This dissertation has been submitted by a student. This is not an example of the work written by our professional dissertation writers.
State of the art in sperm assessment using flow cytometry
Flow cytometry is emerging as a substantial tool in the domain of modern andrology for the routine analysis of spermatozoa. Recent application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, analysis of semen samples was routinely performed by microscopical evaluation and manual techniques by laboratory operators; analysis is inclined due to comprehensive variability among observers, influencing its clinical validity. During last decade, to evaluate farm animal semen, variety of new flow cytometric techniques have been intercalated which made possible a wide spread evaluation of several sperm functionality and characteristics. Here in this paper, an initiative has been taken to explore numerous current flow cytometry developments pressing for andrological tests.
After the invention of flow cytometry, sperm evaluation by traditional (microscopic) means became questioned and avoided due to the robust advantages of flow cytometry over the microscopic methods. By the recent development of diverse fluroscence probes, flow cytometry became capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gametes based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the opportunity of working with small sample sizes, increases the repeatability of data obtained, removes the subjectivity of evaluation and allows simultaneous assessment of multiple fluorochromes. Thus, flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of experiments that are not yet possible with any other technique. Nowadays, semen evaluation using laboratory analyses is very meaningful to the artificial insemination industry to provide the most desired quality product to customers.
Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen attributes that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed.
Up to now, semen evaluation is considered as the most important laboratory test that has enabled us to identify and predict clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (Rodríguez-Martínez, 2007). Determination of the potential fertility of semen sample and, in the long run, of the male from which it has been collected is the ultimate goal of semen evaluations in clinically healthy sires. Now a days, many methods for the estimate the possible fertilizing capacity of a semen sample and, or in the word, of the male (reviewed by Dziuk 1996; Rodríguez-Martínez et al. 1997a; Rodríguez-Martínez and Larsson 1998; Saacke et al. 1998; Larsson and Rodríguez-Martínez 2000; Rodríguez- Martínez 2000, 2003; Popwell and Flowers 2004; Graham and Mocé 2005; Gillan et al. 2005) are existing. The methods routinely accustomed for evaluation of the quality of a semen sample involved an evaluation of general appearance, volume, pH, sperm concentration, viability, morphology and motility. Most of these evaluations are based on microscopic analyses that only measure relatively a few numbers of spermatozoa within a population. In most of the cases, these are time-consuming; results obtained are controversial and are not translatable. It should also be noted that such conventional techniques are apt to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. During this long processes, number of steps like semen extension, fluorophore loading, ultraviolet and laser illumination, high-speed sorting, cooling and cryopreservation are followed, which create a scope to impose different degrees of change in sperm functionality followed by suffer of damage to sperm membranes, organelles or the DNA content. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed by many groups that buck of those so-called procedures are incomplete, time consuming and laborious.
Flow cytometry in diverse technical applications proposes many advantages for the analysis of sperm quality. Flow cytometry is a method where multiple fluorescences and light scattering can be induced allowing single cell or particles illumination in suspension while they flow very rapidly through a sensing area. The increasing use over the past decade of flow cytometry in the leading laboratories in human and veterinary andrology has dramatically increased our knowledge of sperm function under physiological and biotechnological conditions. Flow cytometers is capable to acquire data from several subpopulations within a sample in a few minutes, making it perfect for assessing heterogenous populations in a semen sample. Flow cytometry was initially developed in the 1960's, after that flow cytometry is performing automated separation of cells based on the unique recognition of cellular patterns in a population feasible (Hulett et al., 1969). Likewise, cellular patterns can be recognized by utilizing such a separation approach, in each cells within a population (Baumgarth and Roederer, 2000; Herzenberg et al., 2006).
The first notion of flow cytometry development was for medical and clinical applications such as haematology and oncology. Although still much research is going on these medical areas and account for the vast majority of publications on this robust technique, but during the past few years it is being used in a diverse areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Together with elevated use in many areas, recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 2005, 2006;) stallion ( Kavak et al., 2003; Morrell et al., 2008) indicate that newly developed fluorescent stains and techniques of flow cytometry has made possible a more widespread analysis of semen quality at a biochemical, ultrastructural and functional level. Therefore, flow cytometry is the current technical solution for rapid, precisely reproducible assessment of sperm suspensions.
In this review we have described potentiality and scope of flow cytometry for the evaluation of semen, and the way in which this technique can be used in clinical applications for andrology based on some of our previous experiences.
Definition of flow cytometry
The definition of a flow cytometer is ‘an instrument which measures the properties of cells in a flowing stream' or ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream'.
In other word, cytometry is a method which measure physical and chemical attributes of cells or other particles. Such a measurement is made when cells or other particles pass in single file through some sort of measuring apparatus in a stream of fluid. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other traditional analytical tools, where a single value for each attribute is obtained for the whole population, flow cytometry provides data for each and every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters.
Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light scattering are among the cell properties that can be detected. Flow sorting allows individual cells to be sorted on the basis of their measured properties, and one to three or more global properties of the cell can be measured. Flow cytometers and cell sorters make use of one or more excitation sources and one or two fluorescent dyes to measure and characterize several thousands of cells per second. Flow cytometry presents objective and precise results (Bunthof et al., 2001; Shleeva et al., 2002), which help to overcome the problems with the manual methods described above.
Function and types of flow cytometry
A flow cytometer is made of three main systems, fluidics, optics and electronics. ItI It can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment came in to function when it was used for measuring their DNA content (Evenson et al., 1980) and its application for analyzing semen has been increased rapidly in last decade. Flow cytometry is now applied for the evaluation semen such as sperm viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity, DNA status and so on. Continuous innovation of new fluorescent stains and techniques facilitated the flow cytometric evaluation of spermatozoa.
Flow cytometry allows the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. This feature has an added benefit for semen analysis, as few single sperm parameters show significant correlation with fertility in vivo for semen within the acceptable range of normality (Larsson and Rodriguez-Martinez, 2000) and it is the general statistics that the more sperm parameters can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993).
There are two main types of flow cytometers-analysers and sorters are in use. Together with data collection on cells, sorters have the potentiality to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation using a combination of microspheres, flow cytometry and high speed digital processing.
Advantages of FC compared to other conventional techniques to explore sperm structure and function
Use of authentic assays in the fertility clinic and artificial insemination industries increasing day by day. In this respect, use of flow cytometry might be an important attempt to resolve sustaining problem with so called commonly used manual method for the semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with such techniques. It is worth mentinign here that so called method deal only with few hundred sperm. When we deal with such a few sperm population, there is a possibility that obtained result might not be statistically significant (Russel and Curtis, 1993). The methods which are frequently used are enable to determine sperm concentration (Jorgensen et al., 1997), motility or morphology only (Keel et al., 2002). Objectivity, cell number measured, speed of count and precision are the advantages of flow cytometry to conventional light microscopy techniques (Spano and Evenson, 1993). The technique now a days has been used to determine a number of factors including those of acrosome status, membrane integrity, mitochondrial function as well as multiparameter measurement in human (Garrido et al., 2002). Flow cytometry has the ability to analyze thousands of cells in few minutes. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in a diverse species (boar, bull, stallion etc), and the observed errors were significantly better than those obtained from the so-called manual methods.
Although there are diverse benefits of flow cytometer for the analysis of semen, feasibility of applying flow cytometry sometimes restricted to researcher due to the high outlay and difficulties of operation associated with the requirement of a skilled operator. Further, a flow cytometer is very large and cannot resist shocks associated with movement, and it also requires much space in the laboratory. Whatever may be the limitation, the development of more affordable ‘‘bench-top'' flow cytometers in recent time raised the potential essentialities to semen analysis.
If the further application of flow cytometric analysis is considered further, it might be seen that it is growing popularities as a technique for assessing more than one sperm attribute, simultaneously. Compared to traditional microscopic techniques, flow cytometry analysis is allowing to give a far more simplified and objective method of semen analysis, especially in relation to fertilization with acrosome reactivity potential of spermatozoa (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996).
A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Data from a number of preliminary studies proposed that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). According to the World Health Organization that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, might lead to inaccuracy (World Health Organization, 1999). Data from Tomlinson and colleagues indicate that two proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) can result in a lower concentrations of sperm compared to the hemocytometer method (Tomlinson et al., 2001). In contrast, plenty of reports document unacceptable differences between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong?
Improvement of semen quality testing has been emphasizing in some reports (Jorgensen et al., 1997; Keel et al., 2000). But due to low number of sperm evaluation by the conventional method results in poor reproducibility. These problems might be overcome when using flow cytometry. The validation of method is a challenge due to its essentiality of having specific, precise, objective, and accurate evaluation to establish a correlation of fertility data or to predict potential of a semen sample accurately (Amann, 1989). In a fertility clinic, precision of data in important as the result of semen analysis is frequently used to manage fertility of a patient and treatment of the unfertile couples. Thus, it is important to take into consideration within and between laboratory variations for successful infertility treatments.
Sometimes it's a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate “patterns" of fluorescence rather than the fluorescence intensity. Flow cytometer has the lack of ability to discriminate sperm containing a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is almost no difference between methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the acrosomal region of the sperm (Purvis et al., 1990; Holden and Trounson, 1991) and that other binding sites can be easily distinguished by epifluorescence microscopy, whereas flow cytometry identifies the signal from the entire sperm.
Additionally, conventional light microscopic semen assessment is increasingly being replaced by fluorescent staining techniques, computer-assisted sperm analysis (CASA) systems, and flow cytometry (Pen˜a et al., 2001; Verstegen et al., 2002). Additional advantages over existing techniques are that this approach is faster than the hemacytometer and that cellular debris, fat droplets, and other particulate material in extended semen are not erroneously counted as sperm, as often occurs with electronic cell counters. This method can also be used to determine the number of somatic cells in a semen sample.
Application of flow cytometry for sperm count
Sperm count is an important predominant factor for the evaluation of sperm fertility potential.
Accurate determination of sperm cell concentration is critical especially in AI industry because it provides assurance to customers that straws of extended semen contain the sperm numbers indicated which will help to decide appropriate doze especially for pig. Accuracy of sperm count is a common problem in the andrological laboratories and accurate measure of sperm concentration is particularly important for export in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Every time new and more accurate methods for the sperm count determinations are coming and being replaced by the older ones. Some laboratories are trying the Maklerm counting chamber (Seif- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. Although hemacytometers are routinely used for sperm counts, due to the slow process and need for multiple measurements of each sample, the chance of error increase. Freund and Carol (ref) stated that a difference of 20% were not unusual between the determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (Ref). Spectrophotometer is recently being used in the AI industries to assess sperm concentration by determining turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (Ref). The accuracy of this method depends on the methods used for spectrophotometer calibration. Although, sperm concentration can also be determined by spectrophotometrically, the debris present in the raw semen crease problem with misestimation. Sperm number in the frozen thawed semen is difficult to ascertain as most of the extender contain egg yolk particles, fats and other particles which affect measurement of sperm with electric cell counter or spectrophotometers (Evenson et al., 1993). On the other hand flow cytometry created possibilities of a rapid determination of sperm number in a precise form. It is the flow cytometry which can reduce intra-laboratory and inter-laboratory variation and conflict regarding sperm concentration assessment. Computer assisted semen analyzer is robust technique for analyzing sperm movement which can count sperm as well; but such an analyzer most of the cases use some counting chamber or hemacytometer which itself can generate error. Although, hemacytometer was originally developed for blood cell counting, its use is now diverse including andrological laboratories for sperm counting.
