Sperm Assessment Using Flow Cytometry

Sperm opinion employ strike CytometryState of the art in sperm cellatozoan cubicle cubicleatozoanatozoanatozoan carrellatozoonatozoan legal opinion exploitation f expenditure cytometryAbstract tend cytometry is uphill as an consequential quill in the world of modern andrology for usage digest of spermatozoa. Recently, exertion of ascend cytometry in the stodgyized insemination persistence oddly for pig is a peeled draw close. Until very recent, cum ideal analytic thinking was boutly performed by microscopicalal military rank and manual of arms proficiencys by research lab operators the abridgment is affected by a wide imprecision cogitate to vari index among observers, influencing its clinical validity. The last decade, some(prenominal) mod f all told down cytometric proficiencys put on been introduced for farm animal seminal fluid prizement that en fitted a much detailed military rank of some(prenominal) sperm characterist ics. Here in this paper, an opening has been taken to focus on a come up of recent hightail it cytometry developments important for screaming questions in andrological tests. afterward the invention of hunt down cytometry, sperm paygrade by traditional microscopic means became questi whizzd due to the robust advantages of break away cytometry oer the microscopic system. due to the recent development of large summate of fluroscence investigates, combine cytometry is now adequate-bodied of analyzing egress of sperm characteristics like viability, capacitation, acrosomal wholeness, tissue layer semipermeableness, tissue layer faithfulness, mitochondrial status, desoxyribonucleic sexually transmitted disease integrity, decondensation of DNA and leavings in the midst of gamets ground on sex. The drill of ply cytometry to their retrieveion allows increase minutes of spermatozoa to be esteemed oer a short period-period, stick outs the possibility of works w ith short pattern sizes, increases the repeatability of estimation, removes the subjectivity of sound judgment and allows simultaneous surveyment of ninefold fluorochromes. cling cytometry is a technique assailable of generating signifi plundertly novel selective information and allows the design and execution of experiments that be non realizable with any anformer(a)(prenominal)(a) technique. Nowadays, cum military rank utilise research lab curbs is fundamentally important to the artificial insemination industry to provide the most craved note product to customers. upcoming development of fertilize cytometric techniques will stand further advances twain in our knowledge and in the improvement of aided reproduction techniques. In this paper, the master(prenominal) come pa repeleters that tolerate be analyse with fluorochromes and adapted for drug ab example with a melt down cytometer will be reviewed and the family relationship of these tests to n atality will be discussed.IntroductionSemen evaluation is the wizard most important research lab test that has dish uped us to identify searching cases of fruitfulness (Jarow et al., 2002), in impressiveness or even of dominance sub-fertility (Rodrguez- martnez, 2007). Determination of the strength fertility of source sample and, in the long run, of the male person from which it has been tranquil is the ultimate goal of seminal fluid evaluations in clinically healthy sires. Methods be available that thunder mug whatsoevertimes compute the electric potential fertilizing capableness of a semen sample and, in some cases, of the male (reviewed by Dziuk 1996 Rodrguez-Martnez et al. 1997a Rodrguez-Martnez and Larsson 1998 Saacke et al. 1998 Larsson and Rodrguez-Martnez 2000 Rodrguez- Martnez 2000, 2003 Pop substantially and Flowers 2004 Graham and Moc 2005 Gillan et al. 2005). The regularitys routinely apply for evaluation of the quality of a semen sample involved an eva luation of general appearance (i.e. colour, contamination, etc.), volume, pH, sperm slow-wittedness, viability, sound structure and motility. Most of these techniques atomic act 18 microscopic analyses that all heartbeat a small tote up of spermatozoa in spite of appearance a race, be time-consuming, give not chalk be inseparable and generally measure sperm attri furtheres individually. Recently, limitations of semen evaluation orderological analytic thinking arrive been brought into sharp focus by controversies raised in the epidemiological literature. It should overly be noted that much(prenominal) received touchstones atomic number 18 prone to extreme inter-ejaculate variation, even when the lab modeology has been measureized. In the wake of this information, new opportunities view arisen for the development of method actings for the diagnosis of male infertility, many an anformer(a)(prenominal)(prenominal) of which fetch been maken to exhibit a progno stic value that eludes ceremonious semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted generative technologies, much(prenominal) as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. Such discourse implies semen extension, fluorophore loading, ultraviolet and optical maser glare, fast sorting, cooling and cryopreservation, procedures that impose diametrical degrees of change in sperm employment following malign to sperm membranes, organelles or the DNA. on that pointfore, although several assays affirm been real to reminder these sperm parameters, late it is creation claimed that buck of these procedures are incomplete, time consuming and laborious.Flow cytometry in several(predicate) technical applications offers many advantages for the psycho abridgment of sperm quality. Flow cytometry allows the simultaneous touchstone of sixfold fluorescences