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Flow Cytometry for the Evaluation of Semen

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State of the Art in Sperm Assessment Using Flow Cytometry

Abstract

Flow cytometry is emerging as a substantial tool in the domain of modern andrology for the routine analysis of spermatozoa. Recent application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, analysis of semen samples was routinely performed by microscopical evaluation and manual techniques by laboratory operators; analysis is inclined due to comprehensive variability among observers, influencing its clinical validity. During last decade, to evaluate farm animal semen, variety of new flow cytometric techniques have been intercalated which made possible a wide spread evaluation of several sperm functionality and characteristics. Here in this paper, an initiative has been taken to explore numerous current flow cytometry developments pressing for andrological tests.

After the invention of flow cytometry, sperm evaluation by traditional (microscopic) means became questioned and avoided due to the robust advantages of flow cytometry over the microscopic methods. By the recent development of diverse fluroscence probes, flow cytometry became capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gametes based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the opportunity of working with small sample sizes, increases the repeatability of data obtained, removes the subjectivity of evaluation and allows simultaneous assessment of multiple fluorochromes. Thus, flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not yet possible with any other technique. Nowadays, semen evaluation using laboratory analyses is very meaningful to the artificial insemination industry to provide the most desired quality product to customers.

Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen attributes that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed.

Introduction

Up to now, semen evaluation is considered as the most important laboratory test that has enabled us to identify and predict clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (Rodríguez-Martínez, 2007). Determination of the potential fertility of semen sample and, in the long run, of the male from which it has been collected is the ultimate goal of semen evaluations in clinically healthy sires. Now a days, many methods for the estimate the possible fertilizing capacity of a semen sample and, or in the word, of the male (reviewed by Dziuk 1996; Rodríguez-Martínez et al. 1997a; Rodríguez-Martínez and Larsson 1998; Saacke et al. 1998; Larsson and Rodríguez-Martínez 2000; Rodríguez- Martínez 2000, 2003; Popwell and Flowers 2004; Graham and Mocé 2005; Gillan et al. 2005) are existing. The methods routinely accustomed for evaluation of the quality of a semen sample involved an evaluation of general appearance, volume, pH, sperm concentration, viability, morphology and motility. Most of these evaluations are based on microscopic analyses that only measure relatively a few numbers of spermatozoa within a population. In most of the cases, these are time-consuming; results obtained are controversial and are not translatable. It should also be noted that such conventional techniques are apt to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. During this long processes, number of steps like semen extension, fluorophore loading, ultraviolet and laser illumination, high-speed sorting, cooling and cryopreservation are followed, which create a scope to impose different degrees of change in sperm functionality followed by suffer of damage to sperm membranes, organelles or the DNA content. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed by many groups that buck of those so-called procedures are incomplete, time consuming and laborious.

Flow cytometry in diverse technical applications proposes many advantages for the analysis of sperm quality. Flow cytometry is a method where multiple fluorescences and light scattering can be induced allowing single cell or particles illumination in suspension while they flow very rapidly through a sensing area. The increasing use over the past decade of flow cytometry in the leading laboratories in human and veterinary andrology has dramatically increased our knowledge of sperm function under physiological and biotechnological conditions. Flow cytometers is capable to acquire data from several subpopulations within a sample in a few minutes, making it perfect for assessing heterogenous populations in a semen sample. Flow cytometry was initially developed in the 1960's, after that flow cytometry is performing automated separation of cells based on the unique recognition of cellular patterns in a population feasible (Hulett et al., 1969). Likewise, cellular patterns can be recognized by utilizing such a separation approach, in each cells within a population (Baumgarth and Roederer, 2000; Herzenberg et al., 2006).

The first notion of flow cytometry development was for medical and clinical applications such as haematology and oncology. Although still much research is going on these medical areas and account for the vast majority of publications on this robust technique, but during the past few years it is being used in a diverse areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Together with elevated use in many areas, recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 2005, 2006;) stallion ( Kavak et al., 2003; Morrell et al., 2008) indicate that newly developed fluorescent stains and techniques of flow cytometry has made possible a more widespread analysis of semen quality at a biochemical, ultrastructural and functional level. Therefore, flow cytometry is the current technical solution for rapid, precisely reproducible assessment of sperm suspensions.

In this review we have described potentiality and scope of flow cytometry for the evaluation of semen, and the way in which this technique can be used in clinical applications for andrology based on some of our previous experiences.

Definition of flow cytometry

The definition of a flow cytometer is ‘an instrument which measures the properties of cells in a flowing stream' or ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream'.

In other word, cytometry is a method which measure physical and chemical attributes of cells or other particles. Such a measurement is made when cells or other particles pass in single file through some sort of measuring apparatus in a stream of fluid. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other traditional analytical tools, where a single value for each attribute is obtained for the whole population, flow cytometry provides data for each and every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters.

Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light scattering are among the cell properties that can be detected. Flow sorting allows individual cells to be sorted on the basis of their measured properties, and one to three or more global properties of the cell can be measured. Flow cytometers and cell sorters make use of one or more excitation sources and one or two fluorescent dyes to measure and characterize several thousands of cells per second. Flow cytometry presents objective and precise results (Bunthof et al., 2001; Shleeva et al., 2002), which help to overcome the problems with the manual methods described above.

Function and types of flow cytometry

A flow cytometer is made of three main systems, fluidics, optics and electronics. ItI It can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment came in to function when it was used for measuring their DNA content (Evenson et al., 1980) and its application for analyzing semen has been increased rapidly in last decade. Flow cytometry is now applied for the evaluation semen such as sperm viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity, DNA status and so on. Continuous innovation of new fluorescent stains and techniques facilitated the flow cytometric evaluation of spermatozoa.

