**3.5. Assessment of DNA integrity**

stressor (incubation at 100ºC), which produce a pink-coloured chromogen and is readable at a wavelength of 532 nm. Also, the fluorescent probe BODIPY581/591-C11 (4,4-difluoro-5-(4 phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid) is frequently used in association with flow cytometry to assess LPO in the sperm. BODIPY is a fatty acids sen‐ sitive fluorescent probe that changes fluorescence from red to green in the presence of lipid peroxidation. Its association with a vital probe further allows to evaluate the fluorescence

Additional, currently used methods also include the glutathione peroxidase reaction (where the hydrogen peroxide oxidizes GSH (reduced glutathione) into GSSG (oxidized glutathione) in the presence of glutathione reductase and NADPH results from the con‐ sumption of NADPH in proportion to the peroxide content), by flow cytometry measure‐ ment of the fluorescent intensity of the compounds oxidized by ROS (such as the dichlorofluorescin diacetate- DCFH-DA- or the Hydroethidine- HE), using the gas-liquid chromatography separation of lipid peroxides, followed by its identification by mass spectrometry and by measuring cytotoxic aldehydes through high performance liquid

ROS production can be directly monitored by a luminol or a lucigenin-based chemillumi‐ nescence assay [43,45]. This assay does not distinguish between intracellular and extracel‐ lular ROS, but it differentiates between the production of superoxide and hydrogen peroxide according to the probe used (lucigenin and luminol, respectively for superoxide and hydrogen peroxide). Measurement of chemilluminescence is proportional to ROS ac‐

An important side effect of the oxidative stress is apoptosis [42]. The most important changes associated to sperm apoptosis are the externalization of the phosphatidylserine (PS), a molecule usually confined to the inner leaflet of the plasma membrane, the caspase system activation, the DNA fragmentation, the lost of mitochondrial integrity and the in‐ crease of cell membrane permeability [41]. To assess sperm apoptosis it is frequently used the Annexin V, a Ca2+-dependent PS-binding protein that reacts to the PS, which is translo‐ cated to the outer leaflet of the plasma membrane in damaged sperm. Annexin V can be con‐ jugated to fluorochromes such as FITC (Fluorescein isothiocyanate) in flow cytometry analysis. If a vital staining is used, such as the propidium iodide, the combination allows to distinguish between three sperm sub-populations: viable (Annexin-FITC-PI-negative), early apoptotic (Annexin-FITC-positive and PI-negative) and late apoptotic (Annexin-FITC-PI-

Caspases are molecules associated with the apoptotic pathway and can be classified as ini‐ tiators or executors; caspase 7 and 9 are initiators, while active caspase 3 is an executor. The determination of the caspase enzymatic activity in sperm extracts, in comparison to the one of neutrophils, can also be used to assess apoptosis in sperm, which may be completed by the semiquantitative determination of active caspase 3 and caspase 7 content, by Western blotting. Caspase activity has been shown to be consistently higher in low motility sperm, in

emission ratio in living cells [44,46].

106 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

chromatography (HPLC) [44,45].

cumulation [45].

positive) [7,41].

particular, the active caspase 3 [47].

An association between infertility and the integrity of DNA content in sperm has been sug‐ gested. The integrity of male DNA is of utmost importance for embryo development and offspring production [13,41]. DNA damage is not usually perceived under classic or ad‐ vanced semen assessment, but has been proposed to be at the origin of infertility in normo‐ spermic individuals. DNA damage (abnormal chromatin structure) may arise from different processes: deficient recombination or packaging during spermatogenesis, apoptosis and oxi‐ dative stress. DNA loss of integrity does not always impair fertilization, but compromises sustainable embryo development, predisposing to embryo losses and abortion [9,15]. DNA fragmentation may be associated with various pathological and environmental conditions [51,52], but also with endogenous mechanisms such as the oxidative stress and apoptosis.