Around two-decade ago flow cytometry was suggested for sperm numbers in straws of cryopreserved bull semen. Christensen et al. (----) observed similar results for sperm count with flow cytometry and hemocytometer for a number of species. Now a day a simultaneous determination of sperm viability and sperm concentration is possible which can avoid the chance of occurring differences between ejaculates leading lack of coordination with field fertility and laboratory analyses. Thus the present technology is more precise which can get rid of variation from handling the sperm sample and variation from pipetting and the analysis itself. Further, Prathalingam et al. (2006) concluded that there is similarities for sperm count result between flow cytometry and two newly approached method (image analysis and fluorescent plate reader) for sperm counting. Though, use of fluorescent plate was emphasized due to low cost and allowing large number of cells counting from a large number of ejaculates.
Although flow cytometry has become a valuable instrument for andrological determinations, it is also blamed that sperm concentration by flow cytometry signify a higher value than the real one. The possibility arise might be due to that semen samples often contain some alien materials such as immature germ cells, epithelial cells, blood cells, cytoplasmic droplet, cellular debris etc. In the same way, frozen semen has higher chance to introduce such material as they contain diluents components especially egg yolk particles. These particles and cell debris might have frontal and side light scatter parameters those are similar to spermatozoa. Such sperm-count-overestimation problem arisen in our cases also, especially when we deal with frozen semen. Further it is also claimed that flow cytometry has a tendency to overestimate viable spermatozoa. We are also experienced with such trouble which we guess might be due to that egg particles of extender are considered as viable cell as for its staining pattern. Our yet to publish data indicate that this problem can be mimic by a centrifugation process and by using low concentration sample for evaluation with flow cytometry. Very recently Petrunkina and Harrison (2009) proposed a mathematical equation for fixing this flow cytometric sperm counting. Thus much research is going on and we hope such discrepancy will completely be resolved near future to get advantage from this robust technology for sperm counting.
Flow cytometry for detecting sperm intactness
-Viability of spermatozoa
The viability of spermatozoa is a key determinant of sperm quality and prerequisite for successful fertilization. Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable and subjectively assess only 100 to 200 spermatozoa per ejaculate. Merkies et al. (2000) compared different methods of viability evaluation. They concluded that Eosin-nigrosin overestimate viability while fluorescent microscope and flow cytometry estimate similar trend of viability. Current flow cytometric procedures are able to simultaneously evaluate sperm cell viability together with some other attributes. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (Rodríguez-Martínez, 2007; Garner and Johnson, 1995; Garner et al., 1996), rams (Garner and Johnson, 1995), rabbits (Garner and Johnson, 1995), mice (Garner and Johnson, 1995; Songsasen et al., 1997), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004).
Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method.
One of the first attempts to assess sperm viability utilized rhodamine 123 for determining potentiality of mitochondrial membrane while ethidium bromide for membrane integrity through flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990).
The most generally used sperm viability stain combinations is SYBR-14 and PI at present. This stains are now sold commercially as live/dead viability kit. When these two stains are combinely used, the nuclei of viable sperm take fluoresce green and membrane integrity lost cells take red stain. This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been introduced to evaluate sperm concentration  and viability. Due to the small size and low cost, this instrument has been attracted for field measurements of both concentration and viability. In our hand this instrument was also became useful for the quick measurement of sperm concentration and viability in stallion (Morrell et al., 2010).
Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and obtained an inverse correlation between the damaged cells per cent and the field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility.
Fluorochromes used to assess sperm viability by both approach could be utilized in combination with each other. In that case, when CFDA is used combined with PI, three populations of cells as live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa can be identified. This combination was found useful by Almlid and Johnson (1988) for frozen-thawed boar spermatozoa for monitoring membrane damage at the time of evaluation of various freezing protocols. Further, Harrison and Vickers (1990) also noticed that this combination with a fluorescent microscope is effective indicator of viability of fresh, incubated or cold-shocked spermatozoa in boar and ram. Contrasting to these, Garner et al. (1986) was failed to find a relationship between bull sperm viability and fertility when using combination of CFDA/PI .
Flow cytometry for evaluating sperm viability appears to be a precious tool in the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combined assessment of sperm viability and concentration appears to be useful in the wake of improving quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. But the mechanism and mode of action by which SYBR-14 binds to the DNA of sperm is not known. It is know that PI stains nucleic acids by intercalating between the base pairs (Krishan, 1975). Viability stains can also be used in conjugation with fluorescently labeled plant lectins for simultaneous assessment of the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). It is conceivable that assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) found that insemination of boar sperm stained with SYBR-14 did not compromise fertilization or even the development of flushed porcine embryos in vitro.
Non-viable sperms can be detected using the membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. Plasma membrane which is intact will not permit these stains entering into the spermatozoa and staining the nucleus. Most frequently used stains include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). After a series of comparison between fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality as assessed by flow cytometry using PI, Wilhelm et al. (1996) concluded that viability is the single laboratory assay that correlated with fertility.
-Sperm plasma membrane integrity
Although the sperm plasma membrane covers the entire cell, it consists of three distinct membrane compartments, one which covers the outer acrosomal membrane, one which covers the post acrosomal portion of the sperm head, and one which covers the middle and principal pieces. Sperm membrane is directly or indirectly related with many sperm functions has influence on sperm metabolism for conserving motility, capacitation, acrosome reaction, interactions between sperm and epithelium of the female genital tract, as well as sperm-egg interactions (Rodriguez-Martinez, 2003). Most ‘viability assays' assess whether or not the plasma membrane is intact (the cell is ‘viable') or not (the cell is ‘dead'). However, because the plasma membrane is composed of these different compartments, different viability assays assess the integrity of different plasma membrane compartments. Classical stains, such as eosin-nigrosin and eosin aniline blue, as well as more recent fluorescent stains, such as propidium iodide, ethidium bromide, 4-6-diamidino-2-phenylindole and bisbenzimide (Hoechst dyes), bind to and stain the DNA of sperm that possess a post acrosomal plasma membrane that is not intact. However, these probes will not assess the integrity of the plasma membrane covering the acrosome or principal piece. The integrity of the plasma membrane covering the principal piece can be assessed using sperm motility or the hypo-osmotic swelling test ( Neild et al., 2000; Colenbrander et al., 2003). The integrity of the plasma membrane covering the acrosome is generally assessed in conjunction with the integrity of the outer acrosomal membrane. The acrosomal ridge, present on the sperm of several species, can be used to assess the integrity of this plasma membrane by differential interference phase contrast microscopy, but this anatomical component can only be used for sperm from those species which possess it. Several non-fluorescent and fluorescent staining combinations (reviewed by Cross and Meizel, 1989; Graham, 2001; Colenbrander et al., 2003; Silva and Gadella, 2006) have been developed to permit assessment of acrosomal membrane integrity of fresh and fixed sperm samples using microscopy, fluorometry and flow cytometry.
During the last decade, for dog, many fluorescent dyes were introduced and validated for the assessment of the sperm membrane integrity:combination of carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Pen˜a et al., Rota et al., 1995), SYBR-14 in combination with PI (Rijsselaere et al., 2002; Yu et al., 2002), combination of carboxy-seminaphthorhodfluor (Carboxy-SNARF) with PI (Pen˜a et al., 1999), calcein-AM in combination with ethidium homodimer (Calcein-AM/EthD-1) (Sirivaidyapong et al., 2000) and Hoechst 33258 (Hewitt and England, 1998). The main advantage of these fluorescent staining techniques is the possible analysis of fluorescently labelled sperm with the help of flow cytometry, which enabled evaluation of larger numbers of spermatozoa within few minutes. Pen˜a et al. (1998, 2001) found high correlations between flow cytometry and epifluorescence microscopy for the percentage of live and dead spermatozoa as determined by a CFDA-PI staining.
Apoptosis is a carefully regulated process of cell death that occurs as a normal component of development. During this process, the plasma membrane became slightly permeable and loses asymmetry in one of the earliest stages of apoptosis. When the cell membrane is disturbed the phospholipid results in a translocation of PS (Desagher and Martinou, 2000). Recently, it has been demonstrated in human (Kemal et al., 2001) and bull (Anzar et al., 2002) that freezing-thawing of semen induces membrane PS translocation, thus indicating cryopreservation is a cause of apoptosis (Baust, 2002). At the early stage of hampered plasma membrane activity, asymmetry of membrane PL happens (Ref). In all studied mammalian cell types including spermatozoa, the two leaflets of the plasma membrane bilayer differ in its phospholipid content. Phosphatidylserine and phosphatidylethanolamine these two are concentrated in the inner leaflet, whereas sphyngomyeline and phosphatidylcholine are concentrated in the outer leaflet (Ref).
As the sperm membrane are extravagant susceptible to cryoinjury, the evaluation of sperm membranes is a proper indicator of the success of cryopreservation (Ref). Number of deleterious effects of boar sperm manipulation such as excessive extension, sorting, chilling or cryopreservation that lead to membrane destabilization. The procedure of freezing and thawing adversely affect the lipid composition of boar sperm plasma membranes not only in poor quality sperm but also in good sperm quality (Ref). Lipid phase transition, ice crystallization, as well as membrane-recognizations exert membrane integrity, structure and function which occur due to adverse influence of cooling, freezing and thawing procedure (Hammerstedt et al., 1990). In general plasma membranes of spermatozoa contain the high amount of long-chain PUFA compare to other somatic cells. In recent studies a close relation between differences in PUFAs within plasma membranes with cryotolerance in sperm has been identified in different species (Swain and Miller 2000; Miller et al., 2004; Miller et al., 2005).
Although high cholesterol to phospholipid ratio in sperm membranes is considered to be positively associated with cryotolerance, between species there is a variation. Some animals are good freezer and some are bad freezer rather than any individual ejaculates. Further, variation in cryopreservation-induced injuries has been claimed have genetic basis where plasma membrane and acrosomal membrane have been found disrupted (Thurston et al., 2002). Holt et al.( 2005) suggested that differences between male to male may cause differences in sperm lipid and protein composition, and the underlying mechanism for this genetic diversity associated with cryoinjuries yet to be elucidate. Addition of cryoprotectants prior to freezing, volumetric changes and associated membrane stretching and shrinkage in response to hyperosmotic cryoprotectant solutions as well as freeze-induced dehydration, thermotropic and lyotropic phase transitions in membrane phospholipids, and the well-established effects of elevated solute concentration and intracellular ice formation which are cooling rate-dependent.