Flow cytometry allows the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. This feature has an added benefit for semen analysis, as few single sperm parameters show significant correlation with fertility in vivo for semen within the acceptable range of normality (Larsson and Rodriguez-Martinez, 2000) and it is the general statistics that the more sperm parameters can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993).

There are two main types of flow cytometers-analysers and sorters are in use. Together with data collection on cells, sorters have the potentiality to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation using a combination of microspheres, flow cytometry and high speed digital processing.

Advantages of FC compared to other conventional techniques to explore sperm structure and function

Use of authentic assays in the fertility clinic and artificial insemination industries increasing day by day. In this respect, use of flow cytometry might be an important attempt to resolve sustaining problem with so called commonly used manual method for the semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with such techniques. It is worth mentinign here that so called method deal only with few hundred sperm. When we deal with such a few sperm population, there is a possibility that obtained result might not be statistically significant (Russel and Curtis, 1993). The methods which are frequently used are enable to determine sperm concentration (Jorgensen et al., 1997), motility or morphology only (Keel et al., 2002). Objectivity, cell number measured, speed of count and precision are the advantages of flow cytometry to conventional light microscopy techniques (Spano and Evenson, 1993). The technique now a days has been used to determine a number of factors including those of acrosome status, membrane integrity, mitochondrial function as well as multiparameter measurement in human (Garrido et al., 2002). Flow cytometry has the ability to analyze thousands of cells in few minutes. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in a diverse species (boar, bull, stallion etc), and the observed errors were significantly better than those obtained from the so-called manual methods.

Although there are diverse benefits of flow cytometer for the analysis of semen, feasibility of applying flow cytometry sometimes restricted to researcher due to the high outlay and difficulties of operation associated with the requirement of a skilled operator. Further, a flow cytometer is very large and cannot resist shocks associated with movement, and it also requires much space in the laboratory. Whatever may be the limitation, the development of more affordable ‘‘bench-top'' flow cytometers in recent time raised the potential essentialities to semen analysis.

If the further application of flow cytometric analysis is considered further, it might be seen that it is growing popularities as a technique for assessing more than one sperm attribute, simultaneously. Compared to traditional microscopic techniques, flow cytometry analysis is allowing to give a far more simplified and objective method of semen analysis, especially in relation to fertilization with acrosome reactivity potential of spermatozoa (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996).

A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Data from a number of preliminary studies proposed that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). According to the World Health Organization that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, might lead to inaccuracy (World Health Organization, 1999). Data from Tomlinson and colleagues indicate that two proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) can result in a lower concentrations of sperm compared to the hemocytometer method (Tomlinson et al., 2001). In contrast, plenty of reports document unacceptable differences between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong?

Improvement of semen quality testing has been emphasizing in some reports (Jorgensen et al., 1997; Keel et al., 2000). But due to low number of sperm evaluation by the conventional method results in poor reproducibility. These problems might be overcome when using flow cytometry. The validation of method is a challenge due to its essentiality of having specific, precise, objective, and accurate evaluation to establish a correlation of fertility data or to predict potential of a semen sample accurately (Amann, 1989). In a fertility clinic, precision of data in important as the result of semen analysis is frequently used to manage fertility of a patient and treatment of the unfertile couples. Thus, it is important to take into consideration within and between laboratory variations for successful infertility treatments.

Sometimes it's a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate “patterns" of fluorescence rather than the fluorescence intensity. Flow cytometer has the lack of ability to discriminate sperm containing a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is almost no difference between methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the acrosomal region of the sperm (Purvis et al., 1990; Holden and Trounson, 1991) and that other binding sites can be easily distinguished by epifluorescence microscopy, whereas flow cytometry identifies the signal from the entire sperm.

Additionally, conventional light microscopic semen assessment is increasingly being replaced by fluorescent staining techniques, computer-assisted sperm analysis (CASA) systems, and flow cytometry (Pen˜a et al., 2001; Verstegen et al., 2002). Additional advantages over existing techniques are that this approach is faster than the hemacytometer and that cellular debris, fat droplets, and other particulate material in extended semen are not erroneously counted as sperm, as often occurs with electronic cell counters. This method can also be used to determine the number of somatic cells in a semen sample.

Application of flow cytometry for sperm count

Sperm count is an important predominant factor for the evaluation of sperm fertility potential.

Accurate determination of sperm cell concentration is critical especially in AI industry because it provides assurance to customers that straws of extended semen contain the sperm numbers indicated which will help to decide appropriate doze especially for pig. Accuracy of sperm count is a common problem in the andrological laboratories and accurate measure of sperm concentration is particularly important for export in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Every time new and more accurate methods for the sperm count determinations are coming and being replaced by the older ones. Some laboratories are trying the Maklerm counting chamber (Seif- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. Although hemacytometers are routinely used for sperm counts, due to the slow process and need for multiple measurements of each sample, the chance of error increase. Freund and Carol (ref) stated that a difference of 20% were not unusual between the determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (Ref). Spectrophotometer is recently being used in the AI industries to assess sperm concentration by determining turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (Ref). The accuracy of this method depends on the methods used for spectrophotometer calibration. Although, sperm concentration can also be determined by spectrophotometrically, the debris present in the raw semen crease problem with misestimation. Sperm number in the frozen thawed semen is difficult to ascertain as most of the extender contain egg yolk particles, fats and other particles which affect measurement of sperm with electric cell counter or spectrophotometers (Evenson et al., 1993). On the other hand flow cytometry created possibilities of a rapid determination of sperm number in a precise form. It is the flow cytometry which can reduce intra-laboratory and inter-laboratory variation and conflict regarding sperm concentration assessment. Computer assisted semen analyzer is robust technique for analyzing sperm movement which can count sperm as well; but such an analyzer most of the cases use some counting chamber or hemacytometer which itself can generate error. Although, hemacytometer was originally developed for blood cell counting, its use is now diverse including andrological laboratories for sperm counting.