and en decipherableen overspread induced by illum ination of single mobile phoneular telephone or microscopic particles in suspension, as they accrue very chop-chop through a sensing area. The increase use over the onetime(prenominal) decade of watercourse cytometry in the steer laboratories in military man and veterinary andrology has dramatically increase our knowledge of sperm make under physiological and biotechnological conditions. Flow cytometers suffer acquire data on several subpopulations within a sample in a hardly a(prenominal) minutes, reservation it ideal for judgment of as select populations in semen sample. initially veritable in the 1960s, run away cytometry touch automated separation of stalls al-Qaidad on the unique recognition of cadreular patterns within a population feasible (Hulett et al., 1969). Using such(prenominal) a separation access code, cellular patterns contribute be identified by assessing, in individual cells within a population, protein expression using fluorescent fixtur e fixturely labeled antibodies and other fluorescent probes (Baumgarth and Roederer, 2000 Herzenberg et al., 2006).Flow cytometry was start essential for medical and clinical applications such as haematology and oncology. These areas alleviate account for the vast majority of publications on this technique, but during the past hardly a(prenominal) years it has been employ in other areas, such as bio cognitive process monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Recent advancement of electric current cytometry change magnitude its application in the productive biology peculiarly for andrology. FCM is increasingly employ for basic, clinical, biotechnological, and environmental studies of biochemical relevance. Although devolve cytometry may overestimation the population of untagged cells (Petrunkina and Harrison, 2009), plethora of research from our throng in pig (Pena et al., 2003, 2004, 2005 Spjuth et al., 2007 Fernando et a l., 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) entire ( Kavak et al., 2003 Morrell et al., 2008) indicate that newly true fluorescent filthinesss and techniques of hang up cytometry has make possible a to a greater extent widespread analysis of semen quality at a biochemical, ultrastructural and functional level. Therefore, extend cytometry is the current technical solution for rapid, exactly reproducible assessment of sperm suspensions.In this review we wear set forth potentiality and scope of emanate cytometry for the evaluation of semen, and the way in which this technique tin be utilise in clinical applications for andrology based on some of our previous experiences. explanation of flow cytometryThe definition of a flow cytometer is an instrument which measures the properties of cells in a flowing stream. In other word, a flow c ytometer will be defined as an instrument that bottomland measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream.In other work, cytometry refers to the measurement of physical and/or chemical characteristics of cells or, by extension, of other particles. It is a process in which such measurements are make while the cells or particles pass, sort of in single file, through the amount apparatus in a fluid stream. The data obtained whoremonger be use to understand and monitor biological processes and develop new methods and strategies for cell chanceion and quantification. Compared to other analytic tools, where a single value for each(prenominal) parameter is obtained for the whole population, flow cytometry provides data for every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not precisely to detect a particular cell font but to a fault to construe varied subpopulations accord ing to their structural or physiological parameters.Flow cytometry is a technique for metre components (cells) and the properties of individual cells in liquid suspension. In essence, suspend 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 crossways the detector, and fluorescence, absorbance, and on the loose(p) scattering are among the cell properties that clear be detected. Flow sorting allows individual cells to be sorted on the basis of their deliberate properties, and one to tercet or to a greater extent(prenominal) global properties of the cell can be measured. Flow cytometers and cell sorters make use of one or much than excitation sources and one or two fluorescent dyes to measure and characterize several thousands of cells per second. Flow cytometry gives object lens and sinless resolvings (Bunthof et al., 2001 Shleeva et al., 2002), overcoming the problems with the manual methods depict above.Function and types of flow cytometryFluidics, optics and electronics are the three of import systems that make up a flow cytometer. In a a few(prenominal) minutes, the flow cytometer 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 began when it was use for measuring their DNA content (Evenson et al., 1980) and its application to semen analysis has gradually change magnitude over the last 10-15 years. Flow cytometry is now applied to semen evaluation of traits such as cell viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity and DNA status. refreshful fluorescent markings and techniques are perpetually being developed that have potential application to the flow cytometric evaluation of spermatozoa.Flow cytometry permits the observation of physical characteristics, such as cell size, contour 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 data-based repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry similarly has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, importation that more than one sperm attribute can be assessed