**3.2.7 DNA status of spermatozoa**

DNA integrity has been considered as an important parameter in the determination of spermatozoa ability to withstand the cryopreservation process. It is suggested that chromatin structure should be studied as an independent complementary parameter for the better assessment of the sperm quality (Evenson et al., 2002). The spermatozoal chromatin is much more compact when compared to somatic and spermatogenic cell types (e.g., spermatognia, spermatocytes and spermatids). It appears that during freezing-thawing procedure the integrity of the nuclear DNA, which is related to fertility, could be negatively

progressively damaged (Martin et al., 1995). When the cell membrane is disturbed, the phospholipid PS is translocated from the inner to the outer leaflet of the plasma membrane

It is widely known that the cryopreservation usually causes sublethal cryodamage to spermatozoa, decreasing post-thaw cell viability. The freezing-thawing of human (Glander & Schaller, 1999), bull (Martin et al., 2004), and boar (Pena et al., 2003), stallion (Ortega Ferrusola et al., 2008), and dog (Kim et al., 2010) spermatozoa induces membrane PS translocation, what demonstrates that cryopreservation leads to apoptosis. Therefore, detecting early phases of membrane dysfunction, or initial phases of apoptosis of viable spermatozoa, would be important when evaluating stressed spermatozoa, such as those subjected to freezing and thawing, and would be useful for controlling freezing procedures

Annexin V is calcium-dependent phosphatidylserine (PS) binding protein conjugated with fluorochrome – FITC or Alexa Fluor®. The properties of Annexin V allow for detection of externally exposed PS. In ejaculated spermatozoa PS is confined to the cytoplasmatic side of the plasma membrane (Gadella et al., 1999). Different categories of apoptotic, necrotic and viable cells can then be sorted out using AnnexinV with PI, through flow cytometer (Fig. 6e),

After induction of apoptosis, mitochondrial pores are being opened, leading to a decrease in mitochondrial membrane potential. Therefore, described above JC-1 dye is used for monitoring of apoptotic changes in spermatozoa, too (Ortega Ferrusola et al., 2009). Mentioned above, the opening of mitochondrial pores causes the release of proapoptotic factors into the cytoplasm, where they are activated. These factors – caspases, are central components in the apoptosis signaling cascade. The detection of activated caspases in living spermatozoa can be performed using fluorescence labeled inhibitors of caspases (FLICATM). It allows investigating caspase activation in semen samples with regard to a single cell. The FLICATM reagent is comprised of 3 segments—it includes a green (FAM 5 carboxyfluorescein) fluorescent label; an amino acid peptide inhibitor sequence targeted by the active caspase; and a fluoromethylketone group (FMK), which acts as a leaving group and forms a covalent bond with the active enzyme. Fluorescence labeled inhibitors of caspases are cell permeable and noncytotoxic (cited by Grunewald et al., 2009). Martin et al. (2004) showed that cryopreservation of bovine spermatozoa induced the significant increase in the proportion of cells with active caspases, which were mainly detected in the

DNA integrity has been considered as an important parameter in the determination of spermatozoa ability to withstand the cryopreservation process. It is suggested that chromatin structure should be studied as an independent complementary parameter for the better assessment of the sperm quality (Evenson et al., 2002). The spermatozoal chromatin is much more compact when compared to somatic and spermatogenic cell types (e.g., spermatognia, spermatocytes and spermatids). It appears that during freezing-thawing procedure the integrity of the nuclear DNA, which is related to fertility, could be negatively

(Desagher & Martinou, 2000).

or visually evaluated using fluorescent microscope.

intermediate piece of spermatozoa.

**3.2.7 DNA status of spermatozoa** 

in semen.

affected. Although, spermatozoa with DNA damage may be able to fertilize an oocyte, that could potentially disturb (epi)genetic regulation of the early embryo and block its further development (Lewis & Aitken, 2005).