Around two-decade ago flow cytometry was suggested for sperm numbers in straws of cryopreserved bull semen. Christensen et al. (----) observed similar results for sperm count with flow cytometry and hemocytometer for a number of species. Now a day a simultaneous determination of sperm viability and sperm concentration is possible which can avoid the chance of occurring differences between ejaculates leading lack of coordination with field fertility and laboratory analyses. Thus the present technology is more precise which can get rid of variation from handling the sperm sample and variation from pipetting and the analysis itself. Further, Prathalingam et al. (2006) concluded that there is similarities for sperm count result between flow cytometry and two newly approached method (image analysis and fluorescent plate reader) for sperm counting. Though, use of fluorescent plate was emphasized due to low cost and allowing large number of cells counting from a large number of ejaculates.

Although flow cytometry has become a valuable instrument for andrological determinations, it is also blamed that sperm concentration by flow cytometry signify a higher value than the real one. The possibility arise might be due to that semen samples often contain some alien materials such as immature germ cells, epithelial cells, blood cells, cytoplasmic droplet, cellular debris etc. In the same way, frozen semen has higher chance to introduce such material as they contain diluents components especially egg yolk particles. These particles and cell debris might have frontal and side light scatter parameters those are similar to spermatozoa. Such sperm-count-overestimation problem arisen in our cases also, especially when we deal with frozen semen. Further it is also claimed that flow cytometry has a tendency to overestimate viable spermatozoa. We are also experienced with such trouble which we guess might be due to that egg particles of extender are considered as viable cell as for its staining pattern. Our yet to publish data indicate that this problem can be mimic by a centrifugation process and by using low concentration sample for evaluation with flow cytometry. Very recently Petrunkina and Harrison (2009) proposed a mathematical equation for fixing this flow cytometric sperm counting. Thus much research is going on and we hope such discrepancy will completely be resolved near future to get advantage from this robust technology for sperm counting.

Flow cytometry for detecting sperm intactness

-Viability of spermatozoa

The viability of spermatozoa is a key determinant of sperm quality and prerequisite for successful fertilization. Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable and subjectively assess only 100 to 200 spermatozoa per ejaculate. Merkies et al. (2000) compared different methods of viability evaluation. They concluded that Eosin-nigrosin overestimate viability while fluorescent microscope and flow cytometry estimate similar trend of viability. Current flow cytometric procedures are able to simultaneously evaluate sperm cell viability together with some other attributes. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (Rodríguez-Martínez, 2007; Garner and Johnson, 1995; Garner et al., 1996), rams (Garner and Johnson, 1995), rabbits (Garner and Johnson, 1995), mice (Garner and Johnson, 1995; Songsasen et al., 1997), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004).

Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method.

One of the first attempts to assess sperm viability utilized rhodamine 123 for determining potentiality of mitochondrial membrane while ethidium bromide for membrane integrity through flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990).

The most generally used sperm viability stain combinations is SYBR-14 and PI at present. This stains are now sold commercially as live/dead viability kit. When these two stains are combinely used, the nuclei of viable sperm take fluoresce green and membrane integrity lost cells take red stain. This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been introduced to evaluate sperm concentration [11] and viability. Due to the small size and low cost, this instrument has been attracted for field measurements of both concentration and viability. In our hand this instrument was also became useful for the quick measurement of sperm concentration and viability in stallion (Morrell et al., 2010).

Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and obtained an inverse correlation between the damaged cells per cent and the field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility.

Fluorochromes used to assess sperm viability by both approach could be utilized in combination with each other. In that case, when CFDA is used combined with PI, three populations of cells as live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa can be identified. This combination was found useful by Almlid and Johnson (1988) for frozen-thawed boar spermatozoa for monitoring membrane damage at the time of evaluation of various freezing protocols. Further, Harrison and Vickers (1990) also noticed that this combination with a fluorescent microscope is effective indicator of viability of fresh, incubated or cold-shocked spermatozoa in boar and ram. Contrasting to these, Garner et al. (1986) was failed to find a relationship between bull sperm viability and fertility when using combination of CFDA/PI .

Flow cytometry for evaluating sperm viability appears to be a precious tool in the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combined assessment of sperm viability and concentration appears to be useful in the wake of improving quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. But the mechanism and mode of action by which SYBR-14 binds to the DNA of sperm is not known. It is know that PI stains nucleic acids by intercalating between the base pairs (Krishan, 1975). Viability stains can also be used in conjugation with fluorescently labeled plant lectins for simultaneous assessment of the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). It is conceivable that assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) found that insemination of boar sperm stained with SYBR-14 did not compromise fertilization or even the development of flushed porcine embryos in vitro.

Non-viable sperms can be detected using the membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. Plasma membrane which is intact will not permit these stains entering into the spermatozoa and staining the nucleus. Most frequently used stains include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). After a series of comparison between fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality as assessed by flow cytometry using PI, Wilhelm et al. (1996) concluded that viability is the single laboratory assay that correlated with fertility.