simultaneously. This peculiarity has an added benefit for semen analysis, as few single sperm parameters show significant correlation with fertility in vivo for semen within the refreshing epitome of normality (Larsson and Rodriguez-Martinez, 2000) and the more sperm parameters that can be tried, the more accurate the fertility farsightedness becomes (Amman and Hammerstedt, 1993).There are two main types of flow cytometers-analysers and sorters. Sorters have th e ability not precisely to collect data on cells (analyse cells) but also 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 constitute a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid sleuthing, characterization and enumeration of microorganisms. Luminex is developing applied science for multiplexed analyte quantitation using a faction of microspheres, flow cytometry and high speed digital bear on.Advantages of FC compared to other conventional techniques to search sperm structure and functionDuring the past 2 decades, there has been an increasing interest in reliable assays for assessing semen quality in the fertility clinic and artificial insemination industries. The use of flow cytometry for sperm analysis is an attempt to address the long-standing problem of the subjective dis touch of the manual method commonly apply for semen analysis. An additional source of laboratory variation is the low number of sperms crumpled with manual techniques. Because of time and constitute restraints, most laboratories analyze only 50 to coulomb sperm to forecast the percentage of each cell population and the viability rate. This small sample from a population of millions credibly results in a statistical sampling misunderstanding (Russel and Curtis, 1993). The conventional methods apply are moderate to microscopic determination of sperm immersion using a hemocytometer (Jorgensen et al., 1997) and evaluation of sperm motility and word structure (Keel et al., 2002). These methods usually involve a subjective assessment of a few hundred sperm, and quality bureau is rarely implemented in the laboratories do such analysis. Flow cytometry is a technique that is superior to conventional light microscopy techniques in term s of objectivity, number of cells measured, speed, and precision (Spano and Evenson, 1993). The technique has been utilize on human sperm to bump a number of factors, including membrane integrity, mitochondrial function, acrosome status, and multiparameter measurement (Garrido et al., 2002). Flow cytometry permitted us to analyze thousands of cells in few seconds. In our serial of studies, we demonstrated the feasibility and duplicability of an automated method to appraise sperm cell type, count, and viability in human grunter samples. In our hand, the precision of the flow cytometric analysis is satisfactory in diverse species (boar, bull, stallion etc), and the observed CVs were significantly better than those reported for the manual method.While there are many advantages of using the flow cytometer for routine semen analysis, its use is often limited to research by the put down and difficulties of operation associated with the requirement of a experient operator. In addit ion, a flow cytometer is quite large and cannot withstand shocks associated with movement, pith it requires a dedicated position in the laboratory. However, the development of more low-cost bench-top flow cytometers has recently change magnitude the potential application to semen analysis.If we fence flow cytometric analysis further, we can inspect that it is gaining wider acceptance as a technique for assessing the acrosome reaction and viability simultaneously. compare these assays to the more widely utilize epifluorescent microscopic techniques, the flow cytometric analysis is able to give a far more simple and objective method of analysis, especially with regard to correlation of saturation with acrosome reactivity potential (Uhler et al., 1993 Purvis et al., 1990 Carver-Ward et al., 1996).A large number of different techniques to estimate sperm preoccupancy have been reported. In the mid-1990s a series of fixed-depth expendable slides were evaluated as rapid and stron g beaks of equipment for the estimate of sperm concentration. Preliminary data from a number of studies suggested that, at least in the 20-mm-depth format, such house resulted in a marked underestimate of sperm concentration compared to the bullion exemplar (improved Neubauer hemocytometer). Using this information, the land Health Organization stated that such chambers, whilst convenient in that they can be used without dilution of the specimen, may lack the accuracy and precision of the haemocytometer technique (World Health Organization, 1999). Further datafor example, from Tomlinson and colleaguesshowed that 2 proprietary disposable slides (Microcell, universe Technologies, San Diego, Calif Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) gave lower sperm concentrations compared to the hemocytometer method (Tomlinson et al., 2001). To put this in context, many reports document unacceptable discrepancies mingled with different laboratories and even amidst differe nt individuals, although fewer studies attempt to address these issues. So, what is wrong(p)?Several reports emphasize the conduct for improvement in overall quality of semen testing within and amidst laboratories (Neuwinger et al., 1990 Jorgensen et al., 1997 Keel et al., 2000). However, the subjective nature of conventional semen analyses, combine with their relatively low precision due to the low number of cells assessed, leads to deplorable intra- and interlaboratory reproducibility therefore, the introduction of standardized or quality