-Changes in sperm membrane permeability
Detection of the membrane permeability in different cell types is considered to be able to differentiate different membrane organization status (Cohen, 1993; Ormerod et al., 1993; Castaneda and Kinne, 2000; Reber et al., 2002). Sperm plasma membrane status is of utmost importance due to its role, not only as a cell boundary, but also for its importance for cell-to-cell interactions, e.g. between spermatozoa and the epithelium of the female genital tract and between the spermatozoon and the oocyte and its vestments (for review, see Rodriguez-Martinez, 2001). Membrane integrity and the stability of its semipermeable features are prerequisites for the viability of the spermatozoon (Rodriguez-Martinez, 2006). However, cryopreservation, whose purpose is to warrant sperm survival, causes irreversible damage to the plasma membrane leading to cell death in a large number of spermatozoa (Holt, 2000) or, in the surviving spermatozoa, to changes similar to those seen during sperm capacitation, thus shortening their lifetime (Perez et al., 1996; Cormier et al., 1997; Maxwell and Johnson, 1997; Green and Watson, 2000; Schembri et al., 2000; Watson, 2000). During the freezing process, cells shrink again when cooling rates are slow enough to prevent intracellular ice formation as growing extracellular ice concentrates the solutes in the diminishing volume of non-frozen water, causing intracellular water exosmosis. Though warming and thawing, the cells return to their normal volume. Thus, it is important to know the permeability coefficient of the cells to cryoprotectants, as well as the effect of cryoprotective agents on the membrane hydraulic conductivity.
Classical combination of probes allows discrimination of two or three subpopulations of spermatozoa, i.e. live, dead and damaged depending on the degree of membrane integrity (Eriksson & Rodrı´guez-Martı´nez, 2000). A new, simple and repeatable method to detect membrane changes in all spermatozoa present in a boar semen sample, by use of markers (combination of SNARF-1, YO-PRO-1 and ethidium homodimer) used to track changes in sperm membrane permeability, has been developed recently by our group (Pena et al., 2005). In determined physiological or pathological situations, live cells are unable to exclude YO-PRO-1, but are still not permeable to other dead-cell discriminatory dyes, like propidium iodide or ethidium homodimer. YO-PRO-1 is an impermeable membrane probe and can leak in, only after destabilization of the membrane, under conditions where ethidium homodimer does not. Because several ATP-dependent channels have been detected in spermatozoa (Acevedo et al., 2006), it seems plausible that this is a result of the silencing of a multidrug transporter. This multidrug transporter is involved in transporting amphipathic small molecules like YO-PRO-1, which in intact cells is actively pumped out but not after destabilization of the plasma membrane, maybe because sub-viable cells lack appropriate amounts of ATP to transport YO-PRO-1 back out of the cell (Ormerod et al., 1993). Therefore, the use of a fluorescent probe, such as YO-PRO-1, which penetrates cells as they undergo changes related to cryoinjury, where membranes become slightly permeable, makes YO-PRO-1 a useful tool for detecting early membrane changes (Idziorek et al., 1995; Wronski et al., 2000). This triple staining distinguishes, as in the Annexin assay, four sperm subpopulations. The three probes are easily distinguished both in flow cytometry and in fluorescence microscopy. The absorption and emission maxima for YO-PRO-1 are 491 nm and 509 nm, respectively, and 528 nm and 617 nm, respectively, for ethidium homodimer to be detected in the Flow Cytometer with the FL1 and FL3 photomul-tipliers.
The triple staining technique offers some advantages over the A/PI assay. Whereas in the A/PI assay there is always an unstained subpopulation, the triple stain labels all the spermatozoa in the sample, an obvious advantage when using manual counting in fluorescence microscopy. If a flow cytometer is available, because only sperm cells are stained with the triple staining technique, spermatozoa and debris can be easily separated based not only on scatter properties of the particles but also on their fluorescent properties. This fact is important because in bull semen, it has been demonstrated that egg-yolk particles can be easily misjudged as spermatozoa based only on their scatter properties (Nagy et al., 2003), requiring preliminary washing and centrifugation to cleanse the cells. Centrifugation might cause sperm damage and, therefore, mask other effects caused by the cryopreservation. The subpopulation of live cells using the new triple staining concurs with the subpopulation of live cells using the well validated A/PI assay. In addition, the staining protocol is much easier than the A/PI because the staining is made from stock solutions and is not necessary to use a binding buffer. As the staining of the probes is not dependent on Ca2+, as is the case binding FITC-A, the preparation and using of a Ca2+ enriched buffer is not necessary. The agreement between both techniques (A and YOPRO- 1/Eth/SNARF-1) was good, although the percentage of live spermatozoa was slightly higher in the triple staining method (Pena et al., 2006). Also, the percentage of early damaged spermatozoa was higher with the A/PI assay. This might reflect an increase in membrane permeability, preceeding the transposition of PS in the evolution of the cryodamage, or in a yet to be determined physiological change probably being a very early step of both processes related to changes in cell volume regulation and movement of ions, occurring during the initiation of apoptosis (Bortner and Cidlowski, 1998) or cryoinjury (Paasch et al., 2005). In addition, an earlier inactivation of enzymes involved in maintaining membrane asymmetry than those involved in transporting amphipatic small molecules like YO-PRO-1 might explain this fact.
-Changes in sperm mitochondrial status
Mitochondria, located in the sperm midpiece, are the primary energy generating centers for motility and other processes in the sperm cell as in most other cells. Interference with this function has severe consequences for any cell (Krahenbuhl, 2001). During the apoptosis process, a number of changes occur in the mitochondria (Lui et al., 1996; Petit et al., 1996; Li et al., 1997). Mitochondria enclosed in the midpiece of the spermatozoa is a source of ATP which is utilized for the propulsion of flagellar. Cellular energy production, membrane lipids productions, growth as well as major determinants of sperm life or death largely depends on mitochondria (Arends and Wyllie, 1991). In other words, mitochondria have been confirmed by several groups as the coordinators of apoptosis in a number of cell systems (Kroemer, 1997; Zhyng et al., 1998). Shivaji et al. (2009) focuses on the identity and function of mitochondrial proteins which undergo capacitation-dependent tyrosine phosphorylation in spermatozoa. Hallap et al. (2005) reported that the results obtained from flow cytometric measurements of mitochondrial function were 10-15% lower than the recordings of motility, either subjectively or as measured by CASA. Such a difference between subjective motility evaluations and flow cytometry evaluations of mitochondrial activity is in line with that reported in several other publications (Garner et al., 1997; Gravance et al., 2000; Wu et al., 2003).
Accumulation in mitochondria is characteristic of many fluorescent dyes, such as Rhodamine 123 (R123), MitoTracker Green (MTG), JC-1, MitoTracker Orange (CMTMRos), MitoTracker Red (CMXRos), MitoTracker Red 580, and MitoTracker Deep Red 633 (Cossarizza---; Garner et al., 1997; Gravance et al., 2002; Ericsson et al., 1993). Despite of numerous published protocols, there are several problems connected with most of these fluorophores. There are approximately 100 mitochondria in the mid-piece of the spermatozoon and fluorescent dyes, able to target defined intracellular compartments, can be used to visualize them. Most of these dyes work by diffusing into living cells and accumulating in mitochondria, provided that an internal 100-200 mV negative potential gradient occurs across the mitochondrial membrane (MMP).
The most widely used mitochondrial-specific probe, R123 is a cationic compound that excites at 488 nm and emits at 515-575 nm (green fluorescence). It accumulates in the mitochondria as a function of transmembrane potential (Chen, 1988; Al-Rubeai, 1993), of R123 concentration, and of sperm numbers (Windsor and White, 1993); it is not dependent on time or temperature (Auger et al., 1989). It was historically applied to spermatozoa in combination with ethidium bromide (Evenson et al., 1982). The R123 accumulated in the mitochondria and fluoresced green, thus identifying the sperm that exhibited a mitochondrial membrane potential. The dead spermatozoa, such as those with damaged membranes, were identified by the uptake of ethidium bromide. A similar combination, R123 and propidium iodide (PI), has been shown to readily discriminate between living and dead spermatozoa (Evenson et al., 1982). Although functioning mitochondria stain green with rhodamine123, this stain does not permit one to differentiate between mitochondria that exhibit different respiratory rates. The novel mitochondrial probe, MitoTracker Green FM (MITO), is nonfluorescent in aqueous solution and fluoresces green upon accumulation in the mitochondria regardless of mitochondrial membrane potential (Haugland, 1996). R123 and MITO are transported into actively respiring mitochondria and their accumulation in the mitochondria causes them to fluoresce green. R123 is not suitable for use in experiments in which the spermatozoa are treated with aldehyde fixatives, whereas the MITO probes are well retained during the fixation process. MITO-labeled mouse spermatozoa were placed in the female reproductive tract by AI to trace the distribution of the mitochondria in the developing embryo (Davies and Gardner, 2002). Gadella and Harrison (2002) also used MITO to show that bicarbonate does not affect the mitochondrial potential of boar spermatozoa. Fluorescence-induced flow cytometric assays of Jagg fluorescence is frequently used to notify sperm with high mitochondrial transmembrane potential in sperm of many species (Garner et al., 1997; Gravance et al., 2001; Love et al., 2003). Martinez-Pastor et al. (2004) observed some relationship between JC-1 staining and motility, although correlation with motility is regulated by many factors. A deeper study has been suggested by this research group. The mitochondrial stain 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) does permit a distinction to be made between spermatozoa with poorly and highly functional mitochondria (Garner et al., 1997). In highly functional mitochondria, the concentration of JC-1 inside the mitochondria increases and the stain forms aggregates that fluoresce orange. When human spermatozoa were divided into high, moderate and low mitochondrial potential groups, based on JC-1 fluorescence, the in vitro fertilization rates were higher in the high potential group than in the low potential group (Kasai et al., 2002). JC-1 has also been used successfully to measure mitochondrial function using a fluorometer (Gravance et al., 2000). However, at greater concentrations, the probe aggregates and in the aggregate form fluoresces red-orange (Thomas et al., 1998).
-Changes in sperm chromatine integrity
Sperm DNA integrity is increasingly being recognized as an important determinant of fertilizing efficacy that has better diagnostic and prognostic capabilities than the standard sperm attributes like sperm morphology, sperm concentration or motility potential. In most of the normal semen parameters analyses quality of sperm DNA is not always reflected while early embryo development largely depends on the presence of normal DNA (Rodriguez-Martinez, 2007). Full sperm DNA integrity usually is defined as the absence of DNA nicks or single stranded (ss) breaks, double stranded (ds) breaks, and chemical modifications of the DNA. Of these, ds DNA breaks are the most mutagenic, because in the pronucleus stage zygote, the two genomes are separated, hence DNA template information for error free repair of the ds breaks is absent. Some recent follow-ups of children born after IVF are addressing the question about the possible increase of pathologies connected with these technigues (Schultz and Williams, 2002). DNA fragmentation frequently affecting abnormal spermatozoa present in large numbers in the semen of subjects with low sperm motility (Benchaib et al., 2003; Gandini et al., 2000; Huang et al., 2005; Lui et al., 2004; Marchetti et al., 2002; Varum et al., 2007; Vicari et al., 2002; Zini et al., 2001) may prevent or hinder fertilization and embryo development, and increase the risk of genetic defects (Erenpreiss et al., 2006; Spano et al., 2005; Huang et al., 2005; Borini et al., 2006; Henkel et al., 2003; Lewis and Aitken, 2005; Seli et al., 2004; Tesarik, 2005). It should be emphasized that detection of DNA damage is often beyond the scope of routine seminological analysis, which in many cases is decisive for qualification to assisted fertilization. DNA damage in sperm is promutagenic. It does not impair fertilization or cleavage, as the paternal genome is transcriptionally inactive until two days after fertilization (Agarwal and Allameneni, 2004). However, once the paternal genome is active it results in poor blastocyst development, unequal cleavage, implantation failure or early foetal loss. Small DNA damages in sperm are repaired by pre- and postreplication repair mechanisms, but large DNA damages cannot be repaired.