DNA damage can be evaluated at different levels. One of the usually used methods, developed for detecting changes in the chromatin structure of DNA integrity, is the sperm chromatin structure assay (SCSA) (Chohan et al., 2006). The SCSA is a flow cytometric method for identification of changes in the DNA status. It is based on the assumption that a structurally abnormal sperm chromatin shows a higher susceptibility to acid denaturation (Evenson et al., 2002). The SCSA method utilizes the metachromatic properties of acridine orange (AO). This stain fluoresces in the green band when intercalates into the intact double-stranded DNA helix, and in the red band when associated with single strand denaturated DNA and RNA. After denaturation of chromatin by decreased pH, the spermatozoa with structurally abnormal chromatin fluorescence is detected in the red band (Fig. 6f) (Bochenek et al., 2001). The fertility data have been shown to correlate with the results obtained from the SCSA of human (Evenson et al., 1980), bull (Ballachey et al., 1988; Karabinus et al., 1990), stallion (Love & Kenney, 1998) and boar semen (Evenson et al., 1994). SCSA was also used for dog semen assessment (Garcia-Macias et al, 2006) and for evaluation of freezing-thawing effect on chicken and goose DNA status (Partyka et al. 2010; Partyka et al., 2011b).

Another method to detect DNA defragmentation is TUNEL assay, which allows to incorporate of fluorescent nucleotide analogs by a terminal nucleotide transferase into single stranded DNA areas at the 3-OH termini (Chohan et al., 2006). Ramos & Wetzels (2001) using this method have shown that DNA damage is limited in functional human spermatozoa resulting from a swim-up procedure.

The alternative method for detecting the DNA damage at the level of individual cells is the single-cell DNA gel electrophoresis assay (COMET). Although this method does not use such equipment as flow cytometry, application of fluorescent DNA specific stain is required. In COMET assay spermatozoa are spread on a surface covered with an agarose gel, and treated with a solution that lyses the cell components leaving the DNA immobilized in the agarose. They are then subjected to a DNA denaturation process, followed by electrophoresis, causing DNA fragments to migrate away from the main bulk of nuclear DNA. After staining with propidium iodide or ethidium bromide, cells with DNA strand breaks, display a comet-like shape, with the undamaged DNA located in the head of the comet and the fragmented DNA dispersed through the tail. Image analyses provide information on the extent of strand breaks in the DNA molecule. Several studies, conducted with different techniques, including comet assay, showed a negative relationship between the fertilization potential of spermatozoa and alterations at the level of genetic material. In particular in humans, infertility has been associated with higher levels of DNA damage in sperm compared to fertile subjects (Irvine et al., 2000). Fraser & Strzeżek (2007) have shown that the freezing–thawing process provoked sperm chromatin destabilization rendering the boar spermatozoa more vulnerable to DNA fragmentation. COMET assay has also been recently used for the evaluation of cryopreserved avian semen (Madeddu et al., 2010; Gliozzi et al., 2011).

Methods of Assessment of Cryopreserved Semen 563

moribund sperm (red/green fluorescence), viable sperm (green stained), unstained debris are discarded; b) Dot plot of PNA-AlexaFluor/PI stain. Spermatozoa can be identified as: acrosome - intact/damaged, together with selection - viable/dead, according to their green and red fluorescence; c) Dot plot of JC-1 staining for mitochondrial status analysis. The intensity of orange fluorescence depends on mitochondrial membrane potential (ΔΨm) allowing for differentiation between high, medium and low ΔΨm; d) Dot plot of C11 BODIPY581/591/PI for assessment of lipid peroxidation (LPO). Spermatozoa can be divided into four subpopulations: dead without LPO, dead with LPO, live without LPO and live with LPO; e) Dot plot of Annexin V/PI stain. Spermatozoa can be identified as: viable, necrotic and apoptotic; f) Dot plot of SCSA using acridine orange. The distribution of spermatozoa is based on green (FL1) and red (FL3) fluorescence. Main population includes

sperm without DNA fragmentation, %DFI represents the percentage of sperm with detectable DNA fragmentation and % HDS determines the percentage of immature cells.