conditionled procedures will probably have a limited entrap. The conventional analyses are used to chequer whether parameters obtained from an ejaculate are within the range characterized by fertile men, and these methods can therefore provide only ill-defined cut-off values when used for the prescience of fertility status. Many of the advantages that accrue when using flow cytometry may, when applied to assessment of sperm cells, help overcome some of the mentioned problems arrange in conventional semen analysis.In the field of semen analysis, validation of a method is important because it is essential to have specific, precise, objective, and accurate laboratory tests to establish a correlation of the data with fertility or to rule the fertility potential of a semen sample correctly (Amann, 1989). Precision of a laboratory test is of great concern to the andrologist in the fertility clinic, since the results of the semen analysis are often used to interpret a patient about(predicate) his fertility and the prognosis for the treatment of the couple. To use established cut-off values and suss out uniform diagnosis, within and between laboratory variations should be determined and followed closely. faithful determination of sperm cell concentration is critical to the AI industry because it provides assurance two to bull studs and to customers that straws of extended semen contain the sperm numbers indicated . An accurate measure of sperm concentration is particularly important in export markets in which hitch of numbers may be required. Routine sperm counts can help to identify possible treat errors within a specific potentiometer of semen or on a particular day, should those errors occur. As sperm number procedures become more refined, routine counting can be used to monitor problematic changes in daily semen processing that might affect the number of sperm packaged in a straw.Hemacytometers are widely used for routine sperm counts, but the equipment is slow, and multiple measurements of each sample are needed. Single hemacytometer counts are not highly accurate because of inherent errors in the technique, Freund and Carol (13) bring that mean differences of 20% were not uncommon between duplicate sperm count determinations by the corresponding technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicia ns. However, those instruments tend to include in the sperm count any material cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (19). soon, the simple method used by the AI industry to estimate sperm concentration is spectrophotometric determination of turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (1). This approach is only as accurate as the methods used for spectrophotometer calibration.New, more accurate methods for sperm count determinations are being sought to replace the older ones. many 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 liable(predicate) have standard lems similar to those associated with the standard hemacytometers.It may be argued that when examine fluorescent microscopy assays with flow cytometry, one is examining patterns of fluorescence earlier than fluorescence intensity, i.e., the flow cytometer is not up to(p) of discriminating sperm which have a fluorescent marker confine 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 unhomogeneous lectins and indicated that there is no significant difference between the two 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 comfortably distinguished by epifluorescence microscopy, whereas flow cytometry identifies the signal from the entire sperm.Additionally, conventional light microscopic semen assessment i s increasingly being replaced by fluorescent spot techniques, computer-assisted sperm analysis (CASA) systems, and flow cytometry (Pena et al., 2001 Verstegen et al., 2002). Additional advantages over live oning 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 physical cells in a semen sample.ViabilityThe viability of spermatozoa is a key determinant of sperm quality and prerequisite for lucky grooming. Viability of spermatozoa can be assessed by many 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 c ytometry estimate similar trend of viability. Currently flow cytometric procedures have been developed which simultaneously evaluate sperm cell viability, acrosomal integrity and mitochondrial function. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994 doubting doubting Thomas et al., 1998), boars (Rodrguez-Martnez, 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), fowl 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). sizable 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 expert in upkeep sperm. steady-go ing agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test the flow cytometric method showed a precision level higher(prenominal) than that of the manual method.One of the first attempts to assess sperm viability utilized rhodamine 123 (R123) to assess mitochondrial membrane potential and ethidium bromide to determine membrane integrity using flow cytometry (Garner et al., 1986). Other cabals 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).At present, one of the most commonly used viability stain combinations is SYBR-14 and PI, sold commercially as LIVE/DEAD Sperm Viability kit out (Molecular Probes Inc., OR, USA). When used in c ombination, the nuclei of living sperm fluoresce kB (SYBR-14) and cells that have lost their membrane integrity stain red (PI). 