The organization of sperm DNA in an explicit manner allows the chromatin to keep in the nucleus in a compact and stable form, in an almost crystalline status (Carrell et al., 2003). The histone-to-protamine transition is a multistep process in which, firstly, somatic-type histones are partially exchanged with testis-specific histones, then replaced by transition proteins (Boissenault, 2002) and, finally, by protamines. Sperm chromatin abnormalities and DNA damage could stem from an insult to premeiotic testicular cell compartment or occur at the time of DNA packing at spermiogenesis (Sailer et al., 1995; Haines et al., 2001; Haines et al., 2002; Cordelli et al., 2003). Alternatively, it could be the result of freeradical- induced damage (Aitken et al., 1998; Shen and Ong, 2000; Aitken and Krausz, 2001) or a consequence of apoptosis (Sakkas et al., 2002; Gorczyca et al., 1993). Due to the importance of accurate transmission of genetic information to the offspring, several methods have been developed to detect DNA and chromatin alterations in spermatozoa, and efforts have been done to integrate these tests into conventional semen analysis (Perreault et al., 2003). Flow cytometric assays is one of the most interesting techniques today available which has demonstrated to be an independent fertility predictor either in vivo (Spano et al., 2000) or in vitro (Larson-Cook et al., 2003; Saleh et al., 2003) conditions. The integrity of sperm DNA evaluation with the TUNEL, COMET, acridine orange staining, in situ nick translation, or sperm chromatin structure assay (SCSA) techniques are in use in different laboratories.
Comet assay measures the response of individual cells which enables the study of heterogeneity within a cell population. While it is not possible with other techniques like pulsed-field gel electrophoresis, filter elution or sucrose gradient sedimentation which can estimate only the mean response from a large cell population. Comet assay can detect 200 DNA strand breaks per cell, however with few modifications it may detect up to 50 breaks per cell. In comet assay only a few number of cells are required, data can be collected at the level of individual cells, wide range of eukaryotic cells can be used, the assay is sensitive, simple and can detect non uniform responses within a mixed population. However, DNA damage can be overestimated in comet assay due to the presence of residual RNA which creates background during analysis, or can be underestimated because of proteins which hamper the movement of fragments during electrophoresis. Due to high inter-laboratory variation comet assay is not suitable for clinical use (Olive et al., 1992; Olive et al., 2001).
DNA fragmentation in spermatozoa can also be assessed with TUNEL assay, which can identifies DNA strand breaks with modified nucleotides. These incorporated labelled nucleotides can be detected in spermatozoa by flow cytometry, flourecence microscopy or light microscopy. Duran et al. (2002) found that human semen samples with greater than 12% of the spermatozoa containing DNA fragmentation did not result in pregnancy and Benchaib et al. (2003) obtained no pregnancies if this value was greater than 20%. In enzymatic detection of fragmented DNA bound to the biotinylated nucleotides by TUNEL horseradish peroxidase-labelled peroxidase streptavidin (Streptavidin HRP), which are observed by the hydrogen peroxide, and the stable chromogen, diaminobenzidine (DAB) which produces brown stain (DeadEnd Fluorometric TUNEL System Technical Bulletin, 2007). TUNEL can simultaneously detect single and double strand breaks unlike comet assay which requires different protocols for studying both type of strand breakages. By TUNEL the degree of DNA damage within a cell cannot be quantified, it only reveals the number of cells within a population with DNA damage. It is worth mentioning that such cells can also be identified using flowcytometry or fluorescence microscopy.
In situ nick translation assay (ISNT) is a modified form of TUNEL, which utilizes incorporation of biotinylated-dUTP at the ssDNA in a reaction catalyzed by template dependent enzyme, DNA polymerase 1 (DNA Pol 1), unlike TUNEL which utilizes template independent TdT. Moreover ISNT can only be used for single strand breaks not for both ss and ds breaks as in TUNEL (Irvine et al., 2000). ISNT assay is able to identify endogenous DNA damage; however, Irvine et al. (2000) reported that ISNT assay has limited correlation with fertility in in vivo studies.
Sperm chromatin structure assay (SCSA) is a technique, in which the extent of DNA denaturation following heat or acid treatment is determined by measuring the metachromatic shift from green fluorescence (acridine orange intercalated into double stranded nucleic acid) to red fluorescence (acridine orange associated with single stranded DNA) (Darzynkiewicz et al., 1976). The ratio of the amount of red to green for each individual spermatozoon is assessed by flow cytometry, with sperm containing greater red to green ratios exhibiting more DNA denaturation than sperm exhibiting lesser red to green ratios (Evenson and Wixon, 2006). One benefit of SCSA over other methods that evaluate DNA integrity is that the procedure is relatively easy, quick, and thousands of sperm can be evaluated objectively. However, no term pregnancies have been detected when the DFI fraction was more than 28 percent, which allowed to conclude that SCSA-derived thresholds markedly predicted negative pregnancy outcomes in patients attempting pregnancy via ART (Saleh et al., 2002; Larson et al., 2000; Cook et al., 2003). In clinical applications, the SCSA showed that an increased fraction of abnormal sperm has been found in patients with leukocytospermia, febrile illness and untreated testicular cancer patients (Evenson et al., 2000; Alvarez et al., 2002). The SCSA has also been successfully used to check gamete quality after cell manipulation (Spano et al., 1999).
The most important parameter revealed by SCSA is the DNA fragmentation index (% DFI). It is a simple and less time consuming method for the analysis of human spermatozoa (Frazer, 2005). The SCSA does not give much information about the extent of DNA damage in spermatozoa, since it focuses on measuring the percentage of spermatozoa with dispersed or non dispersed nuclei (Frazer, 2005). Many research groups recently potentially utilized SCSA on domestic animals (deAmbrogi et al 2006, Koonjaenak et al 2007, Morrell et al 2008).
Acridine orange test (AOT) is a simplified microscopic method of SCSA which does not require expensive flowcytometry and relies on visual interpretation of fluorescing spermatozoa and debris that fall into a broad range of colours under microscopic examination. Pretreated sperm samples (citric acid solution pH 2.0) stained with acridine orange (0.2 mg/ml H2O) are washed and covered with coverslip for examination (Spano et al., 1999). Sperms with ds DNA fluoresce green while those with ss DNA fluoresce red under a fluorescence microscope. Indistinct colour, rapidly fading fluorescence and heterogeneous slide staining exacerbate problems with interpretation (Duran et al., 1998).
On the other hand lack of correlation between DNA damage and fertilization rates but rather an association between DNA damage and post fertilization development (Agarwal and Allameneni, 2004).
During spermiogenesis, a decrease in binding capacity for DNA fluorochromes occurs due to the progressive chromatin packaging (Evenson et al., 1986), and FCM techniques have been proposed as measures of chromatin condensation anomalies using different DNA-specific dyes such as DAPI (Spano et al., 1984), ethidium bromide/mithramycin (Engh et al., 1992) and propidium iodide (Molina et al., 1995). Using this approach, associations have been demonstrated between fertility capability and the increase of highstainable abnormal sperm fraction (Hacker-Klom et al., 1999).
Many technical and biological factors determine the accuracy to evaluate the DNA damage in sperm cells. In spite of the variety of different methods available to evaluate sperm chromatin maturity levels, the bulk of evidence points to the pivotal role of chromatin /DNA integrity during spermatogenesis and in the fertilization process. Lower chromatin packaging quality detected in morphologically normal spermatozoa may represent one of the major factors limiting the sperm fertilizing ability, thus stressing the importance of the chromatin packing or DNA integrity as a prerequisite for optimal expression of male fertility potential.
-Decondensationof spermatozoa during fertilization
During mammalian spermiogenesis sperm chromatin undergoes a step-by-step condensation and packaging mainly characterized by replacement of histones with protamines and the formation of S-S and S-Zn-S bonds between cysteine residues (Bedford & Calvin, 1974). Therefore, mammalian spermatozoa nuclei are highly structured, exceptionally stable, condensed organella (Arkhis et al., 1991; Balhorn et al., 1991). Decondensation of sperm chromatin take place after entering into the ovum or injection of the oocyte (ICSI) when disulphide bonds of the protamine of the sperm nucleus are reduced, presumably by glutathione (Perreault et al., 1984, 1988; Perreault, 1992), followed by replacement of the protamine by embryonic histones (Zirkin et al., 1985; Betzalel & Moav, 1987; Philpott et al., 1991; Perreault, 1992; Leno et al., 1996). Although, the ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin in the cytoplasma of the oocyte (Kvist, 1980; Zirkin et al., 1985; Gopalkrishnan et al., 1991).
Methods for assessing sperm chromatin decondensation must therefore be considered useful additions to the arsenal of tests useful for diagnosing patients with male infertility. This is especially likely to prove useful in cases where intracytoplasmic sperm injection is being considered as a therapeutic option. Several groups have measured sperm chromatin decondensation by observing changes in size and shape of the sperm head by microscopic examinations (Jaeger et al., 1990; Lassalle and Testart, 1991; Lipitz et al., 1992; Montag et al., 1992; Banerjee and Hulten, 1994; Cameron and Poccia, 1994; Morcos and Swan, 1994; Chitale and Rathaur, 1995; Reyes and Sanchez-Vazques, 1996). These methods tend to be somewhat subjective because it is difficult to decide what degree of sperm head enlargement is to be the criterion of decondensation and they are time consuming because they require counting many cells in order to achieve statistical significance.
Flow cytometry offers an excellent means to quantitates these events associated with chromatin decondensation. Flow cytometry provides a useful alternative to microscopic examination of cells for determination of sperm decondensation. Zucker et al. (1992) performed decondensation in rat and hamster spermatozoa by using sodium dodecyl sulphate and dithiothreitol as a reducing agent and assessed decondensation by increase in light scatter. Evenson et al. (1980) evaluated decondensation in mouse and human sperm nuclei using Acridine Orange staining and plotting green fluorescence versus pulse width of the green signal, which is related to cell size. In the method reported here the increase in intensity of green and red fluorescence of Acridine Orange-stained spermatozoa after incubation with β-mercaptoethanol served as a measure of the decondensation.