Evaluation of sperm DNA integrity can be achieved by a variety of tests covering different aspects of the DNA damage. Unfortunately, most of the available techniques provide limit‐ ed information regarding the nature of the DNA lesions evidenced, and do not allow to highlight the exact pathogenesis of disrupted sperm DNA [53,54].

fragmentation index - DFI) has been associated to male infertility [13,43,53]. It is possible to score different spermatozoa populations by using SCSA: the sperm without fragmented

The sperm chromatin dispersion test (SCD) is a method based on the principle that sperm with fragmented DNA fail to produce a halo, which is characteristically observed in sperm with non-fragmented DNA, when mixed in aqueous, low melting agarose followed by acid denaturation and removal of nuclear proteins [21,54]. Despite not being necessary, this test can be visualised using a fluorescent dye (such as propidium iodide, DAPI or ethidium bro‐ mide) or simply be stained with Diff-Quick® reagent. Halosperm® is a commercial kit to as‐ sess DNA fragmentation in sperm from different species, before or after semen manipulation. Regarding this kit, sperm presenting a large- and medium-sized halo is con‐ sidered to have no fragmentation, while spermatozoa having a small halo or without halo is

**Assay Parameter Principle Detection method**

Cells with labelled DNA (%) Microscopy (bright or

% Cells with small or no halo Microscopy (bright or

% cells with migration tails (fragmented DNA) and also the length of the tail (% DNA

DFI (%) = cells with red fluorescence divided by the total of cells (red+green).

in the tail)

**Table 1.** Comparison of available methods for assessment of DNA fragmentation is spermatozoa (Adapted from [56]).

fluorescence) Flow cytometry

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 109

Fluorescence microscopy

Flow cytometry

fluorescence)

DNA, the sperm with moderate DFI and the sperm with high DFI.

classified as having DNA fragmentation (Figure 7) [55].

with deoxynucleotidyl transferase to SS

Alkaline conditions denature DNA and reveals SS and DS DNA breaks Neutral conditions reveal mostly DS

TUNEL Addition of labeled dUTP nucleotides

and DS DNA breaks Template independent

Comet Fragmented DNA in sperm cells is

SCSA Mild acid treatment denaturates and

SCD Mild acid denaturation of DNA and lysis

lyses DNA with SS or DS breaks Acridine orange differentially emits fluorescence with DS DNA (Green) or SS

of protamines induce a decondensation halo around sperm head if DNA is intact, and no halo is observed if DNA is

(SS- Single-stranded; DS- Double-stranded; DFI-DNA fragmentation index)

breaks

DNA (Red)

damaged

detected by eletrophoresis

Less expensive methods to assess the sperm chromatin structure uses chromatin structural probes or dyes, such as the acridine orange (measures the susceptibility to conformational changes), the aniline blue (that stains loosely condensed chromatin), chromomycin α (com‐ peting with protamine binding to DNA, it reveals protamination defects on sperm) and the toluidine blue (that stains phosphate residues of fragmented DNA). However, several fac‐ tors modulate the DNA staining of chromatin, decreasing their specificity [52].

Nowadays, the most currently used tests of sperm DNA fragmentation are: the Comet assay (single cell gel electrophoresis), the TUNEL (terminal deoxynucleotidyl transferase-mediat‐ ed dUTP (2´-deoxyuridine, 5´-triphosphate) nick end labelling) assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test. The first three assays focus on the DNA fragmentation detection, while the last assay is a sperm nuclear matrix assay detecting possible deficient DNA repair or chromatin disorganization [43]. On table 1 we compare these methods.

The Comet assay is a fluorescence microscopic test that identifies single (SS) and doublestranded (DS) DNA in single sperm. In this assay, sperm cells are mixed with low-to-moder‐ ate melting agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis, the DNA being visualized with the aid of a fluorochrome dye. DNA damage is quantified by measuring the displacement between the genetic material of the comet nucleus (unbroken DNA) and the resulting tail (damaged DNA) [21,52,53]. The length of the tail is positively correlated with the percentage of DNA fragmentation. Al‐ though highly sensitive, this method is also labour intensive and the comet tail is of difficult standardization. Further, less apparent clinical association exists between the test results and clinical infertility [43], and clinical thresholds were yet to be established.