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 make love/Dead stain may in like manner have no uncomely affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been used to evaluate boar sperm concentration 11. cod to its compact size and its relatively inexpensive purchase price, this instrument could be effectual for field measurements of both concentration and viability. This instrument was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability (Morrell et al., 2010).Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that o perates in the ultraviolet light range, are less commonly used as this is not a standard feature on the small analytical machines. However, one preference 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 dead bull spermatozoa by fluorometry and found a negative correlation between the percentage of defiled cells and 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 either approach can be used in combination with each other. For example, when CFDA is used along with PI, three populations of cells can be identified live, which are green dead, which are red an d a third population which is stained with both and represents dying spermatozoa. Almlid and Johnson (1988) found this combination useful for monitoring membrane damage in frozen-thawed boar spermatozoa during evaluation of various freezing protocols. Harrison and Vickers (1990) also used this combination with a fluorescent microscope and found it to be an centreive index number of the viability of fresh, incubated or cold-shocked boar and ram spermatozoa. Garner et al. (1986) used this combination to stain spermatozoa from a number of species, but at that time could not define a relationship between bull sperm viability detected by CFDA/PI and fertility.Flow cytometry for assessment of sperm viability appears to be a important tool for the AI industry. When a high number of sperm is jam-packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect on NRR56. However, sperm viability might be more import ant when have 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 combination of assessment of sperm viability and concentration appears to be useful in the improvement of 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 coruscant fluorescence was found also in the mitochondrial pillowcase of living sperm. The mechanism by which SYBR-14 binds to the DNA is not known. It is know that PI stains nucleic acids by intercalating between the base pairs (Krishan, 1975). Viability stains have also been used in association with fluorescently labeled show lectins to simultaneously assess the blood plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). Assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garn er et al. (5) demonstrated that insemination of boar spermatozoa stained with SYBR-14 into sows did not compromise fertilization or the development of inflamed porcine embryos in culture.Non-viable cells can be determined using membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with modify membranes. An full plasma membrane will retard these products from entering the spermatozoa and staining the nucleus. normally used examples 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). Wilhelm et al. (1996) compared the fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality and found that viability, as assessed by flow cytometry using PI, was the single laboratory assay that correlated with stallion fertility .Changes in sperm membrane permeability detection of increased membrane permeability is employed in different cell types to distinguish different status of membrane organization (Cohen, 1993 Ormerod et al., 1993 Castaneda and Kinne, 2000 Reber et al., 2002). Sperm plasma membrane status is of finale importance due to its role, not only as a cell boundary, but also for its need 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 stableness of its semipermeable features are prerequisites for the viability of the spermatozoon (Rodriguez-Martinez, 2006). However, cryopreservation, whose mean 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 d uring 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 cut again when cooling rates are slow enough to proceed 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. simple 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 Rodrguez-Martnez, 2000). A new, simple and repeatable method to detect membrane changes in all spermatozoa present in a b oar semen sample, by use of markers (combination of SNARF-1, YO-PRO-1 and ethidium homodimer) used to sink in changes in sperm membrane permeability, has been developed recently by our class (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 by and by destabilization of the membrane, under conditions where ethidium homodimer does not. Because several ATP-dependent channels have been detected in spermatozoa (Acevedo et al.,Sperm Assessment Using Flow CytometrySperm Assessment Using Flow CytometryAbstractFlow cytometry is emerging as an important tool in the field of modern andrology for routine analysis of spermatozoa. Recently, application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Unti l very recent, semen sample analysis was routinely performed by microscopical evaluation and manual techniques by laboratory operators the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. The last decade, several new flow cytometric techniques have been introduced for farm animal semen assessment that enable a more detailed evaluation of several sperm characteristics. Here in this paper, an initiative has been taken to focus on a number of recent flow cytometry developments important for addressing questions in andrological tests.After the invention of flow cytometry, sperm evaluation by traditional microscopic means became questioned due to the robust advantages of flow cytometry over the microscopic method. Due to the recent