During fertilization, a sperm initially binds to the oocyte zona pellucida (ZP), undergoes the acrosome reaction (AR), penetrates the ZP, and fuses with the oolemma to form a zygote. Sperm-ZP interactions are carbohydrate-mediated events in various species, including humans (Benoff, 1997). The ZP of mammalian oocytes is a critical site for sperm-oocyte interaction. The ability of sperm to bind to the ZP indicates many functions of spermatozoa, such as viability, motility, morphology, acrosomal status and the ability to penetrate the oocyte (Liu & Baker, 1994), and for that reason this ability is of a diagnostic relevance.

The assessment of the ability of sperm cells to bind the homologous zona pellucida (ZP) is the useful test for prediction of spermatozoal fertilizing ability (Hermansson et al., 2006). It is assumed that it is reliable test to detect sperm damage at a molecular level, which is not visible by microscopic analysis, because binding is receptor-ligand mediated reaction. The test may be done in two ways: by using intact homologous oocytes (ZP-binding assay, ZBA) and by using bisected hemizonae (hemizona binding assay, HZA) (Kawakami et al., 1998; Rijsselaere et al., 2005). In ZBA spermatozoa are coincubated with oocytes obtained from sliced ovaries. The number of spermatozoa that bound to ZP is counted with contrast-phase microscopy. The disadvantage of ZBA is the fact that the attachment of sperm cells to zona depends on the oocyte. This feature was partly overcome in HZA. Bisected by micromanipulation two parts of ZP are coincubated with spermatozoa. As a result the direct comparison of sperm cells from two origins may be done (Ivanova et al., 1999; Mayenco-

A sublethal damage that occurres during cryopreservation leads to loss of sperm surface proteins, segregation of membrane proteins, inactivation of membrane-bound enzymes and decreased lateral protein diffusion within the membrane (Watson, 1995). Kadirvel et al. (2011) observed significant reduction of the zona binding ability of cryocapacitated buffalo bulls spermatozoa, and further reduction of binding ability of frozen-thawed spermatozoa, after incubation, in either capacitating, or non capacitating medium. Similar results have been obtained in bulls (Fazeli et al., 1997) and humans (Amann et al., 1999) spermatozoa, with significantly reduced binding ability to the zona pellucida after freezing and thawing.

**3.3 In vitro gamete interaction tests** 

**3.3.1 Zona pellucida binding assay** 

Aguirre & Pérez Cortés, 1998).

Fig. 6. Examples of flow cytometry analyses of frozen-thawed spermatozoa: a) Dot plot of SYBR-14/PI stain. Four subpopulations can be distinguished: dead sperm (red stained),

Fig. 6. Examples of flow cytometry analyses of frozen-thawed spermatozoa: a) Dot plot of SYBR-14/PI stain. Four subpopulations can be distinguished: dead sperm (red stained),

moribund sperm (red/green fluorescence), viable sperm (green stained), unstained debris are discarded; b) Dot plot of PNA-AlexaFluor/PI stain. Spermatozoa can be identified as: acrosome - intact/damaged, together with selection - viable/dead, according to their green and red fluorescence; c) Dot plot of JC-1 staining for mitochondrial status analysis. The intensity of orange fluorescence depends on mitochondrial membrane potential (ΔΨm) allowing for differentiation between high, medium and low ΔΨm; d) Dot plot of C11 BODIPY581/591/PI for assessment of lipid peroxidation (LPO). Spermatozoa can be divided into four subpopulations: dead without LPO, dead with LPO, live without LPO and live with LPO; e) Dot plot of Annexin V/PI stain. Spermatozoa can be identified as: viable, necrotic and apoptotic; f) Dot plot of SCSA using acridine orange. The distribution of spermatozoa is based on green (FL1) and red (FL3) fluorescence. Main population includes sperm without DNA fragmentation, %DFI represents the percentage of sperm with detectable DNA fragmentation and % HDS determines the percentage of immature cells.