-Sperm plasma membrane integrity

Although the sperm plasma membrane covers the entire cell, it consists of three distinct membrane compartments, one which covers the outer acrosomal membrane, one which covers the post acrosomal portion of the sperm head, and one which covers the middle and principal pieces. Sperm membrane is directly or indirectly related with many sperm functions has influence on sperm metabolism for conserving motility, capacitation, acrosome reaction, interactions between sperm and epithelium of the female genital tract, as well as sperm-egg interactions (Rodriguez-Martinez, 2003). Most ‘viability assays' assess whether or not the plasma membrane is intact (the cell is ‘viable') or not (the cell is ‘dead'). However, because the plasma membrane is composed of these different compartments, different viability assays assess the integrity of different plasma membrane compartments. Classical stains, such as eosin-nigrosin and eosin aniline blue, as well as more recent fluorescent stains, such as propidium iodide, ethidium bromide, 4-6-diamidino-2-phenylindole and bisbenzimide (Hoechst dyes), bind to and stain the DNA of sperm that possess a post acrosomal plasma membrane that is not intact. However, these probes will not assess the integrity of the plasma membrane covering the acrosome or principal piece. The integrity of the plasma membrane covering the principal piece can be assessed using sperm motility or the hypo-osmotic swelling test ( Neild et al., 2000; Colenbrander et al., 2003). The integrity of the plasma membrane covering the acrosome is generally assessed in conjunction with the integrity of the outer acrosomal membrane. The acrosomal ridge, present on the sperm of several species, can be used to assess the integrity of this plasma membrane by differential interference phase contrast microscopy, but this anatomical component can only be used for sperm from those species which possess it. Several non-fluorescent and fluorescent staining combinations (reviewed by Cross and Meizel, 1989; Graham, 2001; Colenbrander et al., 2003; Silva and Gadella, 2006) have been developed to permit assessment of acrosomal membrane integrity of fresh and fixed sperm samples using microscopy, fluorometry and flow cytometry.

During the last decade, for dog, many fluorescent dyes were introduced and validated for the assessment of the sperm membrane integrity:combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Pen˜a et al., Rota et al., 1995), SYBR-14 in combination with PI (Rijsselaere et al., 2002; Yu et al., 2002), combination of carboxy-seminaphthorhodfluor (Carboxy-SNARF) with PI (Pen˜a et al., 1999), calcein-AM in combination with ethidium homodimer (Calcein-AM/EthD-1) (Sirivaidyapong et al., 2000) and Hoechst 33258 (Hewitt and England, 1998). The main advantage of these fluorescent staining techniques is the possible analysis of fluorescently labelled sperm with the help of flow cytometry, which enabled evaluation of larger numbers of spermatozoa within few minutes. Pen˜a et al. (1998, 2001) found high correlations between flow cytometry and epifluorescence microscopy for the percentage of live and dead spermatozoa as determined by a CFDA-PI staining.

Apoptosis is a carefully regulated process of cell death that occurs as a normal component of development. During this process, the plasma membrane became slightly permeable and loses asymmetry in one of the earliest stages of apoptosis. When the cell membrane is disturbed the phospholipid results in a translocation of PS (Desagher and Martinou, 2000). Recently, it has been demonstrated in human (Kemal et al., 2001) and bull (Anzar et al., 2002) that freezing-thawing of semen induces membrane PS translocation, thus indicating cryopreservation is a cause of apoptosis (Baust, 2002). At the early stage of hampered plasma membrane activity, asymmetry of membrane PL happens (Ref). In all studied mammalian cell types including spermatozoa, the two leaflets of the plasma membrane bilayer differ in its phospholipid content. Phosphatidylserine and phosphatidylethanolamine these two are concentrated in the inner leaflet, whereas sphyngomyeline and phosphatidylcholine are concentrated in the outer leaflet (Ref).

As the sperm membrane are extravagant susceptible to cryoinjury, the evaluation of sperm membranes is a proper indicator of the success of cryopreservation (Ref). Number of deleterious effects of boar sperm manipulation such as excessive extension, sorting, chilling or cryopreservation that lead to membrane destabilization. The procedure of freezing and thawing adversely affect the lipid composition of boar sperm plasma membranes not only in poor quality sperm but also in good sperm quality (Ref). Lipid phase transition, ice crystallization, as well as membrane-recognizations exert membrane integrity, structure and function which occur due to adverse influence of cooling, freezing and thawing procedure (Hammerstedt et al., 1990). In general plasma membranes of spermatozoa contain the high amount of long-chain PUFA compare to other somatic cells. In recent studies a close relation between differences in PUFAs within plasma membranes with cryotolerance in sperm has been identified in different species (Swain and Miller 2000; Miller et al., 2004; Miller et al., 2005).

Although high cholesterol to phospholipid ratio in sperm membranes is considered to be positively associated with cryotolerance, between species there is a variation. Some animals are good freezer and some are bad freezer rather than any individual ejaculates. Further, variation in cryopreservation-induced injuries has been claimed have genetic basis where plasma membrane and acrosomal membrane have been found disrupted (Thurston et al., 2002). Holt et al.( 2005) suggested that differences between male to male may cause differences in sperm lipid and protein composition, and the underlying mechanism for this genetic diversity associated with cryoinjuries yet to be elucidate. Addition of cryoprotectants prior to freezing, volumetric changes and associated membrane stretching and shrinkage in response to hyperosmotic cryoprotectant solutions as well as freeze-induced dehydration, thermotropic and lyotropic phase transitions in membrane phospholipids, and the well-established effects of elevated solute concentration and intracellular ice formation which are cooling rate-dependent.

