Flow Cytometry for the Evaluation of Semen

Flow Cytometry for the Evaluation of Semen State of the Art in Sperm Assessment Using Flow Cytometry Abstract Flow cytometry is emerging as a substantial tool in the domain of modern andrology for the routine analysis of spermatozoa. Recent application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, analysis of semen samples was routinely performed by microscopical evaluation and manual techniques by laboratory operators; analysis is inclined due to comprehensive variability among observers, influencing its clinical validity. During last decade, to evaluate farm animal semen, variety of new flow cytometric techniques have been intercalated which made possible a wide spread evaluation of several sperm functionality and characteristics. Here in this paper, an initiative has been taken to explore numerous current flow cytometry developments pressing for andrological tests. After the invention of flow cytometry, sperm evaluation by traditional (microscopic) means became questioned and avoided due to the robust advantages of flow cytometry over the microscopic methods. By the recent development of diverse fluroscence probes, flow cytometry became capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gametes based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the opportunity of working with small sample sizes, increases the repeatability of data obtained, removes the subjectivity of evaluation and allows simultaneous assessment of multiple fluorochromes. Thus, flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of exper iments that are not yet possible with any other technique. Nowadays, semen evaluation using laboratory analyses is very meaningful to the artificial insemination industry to provide the most desired quality product to customers. Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen attributes that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed. Introduction Up to now, semen evaluation is considered as the most important laboratory test that has enabled us to identify and predict clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (Rodrà ­guez-Martà ­nez, 2007). Determination of the potential fertility of semen sample and, in the long run, of the male from which it has been collected is the ultimate goal of semen evaluations in clinically healthy sires. Now a days, many methods for the estimate the possible fertilizing capacity of a semen sample and, or in the word, of the male (reviewed by Dziuk 1996; Rodrà ­guez-Martà ­nez et al. 1997a; Rodrà ­guez-Martà ­nez and Larsson 1998; Saacke et al. 1998; Larsson and Rodrà ­guez-Martà ­nez 2000; Rodrà ­guez- Martà ­nez 2000, 2003; Popwell and Flowers 2004; Graham and Mocà © 2005; Gillan et al. 2005) are existing. The methods routinely accustomed for evaluation of the quality of a semen sample involved an evaluation of general appearance, volume, pH, sperm concentration, viability, morphology and motility. Most of these evaluations are based on microscopic analyses that only measure relatively a few numbers of spermatozoa within a population. In most of the cases, these are time-consuming; results obtained are controversial and are not translatable. It should also be noted that such conventional techniques are apt to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. During this long processes, number of steps like semen extension, fluorophore loading, ultrav iolet and laser illumination, high-speed sorting, cooling and cryopreservation are followed, which create a scope to impose different degrees of change in sperm functionality followed by suffer of damage to sperm membranes, organelles or the DNA content. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed by many groups that buck of those so-called procedures are incomplete, time consuming and laborious. Flow cytometry in diverse technical applications proposes many advantages for the analysis of sperm quality. Flow cytometry is a method where multiple fluorescences and light scattering can be induced allowing single cell or particles illumination in suspension while they flow very rapidly through a sensing area. The increasing use over the past decade of flow cytometry in the leading laboratories in human and veterinary andrology has dramatically increased our knowledge of sperm function under physiological and biotechnological conditions. Flow cytometers is capable to acquire data from several subpopulations within a sample in a few minutes, making it perfect for assessing heterogenous populations in a semen sample. Flow cytometry was initially developed in the 1960s, after that flow cytometry is performing automated separation of cells based on the unique recognition of cellular patterns in a population feasible (Hulett et al., 1969). Likewise, cellular patterns can be recognized by utilizing such a separation approach, in each cells within a population (Baumgarth and Roederer, 2000; Herzenberg et al., 2006). The first notion of flow cytometry development was for medical and clinical applications such as haematology and oncology. Although still much research is going on these medical areas and account for the vast majority of publications on this robust technique, but during the past few years it is being used in a diverse areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Together with elevated use in many areas, recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 20 05, 2006;) stallion ( Kavak et al., 2003; Morrell et al., 2008) indicate that newly developed fluorescent stains and techniques of flow cytometry has made possible a more widespread analysis of semen quality at a biochemical, ultrastructural and functional level. Therefore, flow cytometry is the current technical solution for rapid, precisely reproducible assessment of sperm suspensions. In this review we have described potentiality and scope of flow cytometry for the evaluation of semen, and the way in which this technique can be used in clinical applications for andrology based on some of our previous experiences. Definition of flow cytometry The definition of a flow cytometer is ‘an instrument which measures the properties of cells in a flowing stream or ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream. In other word, cytometry is a method which measure physical and chemical attributes of cells or other particles. Such a measurement is made when cells or other particles pass in single file through some sort of measuring apparatus in a stream of fluid. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other traditional analytical tools, where a single value for each attribute is obtained for the whole population, flow cytometry provides data for each and every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters. Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light scattering are among the cell properties that can be detected. Flow sorting allows individual cells to be sorted on the basis of their measured properties, and one to three or more global properties of the cell can be measured. Flow cytometers and cell sorters make use of one or more excitation sources and one or two fluorescent dyes to measure and characterize several thousands of cells per second. Flow cytometry presents objective and precise results (Bunthof et al., 2001; Shleeva et al., 2002), which help to overcome the problems with the manual methods described above. Function and types of flow cytometry A flow cytometer is made of three main systems, fluidics, optics and electronics. ItI It can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment came in to function when it was used for measuring their DNA content (Evenson et al., 1980) and its application for analyzing semen has been increased rapidly in last decade. Flow cytometry is now applied for the evaluation semen such as sperm viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity, DNA status and so on. Continuous innovation of new fluorescent stains and techniques facilitated the flow cytometric evaluation of spermatozoa. Flow cytometry allows the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. This feature has an added benefit for semen analysis, as few single sperm parameters show significant correlation with fertility in vivo for semen within the acceptable range of normality (Larsson and Rodriguez-Martinez, 2000) and it is the general statistics that the more sperm parameters can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993). There are two main types of flow cytometers-analysers and sorters are in use. Together with data collection on cells, sorters have the potentiality to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation using a combination of microspheres, flow cytometry and high speed digital processing. Advantages of FC compared to other conventional techniques to explore sperm structure and function Use of authentic assays in the fertility clinic and artificial insemination industries increasing day by day. In this respect, use of flow cytometry might be an important attempt to resolve sustaining problem with so called commonly used manual method for the semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with such techniques. It is worth mentinign here that so called method deal only with few hundred sperm. When we deal with such a few sperm population, there is a possibility that obtained result might not be statistically significant (Russel and Curtis, 1993). The methods which are frequently used are enable to determine sperm concentration (Jorgensen et al., 1997), motility or morphology only (Keel et al., 2002). Objectivity, cell number measured, speed of count and precision are the advantages of flow cytometry to conventional light microscopy techniques (Spano and Evenson, 1993). The technique now a days has been used to determi ne a number of factors including those of acrosome status, membrane integrity, mitochondrial function as well as multiparameter measurement in human (Garrido et al., 2002). Flow cytometry has the ability to analyze thousands of cells in few minutes. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in a diverse species (boar, bull, stallion etc), and the observed errors were significantly better than those obtained from the so-called manual methods. Although there are diverse benefits of flow cytometer for the analysis of semen, feasibility of applying flow cytometry sometimes restricted to researcher due to the high outlay and difficulties of operation associated with the requirement of a skilled operator. Further, a flow cytometer is very large and cannot resist shocks associated with movement, and it also requires much space in the laboratory. Whatever may be the limitation, the development of more affordable ‘‘bench-top flow cytometers in recent time raised the potential essentialities to semen analysis. If the further application of flow cytometric analysis is considered further, it might be seen that it is growing popularities as a technique for assessing more than one sperm attribute, simultaneously. Compared to traditional microscopic techniques, flow cytometry analysis is allowing to give a far more simplified and objective method of semen analysis, especially in relation to fertilization with acrosome reactivity potential of spermatozoa (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996). A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Data from a number of preliminary studies proposed that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). According to the World Health Organization that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, might lead to inaccuracy (World Health Organization, 1999). Data from Tomlinson and colleagues indicate that two proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) can result in a lower concentrations of sperm compared to the hemocytometer method (Tomlinson et al., 2001) . In contrast, plenty of reports document unacceptable differences between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong? Improvement of semen quality testing has been emphasizing in some reports (Jorgensen et al., 1997; Keel et al., 2000). But due to low number of sperm evaluation by the conventional method results in poor reproducibility. These problems might be overcome when using flow cytometry. The validation of method is a challenge due to its essentiality of having specific, precise, objective, and accurate evaluation to establish a correlation of fertility data or to predict potential of a semen sample accurately (Amann, 1989). In a fertility clinic, precision of data in important as the result of semen analysis is frequently used to manage fertility of a patient and treatment of the unfertile couples. Thus, it is important to take into consideration within and between laboratory variations for successful infertility treatments. Sometimes its a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate â€Å"patterns of fluorescence rather than the fluorescence intensity. Flow cytometer has the lack of ability to discriminate sperm containing a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is almost no difference between methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the acrosomal region of the sperm (Purvis et al., 1990; Holden and Trounson, 1991) and that other binding sites can be easily distinguished by epifluorescence microscopy, whereas flow cytometry identifies the signal from the entire sperm. Additionally, conventional light microscopic semen assessment is increasingly being replaced by fluorescent staining techniques, computer-assisted sperm analysis (CASA) systems, and flow cytometry (PenËÅ"a et al., 2001; Verstegen