Spermatozoon chromatin structure has been shown to be altered by cryopreservation-thawing in the mouse (Watson 2000; Kusakabe et al. 2001) and horse (Linfor and Meyers 2002). Although decondensation depends on spermatozoon disulfide bond and chromatin integrity (Lipitz et al. 1992), it is the oocyte's cytoplasm that induces decondensation (Yanagimachi 1994; Sutovsky and Schatten 1997). The study of decondensation often is conducted in vitro using chemicals rather than oocytes (Calvin and Bedford 1971; Perreault et al. 1988b; Samocha-Bone et al. 1998; Reichart et al. 2000; Martin et al. 2003). However, cytoplasmic emulsions from homologous or heterologous oocytes (Yanagimachi 1984; Ohsumi et al. 1986) are most likely to be physiologically meaningful as an in vitro test of spermatozoon decondensation (Yanagimachi 1994; Li and Gui 2003). There also is variability in rate of decondensation among species (Yanagimachi 1994). For example, decondensation of human spermatozoa is generally complete in 1 to 2 h (Dozortsev et al. 1995; Hammadeh et al. 2001), whereas decondensation in the golden hamster is complete within 40 min (Yanagimachi and Noda 1970). Mouse spermatozoa undergo decondensation within 1 h (Urner and Sakkas 1999), although this rate will extend up to 4 h depending on
post-ovulatory age of the oocytes used to induce the response (Fraser, 1979).
Flow cytometry for assessing sperm functionality
-Capacitation changes in spermatozoa
Capacitation of sperm is a prerequisite for successful fertilization. Capacitation is an important, but rather incompletely understood phenomenon that a spermatozoon undergoes before it can fertilize the oocyte. Capacitation is reversible and lasts hours. During this process various cellular changes occur at specific times and locations, including an increase in membrane fluidity due to lipid modifications, an influx of calcium to the sperm head and flagellum, the generation of controlled amounts of reactive oxygen species, as well as the phosphorylation of proteins on serine, threonine and tyrosine residues (De Jonge, 2005; O'Flaherty et al., 2006; Lamirande and O'Flaherty, 2007; Tulsiani et al., 2007). Therefore, we are now able to evaluate the ability of spermatozoa to capacitate under various conditions, which should provide information regarding cell longevity and performance. Capacitation is associated with alterations in a variety of intracellular and sperm surface features, but the precise relationship between these modifications and capacitation is not certain. Therefore, in spite of many published reports describing phenomena that might be correlated with capacitation, there is no general consent as to the assay (s) or techniques that can distinguish capacitated from noncapacitated spermatozoa.
One of the most often used methods for determination of the capacitation status is the CTC (chlortetracycline) assay by using fluorescence microscopy. This fluorescent antibiotic will detect and exhibits enhanced fluorescence over the segments of the membrane where Ca2+ accumulates. Chlortetracycline has been shown to interact with mammalian spermatozoa, showing different binding patterns on the sperm head, which are believed to reflect different stages of the capacitation process (Ward&Storey, 1984; DasGupta et al., 1993; Fraser et al., 1995). Though, CTC is empirically accepted but is laborious to use and its working mechanism is scientifically unexplained.
Although sperm capacitation is not only a destabilization process, early stages of sperm capacitation can be measured by loading spermatozoa with the lipid dye merocyanine-540 (Harrison and Gadella, 2005) and then using flow cytometry to determine any significant increase in fluorescence (related to the degree of lipid disorder in the plasma membrane and indicative of of the beginning of capacitation; Redriguez-Martinez et al., 2001). Flow cytometric detection of capacitation-related changes in membrane architecture using merocyanine 540 as a reporter probe, and acrosome status using FITC-PNA staining, have some clear advantages over the all-compassing CTC staining technique. First, given the clear differences in the intensity of fluorescence between control and capacitated or acrosome-reacted cells, flow cytometry allows for the very rapid and objective discrimination of the status of large numbers of sperm cells. For example, in the current study we analyzed 10 000 sperm cells per data point in only a few seconds. Second, prior to analysis, the sperm suspension requires only simultaneous addition of appropriate amounts of PI and FITC-PNA or Yo-Pro-1 and merocyanine 540, followed by a 10-min incubation for the completion of labeling. Third, the cells can be analyzed in a flow cytometer in the unfixed state and under relatively physiological conditions. This ability to control the ambient conditions minimizes the risk of cell deterioration, especially for the notoriously delicate capacitated sperm cells. Recently it was found that between sperm capacitation and protein tyrosine phosphorylation (PTP) correlation exists which suggests importance of PTP for understanding phenomena of capacitation. A new method has been proposed by Sidhu et al. (2004) to measure levels of PTP in sperm that is undergoing to capacitation. In in vitro condition, the global sperm PTP levels under in vitro induced capacitation were estimated flow cytometrically in permeabilized cells. This newly introduced flow cytometric method is more rapid, simple and reliable than the others.
-Acrosomal integrity of spermatozoa
The acrosome is a membrane enclosed structure covering the anterior part of the sperm nucleus. Powerful hydrolyzing enzymes belongs to that structure, is a basic feature of the sperm head of all mammals (Yanagimachi, 1994). The acrosomal exocytosis of mammalian sperm is to be observed before fertilization, as it is essential for sperm penetration of the zona pellucida and for fusion with the plasma membrane. As a prerequisite of fertilization the content of the acrosome is released into its surroundings during the acrosome reaction. It is assumed that the acrosomal integrity facilite sperm to penetrate the ZP of oocyte and confort in the oocyte-sperm fusion process (Yanagimachi, 1994). Acrosomal loss can also occur in degenerating (dying) spermatozoa, it is considered to be essential to distinguish between the occurrence of acrosome reaction in viable and non viable cells (Cross and Meizel, 1989). To evaluate these multiparametric studies, various dyeing methods have been developed recently to assess acrosomal integrity. Hence, the efficacy of a sperm population to undergo the AR could be expected to influence male fertilizing potential.
Acrosome intactness, a prerequisite for fertilization, is being readily examined in vitro using phase-contrast microscopy (Rodríguez-Martínez et al. 1997a). Most frequently used methods are triple or double staining (Talbot and Chacon, 1980; De Jonge et al., 1989), isothiocyano-fluoresceinated Pisum sativum agglutinin (FITC-PSA; Crosset al., 1986), FITC-concanavalin A (FITC-ConA; Holden et al., 1990), chlortetracycline (Saling and Storey, 1979; Amin et al., 1996), paramagnetic beads (Okabe et al., 1992; Ohashi et al., 1992, 1994), Coomassie Blue (Aarons et al., 1993), anti-acrosin antiserum (Tesarik et al., 1990), mannosylated bovine serum albumin (Benoff et al., 1993), quinacrine (Amin et al., 1996) and monoclonal antibodies (Kallajoki and Suominen, 1984; Wolf et al., 1985; Moore et al., 1987; Fe´nichel et al., 1989; Aitken and Brindle, 1993; Chao et al., 1993). A combined assessment method for human sperm morphology and the acrosomal status using monoclonal antibodies against clusterin was demonstrated by O'Bryan et al. (1994). This glyco-protein is located within the acrosomal cap. Electron microscopic studies have been demonstrated the ultrastructural morphology of acrosomes with the staining patterns of spermatozoa after labelling with the antibodies by the Köhn et al. (1997). There are some limitations of microscopic examination it is only easily accomplished in species such as the hamster and guinea pig which have large acrosomes. However, most mammalian sperm, including those of humans have such small acrosomes thus normal acrosome reactions are not easily observed with the light microscopy. Although epifluorescence microscopy is being used, Pen˜a et al.  observed that epifluorescence microscopy was less precise than flow cytometry for detecting the percentage of spermatozoa with damaged acrosomes, probably due to the difference in sample size. Nonetheless, Miyazaki et al. (1990) stated that rate of acrosome-reacted sperm determined with flow cytometry and fluorescence microscopy showed that these methods have very similar results.
The advent of cellular measurements by flow cytometric analysis of individual attributes within a population of cells is an important step toward the evaluation of acrosomal intactness. Comparing other assays to the more widely used epifluorescent microscopic techniques, the flow cytometric analysis can give a far more simple, speedy, accurate and objective method of analysis, especially with regard to correlation of fertilization with acrosome reactivity potentials (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward, 1996). High levels of green and red fluorescence are characteristic of non-reacted spermatozoa, while the AR produces decreased fluorescence intensity. Flow cytometry analysis has permitted determination of the regions from reacted or non-reacted populations, and thus calculation of the percentage for the each. This determination of integrity is normally corroborated by fluorescence microscopy observations. Acrosomal integrity can be measured by a number of probes, but the most commonly used probe is with a plant lectin labeled by a fluorescent probe. There are a large number of lectins available for assessing acrosomal integrity, some of which (eg. Ricinus communis agglutinin) show toxic effect on sperm. PSA is a lectin from the pea plant that binds to a-mannose and agalactose moieties of the acrosomal matrix. As PSA cannot penetrate an acrosomal membrane which it intact, only acrosome-reacted or damaged spermatozoa will stain (Cross et al., 1986). Arachis hypogaea agglutinin (PNA) is a lectin from the peanut plant that binds to the galactose moieties associated with the outer acrosomal membrane of fixed spermatozoa, indicating acrosome-intact cells (Mortimer et al., 1987). A hypogaea agglutinin is believed to display less non-specific binding to other areas of the spermatozoon, leading some workers to favour this over PSA (Graham, 2001). Carver-Ward et al. (1997) proposed that PNA is the reliable lectin compared to PSA, CD46 and ConA. Thus, PNA is capable of differentiating the acrosome reacted sperm from a given population of spermatozoa. However, in observing that only PNA gives a specific comparison between non-acrosome- reacted and acrosome-reacted sperm, the differences between the two markers are merely a matter of magnitude. In contrast, Tao et al. (Ref) stated that compared to PSA, Con A or SBA, PNA is a more reliable acrosome intactness marker. Petrunkina et al. (2005) observed that PNA-FITC, which was used in this study to evaluate sperm responsiveness, binds to the outer acrosomal membrane (OAM) (Fazeli et al., 1997; Flesch et al., 1998), so that FITC-PNA can be used as a probe to monitor boar sperm acrosomal integrity.
One of the frequently used fluorochrome combinations for the simultaneous assessment of plasma membrane integrity (i.e., viability) and acrosome integrity are fluorescein isothiocyanate- conjugated pea (Pisum sativum) agglutinin (FITC-PSA) and propidium iodide (PI) (Graham et al., 1990). On nonpermeabilized spermatozoa, FITC-PSA provides information regarding the integrity of the sperm acrosome. Sperm cells which have an intact acrosome will have no fluorescence, while cells with a reacted or damaged acrosome will show a green fluorescence. Propidium iodide is a DNA-specific stain that cannot enter the intact plasma membrane and, therefore, is used as a dead-marker counterstain. This double-staining for sperm viability and acrosome integrity is relatively reliable for fresh and in vitro-capacitated sperm, because sperm cell particles can easily be distinguished from nonsperm events by their specific forward- and sideways-scatter properties e.g., in dogs (Szasz et al., 2000). The most problems arisen in case frozen-thawed spermatozoa is that they contain egg yolk particles which have scatter properties similar to those of sperm cells,trouble the elimination of nonsperm events (debris) by scatter gating enormously (Pena et al., 1999). Like live acrosome-intact sperm cells, these egg yolk particles have low fluorescence and, therefore, it will be assessed as live acrosome-intact sperm using the PI/FITCPSA double-labeling method. Thus, it is suggested that remove completely all the egg yolk particles when using the PI/FITC-PSA double-staining protocol for the accuracy of analyses of sperm integrity after cryopreservation. However, it is a matter of argument 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 can identify the signal from the entire spermatozoa. Biologically, plasma membrane and the outer acrosomal membranes both fuse with vesiculate during the acrosome reaction, which would preclude binding to the outer acrosomal membrane on nonpermeabilized sperm. It is also a matter of question as to how one could permeabilize the plasma membrane except affecting the underlying outer acrosomal membrane. Acrosome reaction in spermatozoa can also be recognized cytometrically with the help of monoclonal antibodies against inner acrosomal membrane epitopes such as GB24, MH61 and CD46 (Ref). Tao et al. (1993) examined the use of MH61 and CD46, two monoclonal antibodies for acrosome reaction assessment, while others have suggested CD46 alone (D'Cruz and Haas, 1992; Carver-Ward et al., 1994).
