNA fragmentation has been determined only for humans. Up to 15% sperm with damaged DNA is considered normal, 15–25% is believed to reduce fertility, and a level above 25% indicates a high risk of infertility (Evenson et al., 1999, 2000). In bulls, a reduction in fertility has been observed when the percentage of damaged sperm exceeded 10% (Bochenek et al., 2001). Reference values have not been determined for other species Analysis of disturbances in the protamination process should complement morphological and molecular evaluation of semen. Protamination is considered one of the important parameters of male fertility (Aoki et al., 2006; Zini et al., 2007). Unfortunately, assessment of protamination is carried out primarily in human material. Research on nuclear proteins with respect to infertility demonstrates the importance of normal chromatin structure for sperm function. A more detailed understanding of the complex structure of sperm chromatin is essential for the development of new, more versatile and more accurate diagnostic tools.
population. These studies also showed that embryo mortality was correlated with increased histone content in the spermatozoa (Hammadeh et al., 2001; Kazerooni et al., 2009). Immature sperm or sperm with DNA disorders usually contain histone residues (Auger, 2010; Schulte et al., 2010; Jenkins et al., 2011). Further research is therefore necessary to determine how best to use the aniline blue staining technique. Studies on histones and their potential involvement in early embryonic development are needed to understand the epigenetic factors of male infertility. Numerous researchers are carrying out experiments to establish species reference values. Early abortion and embryonic mortality have been shown to correlate with increased histone content in spermatozoa (Hammadeh et al., 2001; Kazerooni et al., 2009). Higher histone levels have been found in infertile individuals or those with oligospermia – reduced mature sperm count, teratozoospermia – abnormal structure, and asthenozoospermia – impaired sperm motility (Zhang et al., 2006; Zini et al., 2007). In the case of animal sperm, this type of research is scarce and most often limited to laboratory experiments, without practical application. Studies of these unique proteins and their potential involvement in early embryonic development are needed to understand the epigenetic factors of male infertility. Protamines are proteins rich in arginine and cysteine. They form an extremely condensed and transcriptionally silenced conformation of sperm chromatin (Vilfan et al., 2004). In the mammalian genome, incorporation of protamines P1 and P2 is strictly regulated during spermatogenesis. Not only impaired protamination, but also the P1 to P2 ratio is a factor affecting fertility. Abnormalities of both the protamination process and the P1 to P2 ratio have been found in individuals whose semen morphology assessment shows low sperm motility or low sperm concentration. Protamination is indispensable to normal sperm chromatin condensation, which in turn affects sperm function (Aoki et al. 2006). The few published results of animal research evaluating histone retention and impaired protamination have dealt with the effect of age on the structure of sperm chromatin and its relationship to the physical characteristics of the ejaculates of breeding boars (Banaszewska et al. 2015a), and the role of sperm chromatin structure in reproduction in cocks (Banaszewska et al. 2015a). Studies in boars have found a relationship between age and sperm count, with increased histone content and impaired protamination in younger individuals. The percentage of spermatozoa with disturbed chromatin structure was highest in boars under two years old. Unfortunately, the effect of ageing on sperm chromatin quality has only been carried out in humans (Sharma et al., 2015). Animals used for insemination are often young individuals whose sperm production capacity is an indicator for reproduction. In such animals, including boars, the spermiogenesis process may not be fully functional, which results in the presence of spermatozoa with an unsatisfactory chromatin structure in their semen. This leads to an increased presence of spermatozoa with histones that have not been fully replaced and abnormally incorporated protamines, and thus to reduced reproduction (Banaszewska and Kondracki, 2012; Banaszewska et al., 2015a). Abnormal protamination in breeding cocks did not exceed a few percent of the reproductive cells analysed. However, comparison of the two commercial lines showed that the F15 line had better semen quality, motility, and reproductive parameters than the Flex line (Banaszewska et al., 2015b). Sperm staining with aniline blue (AB) and chromomycin A3 (CMA3) is a rapid test of chromatin quality in sperm cells. Aniline blue is a specific dye for proteins with high lysine content. This includes histones, which are replaced by protamines during spermiogenesis. Aniline staining reveals abnormal, excessive histone content in the sperm cell. Such sperm are more susceptible to DNA damage and impaired chromatin stability. Studies have shown early abortion and foetal mortality to be correlated with elevated histone content in sperm (Hammadeh et al., 2001; Kazerooni et al., 2009). Abnormalities in sperm chromatin can occur on several levels: histone replacement with
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