et al., 2002). Additional advantages over existing techniques are that this approach is faster than the hemacytometer and that cellular debris, fat droplets, and other particulate material in extended semen are not erroneously counted as sperm, as often occurs with electronic cell counters. This method can also be used to determine the number of somatic cells in a semen sample. Application of flow cytometry for sperm count Sperm count is an important predominant factor for the evaluation of sperm fertility potential. Accurate determination of sperm cell concentration is critical especially in AI industry because it provides assurance to customers that straws of extended semen contain the sperm numbers indicated which will help to decide appropriate doze especially for pig. Accuracy of sperm count is a common problem in the andrological laboratories and accurate measure of sperm concentration is particularly important for export in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Every time new and more accurate methods for the sperm count determinations are coming and being replaced by the older ones. Some laboratories are trying the Maklerm counting chamber (Se if- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. Although hemacytometers are routinely used for sperm counts, due to the slow process and need for multiple measurements of each sample, the chance of error increase. Freund and Carol (ref) stated that a difference of 20% were not unusual between the determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (Ref). Spectrophotometer is recently being used in the AI industries to assess sperm concentration by determining turbidity of a semen s ample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (Ref). The accuracy of this method depends on the methods used for spectrophotometer calibration. Although, sperm concentration can also be determined by spectrophotometrically, the debris present in the raw semen crease problem with misestimation. Sperm number in the frozen thawed semen is difficult to ascertain as most of the extender contain egg yolk particles, fats and other particles which affect measurement of sperm with electric cell counter or spectrophotometers (Evenson et al., 1993). On the other hand flow cytometry created possibilities of a rapid determination of sperm number in a precise form. It is the flow cytometry which can reduce intra-laboratory and inter-laboratory variation and conflict regarding sperm concentration assessment. Computer assisted semen analyzer is robust technique for analyzing sperm movement which can count sperm as well; but such an a nalyzer most of the cases use some counting chamber or hemacytometer which itself can generate error. Although, hemacytometer was originally developed for blood cell counting, its use is now diverse including andrological laboratories for sperm counting. Around two-decade ago flow cytometry was suggested for sperm numbers in straws of cryopreserved bull semen. Christensen et al. (-) observed similar results for sperm count with flow cytometry and hemocytometer for a number of species. Now a day a simultaneous determination of sperm viability and sperm concentration is possible which can avoid the chance of occurring differences between ejaculates leading lack of coordination with field fertility and laboratory analyses. Thus the present technology is more precise which can get rid of variation from handling the sperm sample and variation from pipetting and the analysis itself. Further, Prathalingam et al. (2006) concluded that there is similarities for sperm count result between flow cytometry and two newly approached method (image analysis and fluorescent plate reader) for sperm counting. Though, use of fluorescent plate was emphasized due to low cost and allowing large number of cells counting from a large number of ejaculates. Although flow cytometry has become a valuable instrument for andrological determinations, it is also blamed that sperm concentration by flow cytometry signify a higher value than the real one. The possibility arise might be due to that semen samples often contain some alien materials such as immature germ cells, epithelial cells, blood cells, cytoplasmic droplet, cellular debris etc. In the same way, frozen semen has higher chance to introduce such material as they contain diluents components especially egg yolk particles. These particles and cell debris might have frontal and side light scatter parameters those are similar to spermatozoa. Such sperm-count-overestimation problem arisen in our cases also, especially when we deal with frozen semen. Further it is also claimed that flow cytometry has a tendency to overestimate viable spermatozoa. We are also experienced with such trouble which we guess might be due to that egg particles of extender are considered as viable cell as for it s staining pattern. Our yet to publish data indicate that this problem can be mimic by a centrifugation process and by using low concentration sample for evaluation with flow cytometry. Very recently Petrunkina and Harrison (2009) proposed a mathematical equation for fixing this flow cytometric sperm counting. Thus much research is going on and we hope such discrepancy will completely be resolved near future to get advantage from this robust technology for sperm counting. Flow cytometry for detecting sperm intactness -Viability of spermatozoa The viability of spermatozoa is a key determinant of sperm quality and prerequisite for successful fertilization. Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable and subjectively assess only 100 to 200 spermatozoa per ejaculate. Merkies et al. (2000) compared different methods of viability evaluation. They concluded that Eosin-nigrosin overestimate viability while fluorescent microscope and flow cytometry estimate similar trend of viability. Current flow cytometric procedures are able to simultaneously evaluate sperm cell viability together with some other attributes. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (Rodrà ­guez-Martà ­nez, 2007; Garner and Johnson, 1995; Garner et al., 1996), rams (Garner and Johnson, 1995), rabbits (Garner and Johnson, 1995), mice (Garner and Johnson, 1995; Songsasen et al., 199 7), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004). Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method. One of the first attempts to assess sperm viability utilized rhodamine 123 for determining potentiality of mitochondrial membrane while ethidium bromide for membrane integrity through flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990). The most generally used sperm viability stain combinations is SYBR-14 and PI at present. This stains are now sold commercially as live/dead viability kit. When these two stains are combinely used, the nuclei of viable sperm take fluoresce green and membrane integrity lost cells take red stain. This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been introduced to evaluate sperm concentration [11] and viability. Due to the small size and low cost, this instrument has been attracted for field measurements of both concentration and viability. In our hand this instrument was also became useful for the quick measurement of sperm concentration an d viability in stallion (Morrell et al., 2010). Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and obtained an inverse correlation between the damaged cells per cent and the field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility. Fluorochromes used to assess sperm viability by both approach could be utilized in combination with each other. In that case, when CFDA is used combined with PI, three populations of cells as live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa can be identified. This combination was found useful by Almlid and Johnson (1988) for frozen-thawed boar spermatozoa for monitoring membrane damage at the time of evaluation of various freezing protocols. Further, Harrison and Vickers (1990) also noticed that this combination with a fluorescent microscope is effective indicator of viability of fresh, incubated or cold-shocked spermatozoa in boar and ram. Contrasting to these, Garner et al. (1986) was failed to find a relationship between bull sperm viability and fertility when using combination of CFDA/PI . Flow cytometry for evaluating sperm viability appears to be a precious tool in the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combined assessment of sperm viability and concentration appears to be useful in the wake of improving quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. But the mechanism and mode of action by which SYBR-14 binds to the DNA of sperm is not known. It is know that PI stains nucleic acids by inte rcalating between the base pairs (Krishan, 1975). Viability stains can also be used in conjugation with fluorescently labeled plant lectins for simultaneous assessment of the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). It is conceivable that assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) found that insemination of boar sperm stained with SYBR-14 did not compromise fertilization or even the development of flushed porcine embryos in vitro. Non-viable sperms can be detected using the membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. Plasma membrane which is intact will not permit these stains entering into the spermatozoa and staining the nucleus. Most frequently used stains include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). After a series of comparison between fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality as assessed by flow cytometry using PI, Wilhelm et al. (1996) concluded that viability is the single laboratory assay that correlated with fertility. -Sperm plasma membrane integrity Although the sperm plasma membrane covers the entire cell, it consists of three distinct membrane compartments, one which covers the outer acrosomal membrane, one which covers the post acrosomal portion of the sperm head, and one which covers the middle and principal pieces. Sperm membrane is directly or ind Flow Cytometry for the Evaluation of Semen Flow Cytometry for the Evaluation of Semen State of the Art in Sperm Assessment Using Flow Cytometry Abstract Flow cytometry is emerging as a substantial tool in the domain of modern andrology for the routine analysis of spermatozoa. Recent application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, analysis of semen samples was routinely performed by microscopical evaluation and manual techniques by laboratory operators; analysis is inclined due to comprehensive variability among observers, influencing its clinical validity. During last decade, to evaluate farm animal semen, variety of new flow cytometric techniques have been intercalated which made possible a wide spread evaluation of several sperm functionality and characteristics. Here in this paper, an initiative has been taken to explore numerous current flow cytometry developments pressing for andrological tests. After the invention of flow cytometry, sperm evaluation by traditional (microscopic) means became questioned and avoided due to the robust advantages of flow cytometry over the microscopic methods. By the recent development of diverse fluroscence probes, flow cytometry became capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gametes based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the opportunity of working with small sample sizes, increases the repeatability of data obtained, removes the subjectivity of evaluation and allows simultaneous assessment of multiple fluorochromes. Thus, flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of exper iments that are not yet possible with any other technique. Nowadays, semen evaluation using laboratory analyses is very meaningful to the artificial insemination industry to provide the most desired quality product to customers. Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen attributes that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed. Introduction Up to now, semen evaluation is considered as the most important laboratory test that has enabled us to identify and predict clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (Rodrà ­guez-Martà ­nez, 2007). Determination of the potential fertility of semen sample and, in the long run, of the male from which it has been collected is the ultimate goal of semen evaluations in clinically healthy sires. Now a days, many methods for the estimate the possible fertilizing capacity of a semen sample and, or in the word, of the male (reviewed by Dziuk 1996; Rodrà ­guez-Martà ­nez et al. 1997a; Rodrà ­guez-Martà ­nez and Larsson 1998; Saacke et al. 1998; Larsson and Rodrà ­guez-Martà ­nez 2000; Rodrà ­guez- Martà ­nez 2000, 2003; Popwell and Flowers 2004; Graham and Mocà © 2005; Gillan et al. 2005) are existing. The methods routinely accustomed for evaluation of the quality of a semen sample involved an evaluation of general appearance, volume, pH, sperm concentration, viability, morphology and motility. Most of these evaluations are based on microscopic analyses that only measure relatively a few numbers of spermatozoa within a population. In most of the cases, these are time-consuming; results obtained are controversial and are not translatable. It should also be noted that such conventional techniques are apt to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed semen, and IVF. During this long processes, number of steps like semen extension, fluorophore loading, ultrav iolet and laser illumination, high-speed sorting, cooling and cryopreservation are followed, which create a scope to impose different degrees of change in sperm functionality followed by suffer of damage to sperm membranes, organelles or the DNA content. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed by many groups that buck of those so-called procedures are incomplete, time consuming and laborious. Flow cytometry in diverse technical applications proposes many advantages for the analysis of sperm quality. Flow cytometry is a method where multiple fluorescences and light scattering can be induced allowing single cell or particles illumination in suspension while they flow very rapidly through a sensing area. The increasing use over the past decade of flow cytometry in the leading laboratories in human and veterinary andrology has dramatically increased our knowledge of sperm function under physiological and biotechnological conditions. Flow cytometers is capable to acquire data from several subpopulations within a sample in a few minutes, making it perfect for assessing heterogenous populations in a semen sample. Flow cytometry was initially developed in the 1960s, after that flow cytometry is performing automated separation of cells based on the unique recognition of cellular patterns in a population feasible (Hulett et al., 1969). Likewise, cellular patterns can be recognized by utilizing such a separation approach, in each cells within a population (Baumgarth and Roederer, 2000; Herzenberg et al., 2006). The first notion of flow cytometry development was for medical and clinical applications such as haematology and oncology. Although still much research is going on these medical areas and account for the vast majority of publications on this robust technique, but during the past few years it is being used in a diverse areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Together with elevated use in many areas, recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 20 05, 2006;) stallion ( Kavak et al., 2003; Morrell et al., 2008) indicate that newly developed fluorescent stains and techniques of flow cytometry has made possible a more widespread analysis of semen quality at a biochemical, ultrastructural and functional level. Therefore, flow cytometry is the current technical solution for rapid, precisely reproducible assessment of sperm suspensions. In this review we have described potentiality and scope of flow cytometry for the evaluation of semen, and the way in which this technique can be used in clinical applications for andrology based on some of our previous experiences. Definition of flow cytometry The definition of a flow cytometer is ‘an instrument which measures the properties of cells in a flowing stream or ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream. In other word, cytometry is a method which measure physical and chemical attributes of cells or other particles. Such a measurement is made when cells or other particles pass in single file through some sort of measuring apparatus in a stream of fluid. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other traditional analytical tools, where a single value for each attribute is obtained for the whole population, flow cytometry provides data for each and every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters. Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light scattering are among the cell properties that can be detected. Flow sorting allows individual cells to be sorted on the basis of their measured properties, and one to three or more global properties of the cell can be measured. Flow cytometers and cell sorters make use of one or more excitation sources and one or two fluorescent dyes to measure and characterize several thousands of cells per second. Flow cytometry presents objective and precise results (Bunthof et al., 2001; Shleeva et al., 2002), which help to overcome the problems with the manual methods described above. Function and types of flow cytometry A flow cytometer is made of three main systems, fluidics, optics and electronics. ItI It can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment came in to function when it was used for measuring their DNA content (Evenson et al., 1980) and its application for analyzing semen has been increased rapidly in last decade. Flow cytometry is now applied for the evaluation semen such as sperm viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity, DNA status and so on. Continuous innovation of new fluorescent stains and techniques facilitated the flow cytometric evaluation of spermatozoa. Flow cytometry allows the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. This feature has an added benefit for semen analysis, as few single sperm parameters show significant correlation with fertility in vivo for semen within the acceptable range of normality (Larsson and Rodriguez-Martinez, 2000) and it is the general statistics that the more sperm parameters can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993). There are two main types of flow cytometers-analysers and sorters are in use. Together with data collection on cells, sorters have the potentiality to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation using a combination of microspheres, flow cytometry and high speed digital processing. Advantages of FC compared to other conventional techniques to explore sperm structure and function Use of authentic assays in the fertility clinic and artificial insemination industries increasing day by day. In this respect, use of flow cytometry might be an important attempt to resolve sustaining problem with so called commonly used manual method for the semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with such techniques. It is worth mentinign here that so called method deal only with few hundred sperm. When we deal with such a few sperm population, there is a possibility that obtained result might not be statistically significant (Russel and Curtis, 1993). The methods which are frequently used are enable to determine sperm concentration (Jorgensen et al., 1997), motility or morphology only (Keel et al., 2002). Objectivity, cell number measured, speed of count and precision are the advantages of flow cytometry to conventional light microscopy techniques (Spano and Evenson, 1993). The technique now a days has been used to determi ne a number of factors including those of acrosome status, membrane integrity, mitochondrial function as well as multiparameter measurement in human (Garrido et al., 2002). Flow cytometry has the ability to analyze thousands of cells in few minutes. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in a diverse species (boar, bull, stallion etc), and the observed errors were significantly better than those obtained from the so-called manual methods. Although there are diverse benefits of flow cytometer for the analysis of semen, feasibility of applying flow cytometry sometimes restricted to researcher due to the high outlay and difficulties of operation associated with the requirement of a skilled operator. Further, a flow cytometer is very large and cannot resist shocks associated with movement, and it also requires much space in the laboratory. Whatever may be the limitation, the development of more affordable ‘‘bench-top flow cytometers in recent time raised the potential essentialities to semen analysis. If the further application of flow cytometric analysis is considered further, it might be seen that it is growing popularities as a technique for assessing more than one sperm attribute, simultaneously. Compared to traditional microscopic techniques, flow cytometry analysis is allowing to give a far more simplified and objective method of semen analysis, especially in relation to fertilization with acrosome reactivity potential of spermatozoa (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996). A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Data from a number of preliminary studies proposed that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). According to the World Health Organization that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, might lead to inaccuracy (World Health Organization, 1999). Data from Tomlinson and colleagues indicate that two proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) can result in a lower concentrations of sperm compared to the hemocytometer method (Tomlinson et al., 2001) . In contrast, plenty of reports document unacceptable differences between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong? Improvement of semen quality testing has been emphasizing in some reports (Jorgensen et al., 1997; Keel et al., 2000). But due to low number of sperm evaluation by the conventional method results in poor reproducibility. These problems might be overcome when using flow cytometry. The validation of method is a challenge due to its essentiality of having specific, precise, objective, and accurate evaluation to establish a correlation of fertility data or to predict potential of a semen sample accurately (Amann, 1989). In a fertility clinic, precision of data in important as the result of semen analysis is frequently used to manage fertility of a patient and treatment of the unfertile couples. Thus, it is important to take into consideration within and between laboratory variations for successful infertility treatments. Sometimes its a matter of argument that compared to flow cytometry, fluorescent microscopy evaluate â€Å"patterns of fluorescence rather than the fluorescence intensity. Flow cytometer has the lack of ability to discriminate sperm containing a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is almost no difference between methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the acrosomal region of the sperm (Purvis et al., 1990; Holden and Trounson, 1991) and that other binding sites can be easily distinguished by epifluorescence microscopy, whereas flow cytometry identifies the signal from the entire sperm. Additionally, conventional light microscopic semen assessment is increasingly being replaced by fluorescent staining techniques, computer-assisted sperm analysis (CASA) systems, and flow cytometry (PenËÅ"a et al., 2001; Verstegen et al., 2002). Additional advantages over existing techniques are that this approach is faster than the hemacytometer and that cellular debris, fat droplets, and other particulate material in extended semen are not erroneously counted as sperm, as often occurs with electronic cell counters. This method can also be used to determine the number of somatic cells in a semen sample. Application of flow cytometry for sperm count Sperm count is an important predominant factor for the evaluation of sperm fertility potential. Accurate determination of sperm cell concentration is critical especially in AI industry because it provides assurance to customers that straws of extended semen contain the sperm numbers indicated which will help to decide appropriate doze especially for pig. Accuracy of sperm count is a common problem in the andrological laboratories and accurate measure of sperm concentration is particularly important for export in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Every time new and more accurate methods for the sperm count determinations are coming and being replaced by the older ones. Some laboratories are trying the Maklerm counting chamber (Se if- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. Although hemacytometers are routinely used for sperm counts, due to the slow process and need for multiple measurements of each sample, the chance of error increase. Freund and Carol (ref) stated that a difference of 20% were not unusual between the determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (Ref). Spectrophotometer is recently being used in the AI industries to assess sperm concentration by determining turbidity of a semen s ample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (Ref). The accuracy of this method depends on the methods used for spectrophotometer calibration. Although, sperm concentration can also be determined by spectrophotometrically, the debris present in the raw semen crease problem with misestimation. Sperm number in the frozen thawed semen is difficult to ascertain as most of the extender contain egg yolk particles, fats and other particles which affect measurement of sperm with electric cell counter or spectrophotometers (Evenson et al., 1993). On the other hand flow cytometry created possibilities of a rapid determination of sperm number in a precise form. It is the flow cytometry which can reduce intra-laboratory and inter-laboratory variation and conflict regarding sperm concentration assessment. Computer assisted semen analyzer is robust technique for analyzing sperm movement which can count sperm as well; but such an a nalyzer most of the cases use some counting chamber or hemacytometer which itself can generate error. Although, hemacytometer was originally developed for blood cell counting, its use is now diverse including andrological laboratories for sperm counting. Around two-decade ago flow cytometry was suggested for sperm numbers in straws of cryopreserved bull semen. Christensen et al. (-) observed similar results for sperm count with flow cytometry and hemocytometer for a number of species. Now a day a simultaneous determination of sperm viability and sperm concentration is possible which can avoid the chance of occurring differences between ejaculates leading lack of coordination with field fertility and laboratory analyses. Thus the present technology is more precise which can get rid of variation from handling the sperm sample and variation from pipetting and the analysis itself. Further, Prathalingam