-Changes in sperm membrane permeability

Detection of the membrane permeability in different cell types is considered to be able to differentiate different membrane organization status (Cohen, 1993; Ormerod et al., 1993; Castaneda and Kinne, 2000; Reber et al., 2002). Sperm plasma membrane status is of utmost importance due to its role, not only as a cell boundary, but also for its importance for cell-to-cell interactions, e.g. between spermatozoa and the epithelium of the female genital tract and between the spermatozoon and the oocyte and its vestments (for review, see Rodriguez-Martinez, 2001). Membrane integrity and the stability of its semipermeable features are prerequisites for the viability of the spermatozoon (Rodriguez-Martinez, 2006). However, cryopreservation, whose purpose is to warrant sperm survival, causes irreversible damage to the plasma membrane leading to cell death in a large number of spermatozoa (Holt, 2000) or, in the surviving spermatozoa, to changes similar to those seen during sperm capacitation, thus shortening their lifetime (Perez et al., 1996; Cormier et al., 1997; Maxwell and Johnson, 1997; Green and Watson, 2000; Schembri et al., 2000; Watson, 2000). During the freezing process, cells shrink again when cooling rates are slow enough to prevent intracellular ice formation as growing extracellular ice concentrates the solutes in the diminishing volume of non-frozen water, causing intracellular water exosmosis. Though warming and thawing, the cells return to their normal volume. Thus, it is important to know the permeability coefficient of the cells to cryoprotectants, as well as the effect of cryoprotective agents on the membrane hydraulic conductivity.

Classical combination of probes allows discrimination of two or three subpopulations of spermatozoa, i.e. live, dead and damaged depending on the degree of membrane integrity (Eriksson & Rodrı´guez-Martı´nez, 2000). A new, simple and repeatable method to detect membrane changes in all spermatozoa present in a boar semen sample, by use of markers (combination of SNARF-1, YO-PRO-1 and ethidium homodimer) used to track changes in sperm membrane permeability, has been developed recently by our group (Pena et al., 2005). In determined physiological or pathological situations, live cells are unable to exclude YO-PRO-1, but are still not permeable to other dead-cell discriminatory dyes, like propidium iodide or ethidium homodimer. YO-PRO-1 is an impermeable membrane probe and can leak in, only after destabilization of the membrane, under conditions where ethidium homodimer does not. Because several ATP-dependent channels have been detected in spermatozoa (Acevedo et al., 2006), it seems plausible that this is a result of the silencing of a multidrug transporter. This multidrug transporter is involved in transporting amphipathic small molecules like YO-PRO-1, which in intact cells is actively pumped out but not after destabilization of the plasma membrane, maybe because sub-viable cells lack appropriate amounts of ATP to transport YO-PRO-1 back out of the cell (Ormerod et al., 1993). Therefore, the use of a fluorescent probe, such as YO-PRO-1, which penetrates cells as they undergo changes related to cryoinjury, where membranes become slightly permeable, makes YO-PRO-1 a useful tool for detecting early membrane changes (Idziorek et al., 1995; Wronski et al., 2000). This triple staining distinguishes, as in the Annexin assay, four sperm subpopulations. The three probes are easily distinguished both in flow cytometry and in fluorescence microscopy. The absorption and emission maxima for YO-PRO-1 are 491 nm and 509 nm, respectively, and 528 nm and 617 nm, respectively, for ethidium homodimer to be detected in the Flow Cytometer with the FL1 and FL3 photomul-tipliers.

The triple staining technique offers some advantages over the A/PI assay. Whereas in the A/PI assay there is always an unstained subpopulation, the triple stain labels all the spermatozoa in the sample, an obvious advantage when using manual counting in fluorescence microscopy. If a flow cytometer is available, because only sperm cells are stained with the triple staining technique, spermatozoa and debris can be easily separated based not only on scatter properties of the particles but also on their fluorescent properties. This fact is important because in bull semen, it has been demonstrated that egg-yolk particles can be easily misjudged as spermatozoa based only on their scatter properties (Nagy et al., 2003), requiring preliminary washing and centrifugation to cleanse the cells. Centrifugation might cause sperm damage and, therefore, mask other effects caused by the cryopreservation. The subpopulation of live cells using the new triple staining concurs with the subpopulation of live cells using the well validated A/PI assay. In addition, the staining protocol is much easier than the A/PI because the staining is made from stock solutions and is not necessary to use a binding buffer. As the staining of the probes is not dependent on Ca2+, as is the case binding FITC-A, the preparation and using of a Ca2+ enriched buffer is not necessary. The agreement between both techniques (A and YOPRO- 1/Eth/SNARF-1) was good, although the percentage of live spermatozoa was slightly higher in the triple staining method (Pena et al., 2006). Also, the percentage of early damaged spermatozoa was higher with the A/PI assay. This might reflect an increase in membrane permeability, preceeding the transposition of PS in the evolution of the cryodamage, or in a yet to be determined physiological change probably being a very early step of both processes related to changes in cell volume regulation and movement of ions, occurring during the initiation of apoptosis (Bortner and Cidlowski, 1998) or cryoinjury (Paasch et al., 2005). In addition, an earlier inactivation of enzymes involved in maintaining membrane asymmetry than those involved in transporting amphipatic small molecules like YO-PRO-1 might explain this fact.

-Changes in sperm mitochondrial status

Mitochondria, located in the sperm midpiece, are the primary energy generating centers for motility and other processes in the sperm cell as in most other cells. Interference with this function has severe consequences for any cell (Krahenbuhl, 2001). During the apoptosis process, a number of changes occur in the mitochondria (Lui et al., 1996; Petit et al., 1996; Li et al., 1997). Mitochondria enclosed in the midpiece of the spermatozoa is a source of ATP which is utilized for the propulsion of flagellar. Cellular energy production, membrane lipids productions, growth as well as major determinants of sperm life or death largely depends on mitochondria (Arends and Wyllie, 1991). In other words, mitochondria have been confirmed by several groups as the coordinators of apoptosis in a number of cell systems (Kroemer, 1997; Zhyng et al., 1998). Shivaji et al. (2009) focuses on the identity and function of mitochondrial proteins which undergo capacitation-dependent tyrosine phosphorylation in spermatozoa. Hallap et al. (2005) reported that the results obtained from flow cytometric measurements of mitochondrial function were 10-15% lower than the recordings of motility, either subjectively or as measured by CASA. Such a difference between subjective motility evaluations and flow cytometry evaluations of mitochondrial activity is in line with that reported in several other publications (Garner et al., 1997; Gravance et al., 2000; Wu et al., 2003).