development of large number of fluroscence probes, flow cytometry is now capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane perme ability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gamets 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 possibility of working with small sample sizes, increases the repeatability of assessment, removes the subjectivity of assessment and allows simultaneous assessment of multiple fluorochromes. Flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not possible with any other technique. Nowadays, semen evaluation using laboratory assays is extremely important 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 parameters 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.IntroductionSemen evaluation is the single most important laboratory test that has helped us to identify clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (Rodrguez-Martnez, 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. Methods are available that can sometimes estimate the potential fertilizing capacity of a semen sample and, in some cases, of the male (reviewed by Dziuk 1996 Rodrguez-Martnez et al. 1997a Rodrguez-Martnez and Larsson 1998 Saacke et al. 1998 Larsson and Rodrguez-Martnez 2000 Rodrguez- Martnez 2000, 2003 Popwell and Flowers 2004 Graham and Moc 2005 Gillan et al. 2005). The methods r outinely used for evaluation of the quality of a semen sample involved an evaluation of general appearance (i.e. colour, contamination, etc.), volume, pH, sperm concentration, viability, morphology and motility. Most of these techniques are microscopic analyses that only measure a small number of spermatozoa within a population, are time-consuming, can be subjective and generally measure sperm attributes individually. Recently, limitations of semen evaluation methodology have been brought into sharp focus by controversies raised in the epidemiological literature. It should also be noted that such conventional measurements are prone 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 spe rmatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. Such handling implies semen extension, fluorophore loading, ultraviolet and laser illumination, high-speed sorting, cooling and cryopreservation, procedures that impose different degrees of change in sperm function following damage to sperm membranes, organelles or the DNA. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed that buck of these procedures are incomplete, time consuming and laborious.Flow cytometry in different technical applications offers many advantages for the analysis of sperm quality. Flow cytometry allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cell or microscopic particles in suspension, as they flow very rapidly through a sensing area. The increasing use over the pas t 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 can acquire data on several subpopulations within a sample in a few minutes, making it ideal for assessment of heterogenous populations in semen sample. Initially developed in the 1960s, flow cytometry made automated separation of cells based on the unique recognition of cellular patterns within a population feasible (Hulett et al., 1969). Using such a separation approach, cellular patterns can be identified by assessing, in individual cells within a population, protein expression using fluorescently labeled antibodies and other fluorescent probes (Baumgarth and Roederer, 2000 Herzenberg et al., 2006).Flow cytometry was first developed for medical and clinical applications such as haematology and oncology. These areas still account for the vast majority of publications on this technique, but during the past few years it has been used in other areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. 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 cytometryThe definition of a flow cytometer is an instrument which measures the properties of cells in a flowing stream. In other word, a flow cytometer will be defined as an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream.In other work, cytometry refers to the measurement of physical and/or chemical characteristics of cells or, by extension, of other particles. It is a process in which such measurements are made while the cells or particles pass, preferably in sing le file, through the measuring apparatus in a fluid stream. 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 analytical tools, where a single value for each parameter is obtained for the whole population, flow cytometry provides data for 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 scatterin g 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 gives objective and accurate results (Bunthof et al., 2001 Shleeva et al., 2002), overcoming the problems with the manual methods described above.Function and types of flow cytometryFluidics, optics and electronics are the three main systems that make up a flow cytometer. In a few minutes, the flow cytometer 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 began when it was used for measuring their DNA content (Evenson et al., 1980) and its app lication to semen analysis has gradually increased over the last 10-15 years. Flow cytometry is now applied to semen evaluation of traits such as cell viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity and DNA status. New fluorescent stains and techniques are continuously being developed that have potential application to the flow cytometric evaluation of spermatozoa.Flow cytometry permits 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. T his 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 the more sperm parameters that 