TUNEL assay is possibly the most common method used to assess sperm damage in sperm. It can be used as another ICC method, in both bright field and fluorescence microscopy, or associated with flow cytometry. In the TUNEL assay, terminal deoxynucleotidyl transferase (TdT) incorporates labelled nucleotides into 3′-OH at single- and double-strand DNA breaks, creating a signal of increasing intensity according to the number of DNA breaks. The fluorescence intensity of each analysed sperm is scored as a "positive" or "negative" on a microscope slide. When conjoined with a flow cytometer, precision of the method increases due to the increased number of cells analysed [43,53]. Proportion of TUNEL positive cells seems to be correlated with decreased pregnancy rates [13]. However, numerous variations for the test exist, which reduces its liability.

The sperm chromatin structure assay measures *in situ* DNA susceptibility to acid-induced DNA denaturation. It uses a flow cytometer and the acridine orange fluorescence, a tradi‐ tional fluorescent dye that shows different colour when bonded to single- (red) or doublestranded (green) DNA [43]. The degree of red fluorescence in a sample (named DNA fragmentation index - DFI) has been associated to male infertility [13,43,53]. It is possible to score different spermatozoa populations by using SCSA: the sperm without fragmented DNA, the sperm with moderate DFI and the sperm with high DFI.

Evaluation of sperm DNA integrity can be achieved by a variety of tests covering different aspects of the DNA damage. Unfortunately, most of the available techniques provide limit‐ ed information regarding the nature of the DNA lesions evidenced, and do not allow to

Less expensive methods to assess the sperm chromatin structure uses chromatin structural probes or dyes, such as the acridine orange (measures the susceptibility to conformational changes), the aniline blue (that stains loosely condensed chromatin), chromomycin α (com‐ peting with protamine binding to DNA, it reveals protamination defects on sperm) and the toluidine blue (that stains phosphate residues of fragmented DNA). However, several fac‐

Nowadays, the most currently used tests of sperm DNA fragmentation are: the Comet assay (single cell gel electrophoresis), the TUNEL (terminal deoxynucleotidyl transferase-mediat‐ ed dUTP (2´-deoxyuridine, 5´-triphosphate) nick end labelling) assay, the sperm chromatin structure assay (SCSA) and the sperm chromatin dispersion (SCD) test. The first three assays focus on the DNA fragmentation detection, while the last assay is a sperm nuclear matrix assay detecting possible deficient DNA repair or chromatin disorganization [43]. On table 1

The Comet assay is a fluorescence microscopic test that identifies single (SS) and doublestranded (DS) DNA in single sperm. In this assay, sperm cells are mixed with low-to-moder‐ ate melting agarose and then placed on a glass slide. The cells are lysed and then subjected to horizontal electrophoresis, the DNA being visualized with the aid of a fluorochrome dye. DNA damage is quantified by measuring the displacement between the genetic material of the comet nucleus (unbroken DNA) and the resulting tail (damaged DNA) [21,52,53]. The length of the tail is positively correlated with the percentage of DNA fragmentation. Al‐ though highly sensitive, this method is also labour intensive and the comet tail is of difficult standardization. Further, less apparent clinical association exists between the test results

TUNEL assay is possibly the most common method used to assess sperm damage in sperm. It can be used as another ICC method, in both bright field and fluorescence microscopy, or associated with flow cytometry. In the TUNEL assay, terminal deoxynucleotidyl transferase (TdT) incorporates labelled nucleotides into 3′-OH at single- and double-strand DNA breaks, creating a signal of increasing intensity according to the number of DNA breaks. The fluorescence intensity of each analysed sperm is scored as a "positive" or "negative" on a microscope slide. When conjoined with a flow cytometer, precision of the method increases due to the increased number of cells analysed [43,53]. Proportion of TUNEL positive cells seems to be correlated with decreased pregnancy rates [13]. However, numerous variations