Accumulation in mitochondria is characteristic of many fluorescent dyes, such as Rhodamine 123 (R123), MitoTracker Green (MTG), JC-1, MitoTracker Orange (CMTMRos), MitoTracker Red (CMXRos), MitoTracker Red 580, and MitoTracker Deep Red 633 (Cossarizza---; Garner et al., 1997; Gravance et al., 2002; Ericsson et al., 1993). Despite of numerous published protocols, there are several problems connected with most of these fluorophores. There are approximately 100 mitochondria in the mid-piece of the spermatozoon and fluorescent dyes, able to target defined intracellular compartments, can be used to visualize them. Most of these dyes work by diffusing into living cells and accumulating in mitochondria, provided that an internal 100-200 mV negative potential gradient occurs across the mitochondrial membrane (MMP).

The most widely used mitochondrial-specific probe, R123 is a cationic compound that excites at 488 nm and emits at 515-575 nm (green fluorescence). It accumulates in the mitochondria as a function of transmembrane potential (Chen, 1988; Al-Rubeai, 1993), of R123 concentration, and of sperm numbers (Windsor and White, 1993); it is not dependent on time or temperature (Auger et al., 1989). It was historically applied to spermatozoa in combination with ethidium bromide (Evenson et al., 1982). The R123 accumulated in the mitochondria and fluoresced green, thus identifying the sperm that exhibited a mitochondrial membrane potential. The dead spermatozoa, such as those with damaged membranes, were identified by the uptake of ethidium bromide. A similar combination, R123 and propidium iodide (PI), has been shown to readily discriminate between living and dead spermatozoa (Evenson et al., 1982). Although functioning mitochondria stain green with rhodamine123, this stain does not permit one to differentiate between mitochondria that exhibit different respiratory rates. The novel mitochondrial probe, MitoTracker Green FM (MITO), is nonfluorescent in aqueous solution and fluoresces green upon accumulation in the mitochondria regardless of mitochondrial membrane potential (Haugland, 1996). R123 and MITO are transported into actively respiring mitochondria and their accumulation in the mitochondria causes them to fluoresce green. R123 is not suitable for use in experiments in which the spermatozoa are treated with aldehyde fixatives, whereas the MITO probes are well retained during the fixation process. MITO-labeled mouse spermatozoa were placed in the female reproductive tract by AI to trace the distribution of the mitochondria in the developing embryo (Davies and Gardner, 2002). Gadella and Harrison (2002) also used MITO to show that bicarbonate does not affect the mitochondrial potential of boar spermatozoa. Fluorescence-induced flow cytometric assays of Jagg fluorescence is frequently used to notify sperm with high mitochondrial transmembrane potential in sperm of many species (Garner et al., 1997; Gravance et al., 2001; Love et al., 2003). Martinez-Pastor et al. (2004) observed some relationship between JC-1 staining and motility, although correlation with motility is regulated by many factors. A deeper study has been suggested by this research group. The mitochondrial stain 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) does permit a distinction to be made between spermatozoa with poorly and highly functional mitochondria (Garner et al., 1997). In highly functional mitochondria, the concentration of JC-1 inside the mitochondria increases and the stain forms aggregates that fluoresce orange. When human spermatozoa were divided into high, moderate and low mitochondrial potential groups, based on JC-1 fluorescence, the in vitro fertilization rates were higher in the high potential group than in the low potential group (Kasai et al., 2002). JC-1 has also been used successfully to measure mitochondrial function using a fluorometer (Gravance et al., 2000). However, at greater concentrations, the probe aggregates and in the aggregate form fluoresces red-orange (Thomas et al., 1998).

-Changes in sperm chromatine integrity

Sperm DNA integrity is increasingly being recognized as an important determinant of fertilizing efficacy that has better diagnostic and prognostic capabilities than the standard sperm attributes like sperm morphology, sperm concentration or motility potential. In most of the normal semen parameters analyses quality of sperm DNA is not always reflected while early embryo development largely depends on the presence of normal DNA (Rodriguez-Martinez, 2007). Full sperm DNA integrity usually is defined as the absence of DNA nicks or single stranded (ss) breaks, double stranded (ds) breaks, and chemical modifications of the DNA. Of these, ds DNA breaks are the most mutagenic, because in the pronucleus stage zygote, the two genomes are separated, hence DNA template information for error free repair of the ds breaks is absent. Some recent follow-ups of children born after IVF are addressing the question about the possible increase of pathologies connected with these technigues (Schultz and Williams, 2002). DNA fragmentation frequently affecting abnormal spermatozoa present in large numbers in the semen of subjects with low sperm motility (Benchaib et al., 2003; Gandini et al., 2000; Huang et al., 2005; Lui et al., 2004; Marchetti et al., 2002; Varum et al., 2007; Vicari et al., 2002; Zini et al., 2001) may prevent or hinder fertilization and embryo development, and increase the risk of genetic defects (Erenpreiss et al., 2006; Spano et al., 2005; Huang et al., 2005; Borini et al., 2006; Henkel et al., 2003; Lewis and Aitken, 2005; Seli et al., 2004; Tesarik, 2005). It should be emphasized that detection of DNA damage is often beyond the scope of routine seminological analysis, which in many cases is decisive for qualification to assisted fertilization. DNA damage in sperm is promutagenic. It does not impair fertilization or cleavage, as the paternal genome is transcriptionally inactive until two days after fertilization (Agarwal and Allameneni, 2004). However, once the paternal genome is active it results in poor blastocyst development, unequal cleavage, implantation failure or early foetal loss. Small DNA damages in sperm are repaired by pre- and postreplication repair mechanisms, but large DNA damages cannot be repaired.