In order to mimic the accumulation of lysophospholipid, Maxwell and Johnson (1997) treated boar spermatozoa with lysophosphatidylcholine, which has been implicated in the acrosomal integrity, and found a significant raise in FITC-PSA spermatozoa while assessing with flow cytometry. Pena et al. (2001) stated that compared to microscopic evaluation, the results demonstrated that flow cytometry is a precise method for evaluating the viability and acrosomal status of fresh samples of dog semen. A newly developed triple staining (carboxy-SNARF-1, propidium iodide and FITC-PSA) procedure was developed and was found as an efficient method for evaluating actosomal integrity of cryopreserved dog spermatozoa. Herrera et al. (2002) used FITC-PSA to determine whether there was an association between the acrosome reaction and the incidence of subfertility of boar spermatozoa. The authors found that the percentage of spontaneous acrosome reaction was not significantly different in fertile and subfertile boars. However, the incidence of a progesterone-induced acrosomal integrity was significantly lower in subfertile (5.75%) compared with fertile boars (10.0%), suggesting that assessment of the induced acrosome reaction may be a useful parameter to assess fertility. In another similar study, attempt was made to determine the kinetics of surface carbohydrate turnover during the in vitro capacitation and the AR in fertile and subfertile boars (Jime´nez et al., 2002). Spermatozoa were exposed to three FITC-labeled lectins: Triticum vulgaris agglutinin (WGA; specific for sialic acid and N-acetylglucosaminyl residues), Concanavalia ensiformis agglutinin (Con- A; specific for D-mannosyl and D-glucosyl residues) and Ulex europaeus agglutinin (UEA; specific for L-fucose), and assessed by flow cytometry. The authors reported differences in lectin patterns across capacitated and acrosome-reacted spermatozoa between fertile and subfertile boars.
Cooper and Yeung (1998) emphasized use of F-fucoidin as vital dye for flow cytometry analysis. They suggested that F-fucoidin binds not only to the plasma membrane of intact cells but also to intraacrosomal components, presumably exposed in cells with damaged membranes or those that have undergone the acrosome reaction.
-Apoptotic-like changes in spermatozoa
Apoptosis is a programmed cell death sequence of events which leads to the destruction of cells without releasing harmful substances into its surrounding area. Apoptosis plays a distinct role in development and maintenance of health by eliminating old and unnecessary cells, as well as unhealthy cells. Apoptosis marker has been detected in spermatozoa of many species but probable role of these markers is not yet elucidated completely (Pena et al., 2003; Marti et al., 2006; Angelopoulou et al., 2007; Moran et al., 2008; Ortega-Ferrusola et al., 2008). Martinez-Pastor et al. (2008) proposed that apoptosis in spermatozoa has a partial similarity with the apoptosis in other cells. Changes of sperm membrane permeability have been considered as a typical event of apoptosis in many studies. Active caspases and proteases functioning have been detected as apoptotic mediators in spermatozoa (Paasch et al., 2003). It was also proposed that mitochondria may play important role in sperm apoptosis (Martin et al., 2007) and due to the deficiency of mitochondria spermatozoa may lose its viability. Flow cytometry has the potentiality to measure apoptotic like change in spermatozoa; previously many studies investigated membrane or mitochondrial changes to apoptotic markers by flow cytometry. However, still we have lack of information regarding apoptosis in sperm; it might be that a number of events occur during the apoptosis process. During cryopreservation, freezing and thawing protocol of spermatozoa cause cryodamage and subtle damage, a number of other changes like phase transition in plasmalemma, oxidative damage, capacitation-like changes contribute to sperm death (Watson, 2000). Recently, Ortega-Ferrusola et al. (2009) concluded that apoptotic markers can be a tool for forecasting semen freezeability and cellular damage occur during cryopreservation seems to be an apoptotic like phenomenon in stallion. They also concluded that JC-1 has the highest diagnostic power. In a flow cytometric apoptosis-like change detection, Martinez-Pastor et al. (2008) proposed that apoptotic spermatozoa may be dying cells, possibly with the activated cell death pathways. They assumed that sequence of sperm death might be loss of mitochondrial membrane potential, membrane change (YO-PRO-1+ and PI-) and membrane damage (PI+). Whereas, it was suggested that apoptotic markers like caspase activation and YO-PRO-1 staining might happen only in a specific subpopulation of spermatozoa in red deer (Martinez-Pastor et al., 2009). Thus, activation of apoptosis markers and the resulting consequence is species specific and depend on inductors. Still we have paucity of information on the apoptosis-like changes and much research is necessary in this area to ascertain the complete phenomena.
-Oxidative stress of spermatozoa
Oxidative stress may be defined as an imbalance between relative oxygen species and the potentiality of the antioxidants as to scavenge these which lead to infertility. Oxidative stress in spermatozoa is a matter of interest and concern due to the potential detrimental effects of high ROS on sperm functionality. From studies of different groups it was found that ROS hamper integrity of DNA in the sperm nucleus, breaking of DNA stand breaks and chromatin cross linking (Said et al., 2005). Recent studies manifest that oxidative stress, sperm DNA damage and apoptosis are associated with the infertility. Spermatozoa under oxidative stress are attacked at the plasma membrane and the DNA integrity. High level of relative oxygen productions is associated with oxidative stress, a major causes of DNA damage (Aitken et al., 1997) and lead high rate (20-40%) of infertility (Gil-Guzman et al., 2001). Relative oxygen species stimulate sperm capacitation, hyperactivation, acrosome reaction and protein tyrosin phosphorylation, in a low concentration, if the concentration raise can cause oxidative stress and induce physiological schanges in spermatozoa (Aitken, 1999). Continuous discovery of new fluorochromes made it possible to analyze semen quality especially oxidative stress by flow cytometry. Dihydroethidium a probe is freely permeable to sperm and oxidized to DNA binding fluorophore can detect superoxide anion in spermatozoa. Mitochondria generate ROS production and MMP depict mitochondrial functionality and high MMP results in high fertility. MMP of spermatozoa can be detected by Rh123 stain, which is actively transponted inside mitochondria, accumulation of Rh123 yields green fluorescence (Troiano et al., 1998). It is also assumed that flow cytometric sorting of spermatozoa generate lipid preoxidation leading to oxidative stress, although there is no direct evidence on this prediction. For the single analysis of spermatozoa by flow cytometry, semen samples are highly diluted before sorting which break the natural defence against oxidation.
Application of flow cytometry on novel sperm sex sorting
Sexing of mammalian spermatozoa in order to produce offspring with the desired sex is one of the supreme important new biotechnological developments available for livestock production as well as for human. This strategy allows various options when seeking the improvement of efficiency in production (Johnson 2000). The practical application of sexing spermatozoa, synergistically with other reproductive techniques, could improve the efficiency of animal production both in biological and economic terms. Flow-cytometry based sex preselection works on sperm DNA content measurement to facilitate sorting of X chromosome from the Y chromosome-bearing sperm. The X chromosome carries more DNA than than that of the Y chromosome and principle of flow cytometric sex sorting is based on this difference. This method is reproducible at various laboratories and with many species at a greater degree of purity. Many offspring of the predetermined sex was born using sperm sorting after the innovation of this novel technology. Thus, sex-sorted spermatozoa might be able to produce embryos to obtain sex-preselected offspring both in animals and humans. The spermatozoa not only must be identified as X- and Y-chromosome-bearing by the flowcytometer, but also, after sorting, their fertilizing ability must be preserved.
Current sex-selection methods can be classified broadly into two general groups; one is those methods which separate sperm based on comprehensive physical or kinetic features, and another which depend on separate nuclear characteristics of X chromosome or Y chromosome bearing spermatozoa. Despite of controversy, in laboratories where sperm are sexed using the first method is in practice for few years. The scope of pre-determine the sex of upcoming offspring prior to conception is a highly desired technology for implementation into assisted breeding programs for both farm animals and wildlife. Selection of sex has important implications for populations in which one sex has more intrinsic value, for instance; stud operations and female dairy replacements. Rapid propagation of rare animal genetic resources. The efficiency of production would be improved by reducing animal wastage and allowing for the dissemination, manipulation and storage of superior genetic stock (Parrilla et al. 2004).
In flow cytometry based sex sorting, firstly sperm are stained with a fluorochrome dye (bisnbenzimide), then pass through a beveled nozzle to invest with a ribbon structure to the stream (Stovel et al., 1978). After that, sperm are oriented in a head first position and stream flow is reduced to a number of droplets. Interrupted stream with a regulated droplet size is then pass into a laser beam, DNA content of sperm determine the amount of bound fluorochrome which directly effect intencity of fluorescent. Sperm which emitting appropriate fluorescent signal accept electrical charge are sorted into collection tubes (Stites, 1984).
Since Hoechst 33342 is a non-intercalating agent, and boar spermatozoa have highly compacted chromatin, which protects their DNA from different kinds of insults [Rodriguez-Martinez et al., 1990], it could be that Hoechst 33343 per se does not exert any damaging effect in the fertilizing capability of boar spermatozoa when used at the concentrations required for sex sorting. Spermatozoa stained with Hoechst 33342 and used for standard AI resulted in the same pregnancy and farrowing rates as unstained spermatozoa. Moreover, staining did not affect the total number of piglets born.
Offspring of pre-determined sex using flow cytometry have been successfully produced using fresh (non-frozen) and frozen-thawed spermatozoa in several mammalian species; pigs (Grossfeld et al. 2005), cattle (Seidel et al. 1999), sheep (Hollinshead et al. 2003; de Graaf et al. 2006; de Graaf et al. 2007b) bottlenose dolphins (O'Brien and Robeck 2006), goat (Parrilla et al., 2004) and humans (Fugger 1999).
