Flow Cytometry for the Evaluation of Semen

Flow Cytometry for the Evaluation of Semen 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 exper iments 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 (Rodrà ­guez-Martà ­nez, 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; Rodrà ­guez-Martà ­nez et al. 1997a; Rodrà ­guez-Martà ­nez and Larsson 1998; Saacke et al. 1998; Larsson and Rodrà ­guez-Martà ­nez 2000; Rodrà ­guez- Martà ­nez 2000, 2003; Popwell and Flowers 2004; Graham and Mocà © 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, ultrav iolet 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 1960s, 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., 20 05, 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 determi ne 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 its a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate â€Å"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 (PenËÅ"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 (Se if- 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 s ample 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 a nalyzer 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 it s 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 (Rodrà ­guez-Martà ­nez, 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., 199 7), 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 an d 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 inte rcalating 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 ind Flow Cytometry for the Evaluation of Semen Flow Cytometry for the Evaluation of Semen 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 exper iments 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 (Rodrà ­guez-Martà ­nez, 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; Rodrà ­guez-Martà ­nez et al. 1997a; Rodrà ­guez-Martà ­nez and Larsson 1998; Saacke et al. 1998; Larsson and Rodrà ­guez-Martà ­nez 2000; Rodrà ­guez- Martà ­nez 2000, 2003; Popwell and Flowers 2004; Graham and Mocà © 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, ultrav iolet 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 1960s, 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., 20 05, 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 determi ne 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 its a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate â€Å"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 (PenËÅ"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 (Se if- 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 s ample 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 a nalyzer 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 it s 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 (Rodrà ­guez-Martà ­nez, 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., 199 7), 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 an d 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 inte rcalating 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 ind