et al. (2006) concluded that there is similarities for sperm count result between flow cytometry and two newly approached method (image analysis and fluorescent plate reader) for sperm counting. Though, use of fluorescent plate was emphasized due to low cost and allowing large number of cells counting from a large number of ejaculates. Although flow cytometry has become a valuable instrument for andrological determinations, it is also blamed that sperm concentration by flow cytometry signify a higher value than the real one. The possibility arise might be due to that semen samples often contain some alien materials such as immature germ cells, epithelial cells, blood cells, cytoplasmic droplet, cellular debris etc. In the same way, frozen semen has higher chance to introduce such material as they contain diluents components especially egg yolk particles. These particles and cell debris might have frontal and side light scatter parameters those are similar to spermatozoa. Such sperm-count-overestimation problem arisen in our cases also, especially when we deal with frozen semen. Further it is also claimed that flow cytometry has a tendency to overestimate viable spermatozoa. We are also experienced with such trouble which we guess might be due to that egg particles of extender are considered as viable cell as for it s staining pattern. Our yet to publish data indicate that this problem can be mimic by a centrifugation process and by using low concentration sample for evaluation with flow cytometry. Very recently Petrunkina and Harrison (2009) proposed a mathematical equation for fixing this flow cytometric sperm counting. Thus much research is going on and we hope such discrepancy will completely be resolved near future to get advantage from this robust technology for sperm counting. Flow cytometry for detecting sperm intactness -Viability of spermatozoa The viability of spermatozoa is a key determinant of sperm quality and prerequisite for successful fertilization. Viability of spermatozoa can be assessed by numerous methods, but many are slow and poorly repeatable and subjectively assess only 100 to 200 spermatozoa per ejaculate. Merkies et al. (2000) compared different methods of viability evaluation. They concluded that Eosin-nigrosin overestimate viability while fluorescent microscope and flow cytometry estimate similar trend of viability. Current flow cytometric procedures are able to simultaneously evaluate sperm cell viability together with some other attributes. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (Rodrà ­guez-Martà ­nez, 2007; Garner and Johnson, 1995; Garner et al., 1996), rams (Garner and Johnson, 1995), rabbits (Garner and Johnson, 1995), mice (Garner and Johnson, 1995; Songsasen et al., 199 7), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004). Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method. One of the first attempts to assess sperm viability utilized rhodamine 123 for determining potentiality of mitochondrial membrane while ethidium bromide for membrane integrity through flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990). The most generally used sperm viability stain combinations is SYBR-14 and PI at present. This stains are now sold commercially as live/dead viability kit. When these two stains are combinely used, the nuclei of viable sperm take fluoresce green and membrane integrity lost cells take red stain. This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been introduced to evaluate sperm concentration [11] and viability. Due to the small size and low cost, this instrument has been attracted for field measurements of both concentration and viability. In our hand this instrument was also became useful for the quick measurement of sperm concentration an d viability in stallion (Morrell et al., 2010). Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and obtained an inverse correlation between the damaged cells per cent and the field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility. Fluorochromes used to assess sperm viability by both approach could be utilized in combination with each other. In that case, when CFDA is used combined with PI, three populations of cells as live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa can be identified. This combination was found useful by Almlid and Johnson (1988) for frozen-thawed boar spermatozoa for monitoring membrane damage at the time of evaluation of various freezing protocols. Further, Harrison and Vickers (1990) also noticed that this combination with a fluorescent microscope is effective indicator of viability of fresh, incubated or cold-shocked spermatozoa in boar and ram. Contrasting to these, Garner et al. (1986) was failed to find a relationship between bull sperm viability and fertility when using combination of CFDA/PI . Flow cytometry for evaluating sperm viability appears to be a precious tool in the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combined assessment of sperm viability and concentration appears to be useful in the wake of improving quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. But the mechanism and mode of action by which SYBR-14 binds to the DNA of sperm is not known. It is know that PI stains nucleic acids by inte rcalating between the base pairs (Krishan, 1975). Viability stains can also be used in conjugation with fluorescently labeled plant lectins for simultaneous assessment of the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). It is conceivable that assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) found that insemination of boar sperm stained with SYBR-14 did not compromise fertilization or even the development of flushed porcine embryos in vitro. Non-viable sperms can be detected using the membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. Plasma membrane which is intact will not permit these stains entering into the spermatozoa and staining the nucleus. Most frequently used stains include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). After a series of comparison between fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality as assessed by flow cytometry using PI, Wilhelm et al. (1996) concluded that viability is the single laboratory assay that correlated with fertility. -Sperm plasma membrane integrity Although the sperm plasma membrane covers the entire cell, it consists of three distinct membrane compartments, one which covers the outer acrosomal membrane, one which covers the post acrosomal portion of the sperm head, and one which covers the middle and principal pieces. Sperm membrane is directly or ind

One to Many Communication Essay