Further refinements to standard flow cytometric technology included replacement of the beveled needle by an orientating nozzle with a ceramic tip giving sperm less time to lose orientation, which improved correct orientation of sperm to 70% (Johnson and Welch, 1999). Conversion to high speed modified flow cytometers operating under increased pressure (40-50psi) improved the accuracy and efficiency of sperm sorting (Maxwell et al. 2004). The difference in genetic sex determination between mammals and birds provided another important clue. In mammals, sex is determined by which spermatozoon fertilizes the ovum, the X- or Y-chromosome-bearing gamete. In avian species, however, sex is determined by the oocyte, not by the fertilizing spermatozoon. Avian species produce only Z sperm. Flow cytometric analyses of cockerel sperm revealed a single peak, whereas those of mammals had two peaks (Garner et al., 1983). Management of sex ratio is of particular importance to species that naturally exist in a female-dominated group. Sex sorting might have potential advantages for the conservation of animal genetic resources, especially those which are extincting and endangered. Production of predominantly female offspring by sperm sorting technique together with assisted reproductive technology. Some difficulties arises when sex are sorted in the cryopreserved samples as spermatozoa go under stress during cooling, freeding and thawing procedures. Introduction of a novel sperm cryopreservation method, directional solidification freezing protocol [Robeck et al., 2005] led us to solve this problem and enhanced improvement in sorted spermatozoa functionality compared to a conventional method of freezing [O'Brien and Robeck, 2006].
However, till the sex sorted embryo success rate is controversial. High dilution rates are considered as one of the main drawbacks of sex-sorting sperm technology (Maxwell and Johnson, 1999; Maxwell et al., 1997). It is fair to say that flow cytometric detection of sperm DNA vary between flowcytometers due to DNA content differences in sperm. In conclusion, the presence of a extended difference in DNA content between the X chromosome and the Y chromosome-bearing sperm allows us for a clear identification of these two sperm populations will make the technology more attractive and feasible for domestic animals including human in future.
Future perspective of flow cytometry
There are no excuses for not improving the standards in laboratory andrology: detailed descriptions of robust, reliable techniques and procedures already exist. Although improvements can be made in the existing guidelines, protocols, and quality control systems, flow cytometry systems provide much better tools than other, nonstandardized procedures ever can. Incorporation of molecular techniques would appear to be the key to elucidation of mechanisms that control spermatozoal development and function in pig and AI industries.
A better understanding of the molecular mechanisms regulating spermatozoal development and function will likely lead to new diagnostic techniques and therapeutic strategies for reduced fertility in stallions and may possibly translate to methodologies designed for human. Nonetheless, incorporation of some of these diagnostic tools into a standard semen analysis may yield improved discrimination ability regarding the competence of these complex, yet intriguing, cells. Similar applications would be valuable for critical evaluation of laboratory techniques applied to liquid preservation or cryopreservation of spermatozoa. In order to produce tests that are predictive of fertility on a routine basis, banks of tests, including microscopic analysis, flow cytometric assessments and functional tests, are currently required to formulate a predictive index.
It is also important to emphasize that since a spermatozoon requires many attributes to fertilize an oocyte, relying on an assay which measures a single attribute for culling poor semen samples or to attempt to determine possible reasons for male infertility can lead to frustration, because poor fertilizing samples will still be sent into the field, and specific causes for infertility will remain undiagnosed. Application of flow cytometry waives, or at least, minimizes this problem when thousands of spermatozoa are counted instead of 100 or 200 counted on the microscope (Rodriguez-Martinez, 2003). However, effectively culling poor semen samples should be accomplished if several assays are used which evaluate different sperm variables. Despite of some criticism, flow cytometry is still an efficient method of semen analyses.
The flow cytometric methods will probably further revolutionize our understanding of the sperm physiology and their functionality and will undoubtedly extend its application in isolating many uncharacterized features of spermatozoa.
Acevedo JJ, Mendoza-Lujambio I, de la Vega-Beltrán JL, Treviño CL, Felix R, Darszon A. KATP channels in mouse spermatogenic cells and sperm and their role in capacitation. Dev Biol 2006; 298: 395-405. Dev Biol 2006; 298: 395-405.
Agarwal A, Allameneni SSR. The effect of sperm DNA damage in assisted reproduction outcomes. Mineva Ginecologica 2004: 56 : 235-45.
Aitken RJ, Gordon E, Harkiss D, Twigg GP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998; 59: 1037-1049.
Arkhis, A., Martinage, A., Sautiere, P. et al. (1991) Molecular structure of human protamine 4 (HP4), a minor basic protein of human sperm nuclei. European Journal of Biochemistry 200, 387-392. Aitken RJ, Krausz C. Oxidative stress, DNA damage and the Y chromosome. Reproduction 2001; 122: 497-506.
Almid T, Johnson LA. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J Anim Sci 1988;66: 2899-2905.
Althouse GC, Hopkins SM. Assessment of boar sperm viability using a combination of two fluorophores. Theriogenology 1995;43: 595-603.
Alvarez JG, Sharma RK, Ollero M, et al. Increased DNA dagage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay. Fertil Steril 2002; 78: 319-329.
Amman RP, Hammerstedt RH. In vitro evaluation of sperm quality: an opinion. J Androl 1993; 14: 397- 406.
Betzalel, M. & Moav, B. (1987) Degradation of sperm histones in vitro by cytoplasm of the sea urchin egg. Cell Differentiation 20, 125-136.
Balhorn, R., Corzett, M., Mazrimas, J. et al. (1991) Identification of bull protamine disulphides. Biochemistry 30, 175-185.
Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003;18:1023-1028.
Bedford, J.M, Calvin, H.I., 1974. The occurrence and possible functional significance of -S-S- cross links in sperm heads, with particular reference to eutherian mammals. J. Exp. Zool. 188, 137-155.
Boissenault G. Chromatin remodeling during spermatogenesis: a possible role the transition proteins in DNA strand break repair. FEBS Lett 2002; 514: 111-114.
Bortner CD, Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 1998; 56: 1549-1559.
Banerjee, S. and Hulten, M.A.J. (1994) Sperm nuclear chromatin transformations in somatic cell-free extracts. Mol. Reprod. Dev. 37, 305-317.
Bunthof CJ, Bloemen K, Breeuwer P, Rombouts FM, Abee T. Flow cytometric assessment of the viability of lactic acid bacteria. Appl Environ Microbiol 2001; 67: 2326-2335.
Benchaib M, Braun V Lornage J. et al. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod 2003;18: 1023-1028.
Baumgarth N and Roederer M. A practical approach to multicolor flow cytometry for immunophenotyping. J Immunol Methods 243: 77-97, 2000.
Burkman, L.J, Coddington, C.C., Franken, D.R. et al. (1988) The hemizona assay (HZA): development of a diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil. Steril., 49, 688-697.
Li, C., and Gui, J. (2003). Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish. Cell Res. 13, 159-169.
Liu CH, Tsao HM, Cheng TC. Et al. DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males. J Assist Reprod Genet 2004; 21: 119-126.
Larsson B, Rodriguez-Martinez H. Can we use in vitro fertilization tests to predict semen fertility? Anim Reprod Sci 2000; 60-61: 327-336.
Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor quality semen samples and correlates with failed fertilization in a cytoplasmic sperm injection. Fertil Steril 1998; 69: 528-532.
Lassalle, B. and Testart, J. (1991) Sequential transformation of human sperm nucleus in human egg. J. Reprod. Fertil., 91, 393-402.
Maxwell WMC, Johnson LA. Physiology of spermatozoa at high dilution rates: the influence of seminal plasma. Theriogenology 1999;52:1353-62.
Maxwell WMC, Johnson LA. Chlortetracycline analysis of boar spermatozoa after incubation, flow cytometric sorting, cooling, or cryopreservation. Mol Reprod Dev 1997;46:408-418.
Maxwell, W.M.C., Evans, G., Hollinshead, F.K., Bathgate, R., De Graaf, S.P., Eriksson, B.M., Gillan, L., Morton, K.M., O'Brien, J.K., 2004. Integration of sperm sexing technology into the ART toolbox. Anim. Reprod. Sci. 82-83, 79-95.
Maxwell WM,Welch GR, Johnson LA. Viability and membrane integrity of spermatozoa after dilution and cytometric sorting in the presence or absence of seminal plasma. Reprod Fertil Dev 1997;8:1165-78.
Marchetti C, Obert G, Deffosez A, Formstecher P, Marchwtti P. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum Reprod 2002; 17: 1257-1265.
Molina J, Castilla JA, Gil T, Hortas ML, Vergara F, Herruzo A. Influence of incubation on the chromatin condensation and nuclear stability human spermatozoa by flow cytometry. Hum Reprod 1995;10:1280- 1286.
Mortimer D, Curtis EF, Dravland JE. Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J Reprod Fertil 1987; 81: 127-135.
Matyus L, Szabo Jr G, Resli I, Gaspar Jr R, Damjanovich S. Flow cytometric analysis of viability of bull sperm cells. Acta Biochim Biophys Acad Sci Hung 1984; 19: 209-214.
Montag, M., Tok, V., Liow, S.-L. et al. (1992) In vitro decondensation of mammalian sperm and subsequent formation of pronuclei-like structures for micromanipulation. Mol. Reprod. Devel., 33, 338-346.
Mortimer D, Camenzind AR:The role of follicular fluid in inducing the acrosome reaction of human spermatozoa incubated in vitro. Hum Reprod 1989;4:169-174.
Mortimer D, Curtis EF, Miller RG: Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J Reprod Fert 1987;81: ! 27-135
Morcos, Y. and Swan, M.A. (1994) Reactivated movement of decondensed rat sperm models and a description of their ultrastructure. J. Exp. Zool., 270, 388-398.
Martin, R., Greene, C., Rademaker, A., Ko, E., and Chernos, J. (2003). Analysis of aneuploidy in spermatozoa from testicular biopsies from men with nonobstructive azoospermia. J. Androl. 24, 100- 103.
Nagy S, Jansen J, Topper EK, Gadella BM. A triple stain flow cytometric method to assess plasma and acrosome membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol Reprod 2003; 68: 1828-1835.
O'Brien, J.K. and Robeck, T.R. (2006) Development of sperm sexing and associated reproductive technology for sex preselection of captive bottlenose dolphins. Reprod. Fert. Dev., 18; 319-329.
Ormerod MG, Sun XM, Snowden RT, Davies R, Fearnhead H, Cohen GM. Increased membrane permeability of apoptotic thymocytes: a flow cytometric study. Cytometry 1993; 14: 595-602.
Olive PL, Wlodek D, Durand RE, Banath JP. Factors influencing DNA migration from individual cells subjected to gel electrophoresis. Exp Cell Res 1992; 198 : 259-267.
Olive PL, Durand RE, Banath JP, Johnson PJ. Analysis of DNA damage in individual cells. Methods Cell Biol 2001; 64: 235-249.
Ohsumi, K., Katagiri, C., andYanagimachi, R. (1986). Development of pronuclei from human spermatozoa injected microsurgically into frog (Xenopus) eggs. J. Exp. Zool. 237, 319-325.
Perreault SD, Aitken RJ, Baker HW, et al. Integrating new tests of sperm genetic integrity into semen analysis: breakout group discussion. Adv Exp Med Biol 2003; 518: 253-268.
Paasch U, Grunewald S, Wuendrich K, Jope T, Glander HJ. Inmunomagnetic removal of cryo-damaged human spermatozoa. Asian J Androl 2005; 7: 61-69.
Parinaud J, Labal B, Vieitez G, Richoilley G, Grandjean H: Comparison between fluorescent peanut agglutinin lectin and GB24 antibody techniques for the assessment of acrosomal status. Hum Reprod 1993;8:1685-1688.