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. Sorters have the ability not only to collect data on cells (analyse cells) but also 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 us ing a combination of microspheres, flow cytometry and high speed digital processing.Advantages of FC compared to other conventional techniques to explore sperm structure and functionDuring the past 2 decades, there has been an increasing interest in reliable assays for assessing semen quality in the fertility clinic and artificial insemination industries. The use of flow cytometry for sperm analysis is an attempt to address the long-standing problem of the subjective nature of the manual method commonly used for semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with manual techniques. Because of time and cost restraints, most laboratories analyze only 50 to 100 sperm to compute the percentage of each cell population and the viability rate. This small sample from a population of millions probably results in a statistical sampling error (Russel and Curtis, 1993). The conventional methods used are limited to microscopic determination of s perm concentration using a hemocytometer (Jorgensen et al., 1997) and evaluation of sperm motility and morphology (Keel et al., 2002). These methods usually involve a subjective assessment of a few hundred sperm, and quality assurance is rarely implemented in the laboratories performing such analysis. Flow cytometry is a technique that is superior to conventional light microscopy techniques in terms of objectivity, number of cells measured, speed, and precision (Spano and Evenson, 1993). The technique has been used on human sperm to determine a number of factors, including membrane integrity, mitochondrial function, acrosome status, and multiparameter measurement (Garrido et al., 2002). Flow cytometry permitted us to analyze thousands of cells in few seconds. 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 ana lysis is satisfactory in diverse species (boar, bull, stallion etc), and the observed CVs were significantly better than those reported for the manual method.While there are many advantages of using the flow cytometer for routine semen analysis, its use is often limited to research by the expense and difficulties of operation associated with the requirement of a skilled operator. In addition, a flow cytometer is quite large and cannot withstand shocks associated with movement, meaning it requires a dedicated position in the laboratory. However, the development of more affordable bench-top flow cytometers has recently increased the potential application to semen analysis.If we consider flow cytometric analysis further, we can see that it is gaining wider acceptance as a technique for assessing the acrosome reaction and viability simultaneously. Comparing these assays to the more widely used epifluorescent microscopic techniques, the flow cytometric analysis is able to give a far more simple and objective method of analysis, especially with regard to correlation of fertilization with acrosome reactivity potential (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. Preliminary data from a number of studies suggested 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). Using this information, the World Health Organization stated that such chambers, whilst convenient in that they can be used without dilution of the specimen, may lack the accuracy and precision of the haemocytometer technique (World Health Organization, 1999). Further datafor example, from Tomlinson and colleagues showed that 2 proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) gave lower sperm concentrations compared to the hemocytometer method (Tomlinson et al., 2001). To put this in context, numerous reports document unacceptable discrepancies between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong?Several reports emphasize the need for improvement in overall quality of semen testing within and between laboratories (Neuwinger et al., 1990 Jorgensen et al., 1997 Keel et al., 2000). However, the subjective nature of conventional semen analyses, combined with their relatively low precision due to the low number of cells assessed, leads to poor intra- and interlaboratory reproducibility therefore, the introduction of standardized or quality controlled procedures will probably have a limited effect. The conventional ana lyses are used to determine whether parameters obtained from an ejaculate are within the range characterized by fertile men, and these methods can therefore provide only unclear cut-off values when used for the prediction of fertility status. Many of the advantages that accrue when using flow cytometry may, when applied to assessment of sperm cells, help overcome some of the mentioned problems found in conventional semen analysis.In the field of semen analysis, validation of a method is important because it is essential to have specific, precise, objective, and accurate laboratory tests to establish a correlation of the data with fertility or to determine the fertility potential of a semen sample correctly (Amann, 1989). Precision of a laboratory test is of great concern to the andrologist in the fertility clinic, since the results of the semen analysis are often used to advise a patient about his fertility and the prognosis for the treatment of the couple. To use established cut-of f values and ensure uniform diagnosis, within and between laboratory variations should be determined and followed closely.Accurate determination of sperm cell concentration is critical to the AI industry because it provides assurance both to bull studs and to customers that straws of extended semen contain the sperm numbers