The organization of sperm DNA in an explicit manner allows the chromatin to keep in the nucleus in a compact and stable form, in an almost crystalline status (Carrell et al., 2003). The histone-to-protamine transition is a multistep process in which, firstly, somatic-type histones are partially exchanged with testis-specific histones, then replaced by transition proteins (Boissenault, 2002) and, finally, by protamines. Sperm chromatin abnormalities and DNA damage could stem from an insult to premeiotic testicular cell compartment or occur at the time of DNA packing at spermiogenesis (Sailer et al., 1995; Haines et al., 2001; Haines et al., 2002; Cordelli et al., 2003). Alternatively, it could be the result of freeradical- induced damage (Aitken et al., 1998; Shen and Ong, 2000; Aitken and Krausz, 2001) or a consequence of apoptosis (Sakkas et al., 2002; Gorczyca et al., 1993). Due to the importance of accurate transmission of genetic information to the offspring, several methods have been developed to detect DNA and chromatin alterations in spermatozoa, and efforts have been done to integrate these tests into conventional semen analysis (Perreault et al., 2003). Flow cytometric assays is one of the most interesting techniques today available which has demonstrated to be an independent fertility predictor either in vivo (Spano et al., 2000) or in vitro (Larson-Cook et al., 2003; Saleh et al., 2003) conditions. The integrity of sperm DNA evaluation with the TUNEL, COMET, acridine orange staining, in situ nick translation, or sperm chromatin structure assay (SCSA) techniques are in use in different laboratories.

Comet assay measures the response of individual cells which enables the study of heterogeneity within a cell population. While it is not possible with other techniques like pulsed-field gel electrophoresis, filter elution or sucrose gradient sedimentation which can estimate only the mean response from a large cell population. Comet assay can detect 200 DNA strand breaks per cell, however with few modifications it may detect up to 50 breaks per cell. In comet assay only a few number of cells are required, data can be collected at the level of individual cells, wide range of eukaryotic cells can be used, the assay is sensitive, simple and can detect non uniform responses within a mixed population. However, DNA damage can be overestimated in comet assay due to the presence of residual RNA which creates background during analysis, or can be underestimated because of proteins which hamper the movement of fragments during electrophoresis. Due to high inter-laboratory variation comet assay is not suitable for clinical use (Olive et al., 1992; Olive et al., 2001).

DNA fragmentation in spermatozoa can also be assessed with TUNEL assay, which can identifies DNA strand breaks with modified nucleotides. These incorporated labelled nucleotides can be detected in spermatozoa by flow cytometry, flourecence microscopy or light microscopy. Duran et al. (2002) found that human semen samples with greater than 12% of the spermatozoa containing DNA fragmentation did not result in pregnancy and Benchaib et al. (2003) obtained no pregnancies if this value was greater than 20%. In enzymatic detection of fragmented DNA bound to the biotinylated nucleotides by TUNEL horseradish peroxidase-labelled peroxidase streptavidin (Streptavidin HRP), which are observed by the hydrogen peroxide, and the stable chromogen, diaminobenzidine (DAB) which produces brown stain (DeadEnd Fluorometric TUNEL System Technical Bulletin, 2007). TUNEL can simultaneously detect single and double strand breaks unlike comet assay which requires different protocols for studying both type of strand breakages. By TUNEL the degree of DNA damage within a cell cannot be quantified, it only reveals the number of cells within a population with DNA damage. It is worth mentioning that such cells can also be identified using flowcytometry or fluorescence microscopy.

In situ nick translation assay (ISNT) is a modified form of TUNEL, which utilizes incorporation of biotinylated-dUTP at the ssDNA in a reaction catalyzed by template dependent enzyme, DNA polymerase 1 (DNA Pol 1), unlike TUNEL which utilizes template independent TdT. Moreover ISNT can only be used for single strand breaks not for both ss and ds breaks as in TUNEL (Irvine et al., 2000). ISNT assay is able to identify endogenous DNA damage; however, Irvine et al. (2000) reported that ISNT assay has limited correlation with fertility in in vivo studies.

Sperm chromatin structure assay (SCSA) is a technique, in which the extent of DNA denaturation following heat or acid treatment is determined by measuring the metachromatic shift from green fluorescence (acridine orange intercalated into double stranded nucleic acid) to red fluorescence (acridine orange associated with single stranded DNA) (Darzynkiewicz et al., 1976). The ratio of the amount of red to green for each individual spermatozoon is assessed by flow cytometry, with sperm containing greater red to green ratios exhibiting more DNA denaturation than sperm exhibiting lesser red to green ratios (Evenson and Wixon, 2006). One benefit of SCSA over other methods that evaluate DNA integrity is that the procedure is relatively easy, quick, and thousands of sperm can be evaluated objectively. However, no term pregnancies have been detected when the DFI fraction was more than 28 percent, which allowed to conclude that SCSA-derived thresholds markedly predicted negative pregnancy outcomes in patients attempting pregnancy via ART (Saleh et al., 2002; Larson et al., 2000; Cook et al., 2003). In clinical applications, the SCSA showed that an increased fraction of abnormal sperm has been found in patients with leukocytospermia, febrile illness and untreated testicular cancer patients (Evenson et al., 2000; Alvarez et al., 2002). The SCSA has also been successfully used to check gamete quality after cell manipulation (Spano et al., 1999).