Petrunkina AM and Harrison RAP. Systematic misestimation of cell subpopulations by flow cytometry: A mathematical analysis. Theriogenology 2009; doi:10.1016/j.theriogenology.2009.09.007
Perreault, S. D. (1992) Chromatin remodeling in mammalian zygotes. Mutation Research 296, 43±55.
Pen˜a AI, Quintela LA, Herradon PG. Flow cytometric assessment of acrosomal status and viability of dog spermatozoa. Reprod Domest Anim 1999;34:495-502.
Pena A, Johannisson A, Linde-Forsberg C. Post-thaw evaluation of dog spermatozoa using new triple fluorescent staining and flow cytometry. Theriogenology 1999 52:965-980.
Pena FJ, Saravia F, Johannisson A, Walgren M, Rodriguez-Martinez H. A new and simple method to evaluate early membrane changes in frozen-thawed boar spermatozoa. International Journal of Andrology, 2005; 28: 107-114.
Peña FJ, Núñez-Martínez I, Morán J. Semen technologies in dog breeding: an update. Reprod Domest Anim 2006; 41(S2): 21-29.
Perreault, S. D., Barbee, R. R., and Slott, V. L. (1988b). Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev. Biol. 125, 181-186.
Pen˜a A, Johannisson A, Linde-Forsberg C. Validation of flow cytometry for assessment of viability and acrosomal integrity of dog spermatozoa and for evaluation of different methods of cryopreservation. J Reprod Fertil Suppl 2001;57:371-6.
Parrilla I, Vazquez JM, Roca J and Martinez EA. Flow Cytometry Identification of X- and Y- Chromosome-Bearing Goat Spermatozoa. Reprod Dom Anim, 2004; 39:68-60.
Purvis K, Rui H, Scholberg A, Hesla S, Clausen OPF: Application of flow cytometry to studies on the human acrosome. J Androl 1990;11: 361-366.
Perreault, S. D., Wolff, R. A. & Zirkin, B. R. (1984) The role of disulfide bond reduction during mammalian sperm nuclear decondensation in vivo. Developmental Biology 101, 160-167.
Perreault, S. D., Barbee, R. R. & Slott, V. L. (1988) Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensation activity in maturing hamster oocyte. Developmental Biology 125, 181-186.
Philpott, A., Leno, G. H. & Laskey, R. A. (1991) Sperm decondensation in Xenopus egg cytoplasma is mediated by nucleoplasmin. Cell 65, 569-578.
Rodriguez-Martinez H, Courtens JL, Kvist U, Plo¨en L. Immunocytochemical localization of nuclear protamine in boar spermatozoa during epididymal transit. J Reprod Fertil 1990;89: 591-5.
Rodriguez-Martinez, H. (2003) Laboratory semen assessment and prediction of fertility: still utopia? Reproduction in Domestic Animals 38, 312-318.
Rodríguez-Martínez, H. (2000). Evaluation of frozen semen: traditional and new approaches. In ‘Topics in Bull Fertility'. (Ed. P. J. Chenoweth.) Document no. A0502.0600, 2000a. (Recent Advances in
Veterinary Medicine, International Veterinary Information Services (IVIS): New York.) Available at http://www.ivis.org [Verified 12 September 2006].
Rodríguez-Martínez, H., and Larsson, B. (1998). Assessment of sperm fertilizing ability in farm animals. Acta Agr. Scand. A. (An.) 29(Suppl.), 12-18.
Rodríguez-Martínez, H., and Larsson, B. (1998). Assessment of sperm fertilizing ability in farm animals. Acta Agr. Scand. A. (An.) 29(Suppl.), 12-18.
Rodríguez-Martínez, H., Larsson, B., Zhang, B. R., and Söderquist, L. (1997a). In vitro assessment of viability and fertilizing capacity of bull spermatozoa. J. Reprod. Dev. 43, 1-11.
Rodriguez-Martinez, H. (2007) State of the art in farm animal sperm evaluation. Reproduction, Fertility and Development, 19: 91-101.
Russel OD, Curtis GG. Standardization of specimen preparation, staining and sampling methods improves automated sperm head morphometry analysis. Fertil Steril 1993;59: 412-417.
Reyes, R., Sanchez-Vazquez, M.L., Merchant-Larios, H. et al. (1996) Effect of heparin-reduced glutathione on hamster sperm DNA unpacking and nuclear swelling. Arch. Androl., 37, 33-45.
Reichart, M., Kahane, I., and Bartoov, B. (2000). In vivo and in vitro impairment of human and ram sperm nuclear chromatin integrity by sexually transmitted ureaplasma urealyticum infection. Biol. Reprod. 63, 1041-1048. Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and its possible involvement of apoptosos. Biol Reprod 2002; 66: 1061-1067.
Soffer, Y., Golan, A., Herman, A. et al. (1992) Prediction of in vivo fertilization outcome by sperm penetration assay with TEST-yolk buffer preincubation. Fertil. Steril., 58, 556-562.
Schultz RM, Williams CJ. The science of ART. Science 2002;296: 2188-2190.
Sailer BL, Jost LK, Erickson KR, Tajiran MA, Evenson DP. Effects of X-irradiation on mouse testicular cells and sperm chromatin structure. Emviron Mol Mutagen 1995; 25: 23-30.
Shen HM, Ong CN. Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility. Fee Radic Biol Med 2000; 28: 529-536.
Samocha-Bone, D., Lewin, L. M., Weissenberg, R., Madgar, Y., Soffer, Y., Shochat, L., and Golan, R. (1998). In vitro human spermatozoa nuclear decondensation assessed by flow cytometry. Mol. Hum. Reprod. 4, 133-137.
Saleh RA, Agarwal A, Nelson DR et al. Increased sperm nuclear DNA damage in normospermic infertile men: a prospective study. Fert Steril 2002; 78: 313-313.
Stites DP (1984) Clinical laboratory methods for detection of cellular immune function. In: Stites DB, Stobo JD, Wells JV (eds) Basic and clinical immunology. Lange Medical Publications, Los Altos, CA, pp 353-372
Seidel, G.E. Jr., Schenk, J.L., Herickhoff, L.A., Doyle, S.P., Brink, Z., Green, R.D. and Cran D.G. (1999) Insemination of heifers with sexed sperm. Theriogenology 52; 1407-1420.
Stovel RT, Sweet RG, Herzenberg LA (1978) A means for orienting flat cells in flow systems. Biophys J 23:1-5.
Sutovsky, P., and Schatten, G. (1997). Depletion of glutathione during bovine oocyte maturation reversibly blocks the decondensation of the male pronucleus and pronuclear apposition during fertilization. Biol. Reprod. 56, 1503-1512.
Pinkel D, Gledhill BL, Van Dilla Ma, Stephenson D, Watchmaker G (1982) High resolution DNA measurements of mammalian sperm. Cytometry 3:1-9.
Shleeva MO, Bragamyan K, Telkov MV, Mukamolova GV, Young M, Bell DB, Kaprelyanti AS. Formation and resuscitation of non-cultivable cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase. Microbiol 2002, 148:1581-1591.
Szasz F, Sirivaidyapong S, Cheng FP, Voorhout WF, Marks A, Colenbrander B, Solti L, Gadella BM. Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen. Mol Reprod Dev 2000; 55: 289-298.
Spano` M, Calugi A, Capuano V, et al. Andrological sperm analysis by flow cytometry and sizing. Andrologia 1984;16: 367-375.
Sakkas D, Moffatt O, Manicardi GC, Mariethoz E, Tarozzi N, Bizzaro D. Nature of DNA damage in ejaculated human spermatozoa and the possible involvement of apoptosis. Biol Reprod 2002;66: 1061- 1067.
Thomas CA, Garner DL. Post-thaw bovine spermatozoal quality estimated from fresh samples. J Androl 1994; 15: 305-340.
Tao J, Du J, Critser ES, Critser JK. Assessment of the acrosomal status and viability of human spermatozoa simultaneously using flow cytometry. Hum Reprod 1993;8:1879-I 895.
Talbot P, Chacon R (1980) A new procedure for rapidly scoring acrosome reactions of human sperm. Gamete Res 3:211
Talbot P, Chacon RS (1981) A triple-stain technique for evaluating normal acrosome reactions of human sperm. J Exp Zool 215: 201-208
Uhler ML, Leung A, Chan SYW, Schmid I, Wang C: Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting. Fertil Steril 1993;60:1076-1081
Urner, F., and Sakkas, D. (1999). Characterization of glycolysis and pentose phosphate pathway activity during sperm entry into the mouse oocyte. Biol. Reprod. 60, 973-978.
Varum S, Bento C, Sousa APM. Et al. Characterization of human sperm populations using conventional parameters, surface ubiquitination, and apoptotic markers. Fert Steril 2007; 87: 572-583.
Vicari E, Perdichizzi A, De Palma A, Burrello N, D`Agata R, Calogero AE. Globozoospermia is associated with chromatin structure abnormalities. Hum Reprod 2002; 17: 2128-2133.
Verstegen J, Iguer-ouada M, Onclin K. Computer assisted semen analyzers in andrology and veterinary practice. Theriogenology 2002;57:149-79.
Wu CT, Bollwein H, Weber F, Stolla R. Effects of scrotal insulation on sperm mitochondrial membrane potential, motility, head abnormality and chromatin structure in bulls. Reprod Dom Anim 2003; 38. 329 (abstruct).
Watson PF, Kunze E, Cramer P, Hammerstedt RH. A comparison of critical osmolarity and hydraulic conductivity and its activation energy in fowl and bull spermatozoa. J Androl 1992; 13: 131-138.
Wilhelm KM, Graham JK, Squires EL. Comparison of the fertility of cryopreserved stallion spermatozoa with sperm motion analyses, flow cytometric evaluation, and zona-free hamster oocyte penetration. Theriogenology 1996;46: 559-578.
World Health Organization (1987) Laboratory Manual for the Examination of Human Semen and Cervical-Mucus Interaction. Cambridge University Press, Cambridge, U.K.
Watson, P. (2000). The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60-61, 481-492.
Yanagimachi, R., 1994.Mammalian fertilization. In: Knobil, E., Neill, J.D. (Eds.), The Physiology of Reproduction. Raven Press Ltd., New York, pp. 189-317.
Yanagimachi, R., and Noda, Y. (1970). Electron microscopy studies of spermincorporation into the hamster egg.Am. J.Anat. 128, 429-462.
Zirkin, B. R., Soucek, D. A. & Chang, T. S. K. (1985) In vitro and in vivo studies of mammalian sperm nuclear decondensation. Gamete Research 11, 349±365.
Zucker RM, Perreault SD, Elstein KH. Utility of light scatter in the morphological analysis of sperm. Cytometry 1992; 13: 39-47.
Zucker, R.M., Perreault, S.D. and Elstein, K.H. (1992) Utility of light scatter in the morphological analysis of sperm. Cytometry, 13, 39-47.
Zirkin, B. R., Soucek, D. A. & Chang, T. S. K. (1985) In vitro and in vivo studies of mammalian sperm nuclear decondensation. Gamete Research 11, 349-365.