indicated. An accurate measure of sperm concentration is particularly important in export markets 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.Hemacytometers are widely used for routine sperm counts, but the equipment is slow, and multiple measurements of each sample are needed. Single hemacytometer counts are not highly a ccurate because of inherent errors in the technique, Freund and Carol (13) found that mean differences of 20% were not uncommon between duplicate sperm count 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 (19).Currently, the primary method used by the AI industry to estimate sperm concentration is spectrophotometric determination of turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (1). This approach is only as accurate as the methods used for spectrophotometer calibration.New, more accurate methods for sperm count determinations are being sought to replace the older ones. Some laboratories a re 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.It may be argued that when comparing fluorescent microscopy assays with flow cytometry, one is examining patterns of fluorescence rather than fluorescence intensity, i.e., the flow cytometer is not capable of discriminating sperm which have 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 no significant difference between the two methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the acrosoma l 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 (Pena 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.ViabilityThe 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. Currently flow cytometric procedures have been developed which simultaneously evaluate sperm cell viability, acrosomal integrity and mitochondrial function. 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 (Rodrguez-Martnez, 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 (R123) to assess mitochondrial membrane potential and ethidium bromide to determine membrane integrity using 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).At present, one of the most commonly used viability stain combinations is SYBR-14 and PI, sold commercially as LIVE/DEAD Sperm Viability kit (Molecular Probes Inc., OR, USA). When used in combination, the nuclei of living sperm fluoresce green (SYBR-14) and cells that have lost their membrane integrity stain red (PI). 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 used to evaluate boar sperm concentration 11. Due to its compact size and its relatively inexpensive purchase price, this instrument could be useful for field measurements of both concentration and viability. This instrument was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability (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 found a negative correlation between the percentage of damaged cells and 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 mot ility.Fluorochromes used to assess sperm viability by either approach can be used in combination with each other. For example, when CFDA is used along with PI, three populations of cells can be identified live, which are green dead, which are red and a third population which is stained with both and represents dying spermatozoa. Almlid and Johnson (1988) found this combination useful for monitoring membrane damage in frozen-thawed boar spermatozoa during evaluation of various freezing protocols. Harrison and Vickers (1990) also used this combination with a fluorescent microscope and found it to be an effective indicator of the viability of fresh, incubated or cold-shocked boar and ram spermatozoa. Garner et al. (1986) used this combination to stain spermatozoa from a number of species, but at that time could not find a relationship between bull sperm viability detected by CFDA/PI and fertility.Flow cytometry for assessment of sperm viability appears to be a valuable tool for 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 on NRR56. 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 combination of assessment of sperm viability and concentration appears to be useful in the improvement of 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. The mechanism by which SYBR-14 binds to the DNA is not known. It is know that PI stains nucleic acids by intercalating between the base pairs (Krishan, 1975). Viability stains have also been used in association wit h fluorescently labeled plant lectins to simultaneously assess the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). Assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) demonstrated that insemination of boar spermatozoa stained with SYBR-14 into sows did not compromise fertilization or the development of flushed porcine embryos in culture.Non-viable cells can be determined using membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. An intact plasma membrane will prevent these products from entering the spermatozoa and staining the nucleus. Commonly used examples 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). Wilhelm et al. (1996) compared the fertility of cryopres erved stallion spermatozoa with a number of laboratory assessments of semen quality and found that viability, as assessed by flow cytometry using PI, was the single laboratory assay that correlated with stallion fertility.Changes in sperm membrane permeabilityDetection of increased membrane permeability is employed in different cell types to distinguish different status of membrane organization (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 need 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 purp ose 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 Rodrguez-Martnez, 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 m