The sperm chromatin structure assay measures *in situ* DNA susceptibility to acid-induced DNA denaturation. It uses a flow cytometer and the acridine orange fluorescence, a tradi‐ tional fluorescent dye that shows different colour when bonded to single- (red) or doublestranded (green) DNA [43]. The degree of red fluorescence in a sample (named DNA

tors modulate the DNA staining of chromatin, decreasing their specificity [52].

and clinical infertility [43], and clinical thresholds were yet to be established.

highlight the exact pathogenesis of disrupted sperm DNA [53,54].

108 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

we compare these methods.

for the test exist, which reduces its liability.

The sperm chromatin dispersion test (SCD) is a method based on the principle that sperm with fragmented DNA fail to produce a halo, which is characteristically observed in sperm with non-fragmented DNA, when mixed in aqueous, low melting agarose followed by acid denaturation and removal of nuclear proteins [21,54]. Despite not being necessary, this test can be visualised using a fluorescent dye (such as propidium iodide, DAPI or ethidium bro‐ mide) or simply be stained with Diff-Quick® reagent. Halosperm® is a commercial kit to as‐ sess DNA fragmentation in sperm from different species, before or after semen manipulation. Regarding this kit, sperm presenting a large- and medium-sized halo is con‐ sidered to have no fragmentation, while spermatozoa having a small halo or without halo is classified as having DNA fragmentation (Figure 7) [55].


**Table 1.** Comparison of available methods for assessment of DNA fragmentation is spermatozoa (Adapted from [56]). (SS- Single-stranded; DS- Double-stranded; DFI-DNA fragmentation index)

tion. Nevertheless, putative molecular markers that may be used for sperm quality assess‐ ment were not exhausted in this review. Further efforts must be focused on understanding how these biomarkers correlate with transient impairments of male infertility caused by heat stress, malnutrition, diseases or trauma. Finally, the adjunctive evaluation of spermato‐

This work was supported by the project from CECAV/UTAD with the reference PEst-

1 CECAV [Veterinary and Animal Research Centre] – University of Trás-os-Montes and

2 CERCA (Centre d'Études en Reproduction des Carnivores), Animal Reproduction, National

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[2] Sutovsky P, Lovercamp K. Molecular markers of sperm quality. Soc Reprod Fertil

[3] Dyck MK, Foxcroft GR, Novak S, Ruiz-Sanchez A, Patterson J, Dixon WT. Biological

[4] Mortimer ST. A critical review of the physiological importance and analysis of sperm

[5] Payan-Carreira R, Miranda S and Nizanski W. Artificial Insemination in Dogs. In: Artificial Insemination in Farm Animals. Milad Manafi (Ed.), 2011. ISBN: 978-953-307-312-5, InTech, Available from: http://www.intechopen.com/books/artifi‐

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cial-insemination-in-farm-animals/artificial-insemination-in-dogs

and Alain Fontbonne2

Molecular Markers in Sperm Analysis http://dx.doi.org/10.5772/52231 111

OE/AGR/UI0772/2011, by the Portuguese Science and Technology Foundation.

Rita Payan-Carreira1\*, Paulo Borges1,2, Fernando Mir2

\*Address all correspondence to: rtpayan@gmail.com

Veterinary School of Alfort, Paris-East University, France

zoa functions is particular important when considering sperm storage.

**Acknowledgments**

**Author details**

**References**

Alto Douro, Vila Real, Portugal

Suppl. 2010, 67: 247-56.

**Figure 7.** Image of the Halosperm® test for DNA fragmentation in horses and dogs. The existence of a large halo is indicative of DNA integrity (Adapted from [56]).