The most important parameter revealed by SCSA is the DNA fragmentation index (% DFI). It is a simple and less time consuming method for the analysis of human spermatozoa (Frazer, 2005). The SCSA does not give much information about the extent of DNA damage in spermatozoa, since it focuses on measuring the percentage of spermatozoa with dispersed or non dispersed nuclei (Frazer, 2005). Many research groups recently potentially utilized SCSA on domestic animals (deAmbrogi et al 2006, Koonjaenak et al 2007, Morrell et al 2008).

Acridine orange test (AOT) is a simplified microscopic method of SCSA which does not require expensive flowcytometry and relies on visual interpretation of fluorescing spermatozoa and debris that fall into a broad range of colours under microscopic examination. Pretreated sperm samples (citric acid solution pH 2.0) stained with acridine orange (0.2 mg/ml H2O) are washed and covered with coverslip for examination (Spano et al., 1999). Sperms with ds DNA fluoresce green while those with ss DNA fluoresce red under a fluorescence microscope. Indistinct colour, rapidly fading fluorescence and heterogeneous slide staining exacerbate problems with interpretation (Duran et al., 1998).

On the other hand lack of correlation between DNA damage and fertilization rates but rather an association between DNA damage and post fertilization development (Agarwal and Allameneni, 2004).

During spermiogenesis, a decrease in binding capacity for DNA fluorochromes occurs due to the progressive chromatin packaging (Evenson et al., 1986), and FCM techniques have been proposed as measures of chromatin condensation anomalies using different DNA-specific dyes such as DAPI (Spano et al., 1984), ethidium bromide/mithramycin (Engh et al., 1992) and propidium iodide (Molina et al., 1995). Using this approach, associations have been demonstrated between fertility capability and the increase of highstainable abnormal sperm fraction (Hacker-Klom et al., 1999).

Many technical and biological factors determine the accuracy to evaluate the DNA damage in sperm cells. In spite of the variety of different methods available to evaluate sperm chromatin maturity levels, the bulk of evidence points to the pivotal role of chromatin /DNA integrity during spermatogenesis and in the fertilization process. Lower chromatin packaging quality detected in morphologically normal spermatozoa may represent one of the major factors limiting the sperm fertilizing ability, thus stressing the importance of the chromatin packing or DNA integrity as a prerequisite for optimal expression of male fertility potential.

-Decondensationof spermatozoa during fertilization

During mammalian spermiogenesis sperm chromatin undergoes a step-by-step condensation and packaging mainly characterized by replacement of histones with protamines and the formation of S-S and S-Zn-S bonds between cysteine residues (Bedford & Calvin, 1974). Therefore, mammalian spermatozoa nuclei are highly structured, exceptionally stable, condensed organella (Arkhis et al., 1991; Balhorn et al., 1991). Decondensation of sperm chromatin take place after entering into the ovum or injection of the oocyte (ICSI) when disulphide bonds of the protamine of the sperm nucleus are reduced, presumably by glutathione (Perreault et al., 1984, 1988; Perreault, 1992), followed by replacement of the protamine by embryonic histones (Zirkin et al., 1985; Betzalel & Moav, 1987; Philpott et al., 1991; Perreault, 1992; Leno et al., 1996). Although, the ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin in the cytoplasma of the oocyte (Kvist, 1980; Zirkin et al., 1985; Gopalkrishnan et al., 1991).

Methods for assessing sperm chromatin decondensation must therefore be considered useful additions to the arsenal of tests useful for diagnosing patients with male infertility. This is especially likely to prove useful in cases where intracytoplasmic sperm injection is being considered as a therapeutic option. Several groups have measured sperm chromatin decondensation by observing changes in size and shape of the sperm head by microscopic examinations (Jaeger et al., 1990; Lassalle and Testart, 1991; Lipitz et al., 1992; Montag et al., 1992; Banerjee and Hulten, 1994; Cameron and Poccia, 1994; Morcos and Swan, 1994; Chitale and Rathaur, 1995; Reyes and Sanchez-Vazques, 1996). These methods tend to be somewhat subjective because it is difficult to decide what degree of sperm head enlargement is to be the criterion of decondensation and they are time consuming because they require counting many cells in order to achieve statistical significance.

Flow cytometry offers an excellent means to quantitates these events associated with chromatin decondensation. Flow cytometry provides a useful alternative to microscopic examination of cells for determination of sperm decondensation. Zucker et al. (1992) performed decondensation in rat and hamster spermatozoa by using sodium dodecyl sulphate and dithiothreitol as a reducing agent and assessed decondensation by increase in light scatter. Evenson et al. (1980) evaluated decondensation in mouse and human sperm nuclei using Acridine Orange staining and plotting green fluorescence versus pulse width of the green signal, which is related to cell size. In the method reported here the increase in intensity of green and red fluorescence of Acridine Orange-stained spermatozoa after incubation with β-mercaptoethanol served as a measure of the decondensation.

Spermatozoon chromatin structure has been shown to be altered by cryopreservation-thawing in the mouse (Watson 2000; Kusakabe et al. 2001) and horse (Linfor and Meyers 2002). Although decondensation depends on spermatozoon disulfide bond and chromatin integrity (Lipitz et al. 1992), it is the oocyte's cytoplasm that induces decondensation (Yanagimachi 1994; Sutovsky and Schatten 1997). The study of decondensation often is conducted in vitro using chemicals rather than oocytes (Calvin and Bedford 1971; Perreault et al. 1988b; Samocha-Bone et al. 1998; Reichart et al. 2000; Martin et al. 2003). However, cytoplasmic emulsions from homologous or heterologous oocytes (Yanagimachi 1984; Ohsumi et al. 1986) are most likely to be physiologically meaningful as an in vitro test of spermatozoon decondensation (Yanagimachi 1994; Li and Gui 2003). There also is variabilit


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