What is wi fi ? Wi-Fi is the name of a popular wireless networking technology that uses radio waves to provide wireless high-speed internet and network connections. A common misconception is that the term Wi-Fi is short for â€Å"wireless fidelity,† however this is not the case. Wi-Fi is simply a trademarked term meaning IEEE 802.11x. The wi fi alliance, the organization that owns the Wi-Fi (registered trademark) term specifically defines Wi-Fi as any â€Å"wireless local area network (WLAN) products that are based on the Institute of Electrical and Electronics Engineers’ (IEEE) 802.11 standards.† Initially, Wi-Fi was used in place of only the 2.4GHz 802.11B standard, however the wi fi alliance has expanded the generic use of the Wi-Fi term to include any type of network or WLAN product based on any of the802.11 standerds, including 802.11b,802.11a, dual band ,and so on,in an attempt to stop confusion about wireless LAN interoperability. How Wi-Fi Works? Wi-Fi works with no physical wired connection between sender and receiver by using radio frequency (RF) technology, a frequency within the electromagnetic spectrum associated with radio wave propagation. When an RF current is supplied to an antenna, an electromagnetic field is created that then is able to propagate through space. The cornerstone of any wireless network is an access point (AP)The primary job of an access point is to broadcast a wireless signal that computers can detect and â€Å"tune† into. In order to connect to an access point and join a wireless network, computers and devices must be equipped with wireless network adapters Wi-Fi Support? Wi-Fi is supported by many applications and divecesincludingvidio game consoles, home network, major operating system, and other types of consumer electronic .any products that are tested and approved as â€Å"Wi-Fi Certified† (a registered trademark) by the WI FI alliance are certified as interoperable with each other, even if they are from different manufacturers. For example, a user with a Wi-Fi Certified product can use any brand of acess point with any other brand of client hardware that also is also â€Å"Wi-Fi Certified†. Wi-Fi Alliance? The Wi-Fi Alliance is a trade association that promotes Wriless LAN technology and certifies products if they conform to certain standards of interoperability. Not every IEEE 802.11-compliant device is submitted for certification to the Wi-Fi Alliance, sometimes because of costs associated with the certification process. The lack of the Wi-Fi logo does not necessarily imply a device is incompatible with Wi-Fi devices. The Wi-Fi Alliance owns the Wi-Fi trademark. Manufacturers may use the trademark to brand certified products that belong to a class of wireless local area network (WLAN) devices based on the IEEE 802.11 standards. Advantages: †¢ Flexible if there is ad-hoc situation when additional workstation was required. †¢ Implementation cost is cheaper than wired network. †¢ Ideal for the non-reachable places such as across river or mountain or rural area. †¢ Ideal for temporary network setups. Disadvantages: †¢ Lower speed compared to wired network. (of course!) †¢ Less secure because hacker’s laptop can act as Access Point. If you connected to their laptop, they’ll read all your information (username, password.. bla..bla..bla..). †¢ More complex to configure than wired network. †¢ Affected by surrounding. E.g: walls (blocking), microwave oven (interference), far distance (attenuation)

Essay about Great Leaders - 703 Words

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Definition and Examples of Double Entendres

A double entendre is a  figure of speech in which a word or phrase can be understood in two ways, especially when one meaning is risquà ©. Also called innuendo. One of the most famous double entendres in American advertising is the slogan created by Shirley  Polykoff to promote Clairol hair coloring: Does she or doesnt she? The phrase double entendre  (from the French, now obsolete, for double meaning) is sometimes hyphenated and sometimes italicized. Examples and Observations Rebecca Kordecki . . . created little booties and a slide kit to use while performing moves that strengthen and lengthen the body. The name Booty Slide is a double entendre, she explains: We wear the booties on our feet, but the workout also lifts your booty.(Carlene Thomas-Bailey, American Fitness Crazes Hit the UK. The Guardian, Dec. 28, 2010)While many mento songs are about traditional folksong subjects, from political commentary to simple day-to-day life, a disproportionately large number of the songs are bawdy songs, often featuring poorly-veiled (and delightfully funny) sexual double-entendres. Popular mento songs include references to Big Bamboo, Juicy Tomatoes, Sweet Watermelon, and so on.(Megan Romer, Jamaican Mento Music 101, About.com World Music)Mrs. Slocombe: Before we go any further, Mr. Rumbold, Miss Brahms and I would like to complain about the state of our drawers. Theyre a positive disgrace.Mr. Rumbold: Your what, Mrs. Slocombe?Mrs. Slocombe: Our drawers. Theyre sti cking. And its always the same in damp weather.Mr. Rumbold: Really.Mrs. Slocombe: Miss Brahms could hardly shift hers at all just now.Mr. Lucas: No wonder she was late.Mrs. Slocombe: They sent a man who put beeswax on them, but that made them worse.Mr. Rumbold: Im not surprised.Miss Brahms: I think they need sandpapering.(Mollie Sugden, Nicholas Smith, Trevor Bannister, and Wendy Richard in Are You Being Served?)She touched his organ, and from that bright epoch, even it, the old companion of his happiest hours, incapable as he had thought of elevation, began a new and deified existence.(Charles Dickens, Martin Chuzzlewit, 1844)Nurse: God ye good morrow, gentlemen.Mercutio: God ye good den, fair gentlewoman.Nurse: Is it good den?Mercutio: ’Tis no less, I tell you; for the bawdy hand of the dial is now upon the prick of noon.Nurse: Out upon you! what a man are you!(William Shakespeare, Romeo and Juliet, Act II, scene three)Its impossible to ignore the prominence of water as a p rimary motif in black spiritual culture—from the debilitated Gospel pleas to be washed white as snow to the rebellion-coded double entendre wade in the water, which referenced both baptism and escape routes from slavery.(William J. Cobb, To the Break of Dawn: A Freestyle on the Hip Hop Aesthetic.  NYU Press, 2006)Womens Use of the Double Entendre in 18th-Century EnglandOf all the improvements in polite conversation, I know of nothing that is half so entertaining and significant as the double entendre. It is a figure in rhetoric, which owes its birth, as well as its name, to our inventive neighbours the French; and is that happy art, by which persons of fashion may communicate the loosest ideas under the most innocent expressions. The ladies have adopted it for the best reason in the world: they have long since discovered, that the present fashionable display of their persons is by no means a sufficient hint to the men that they mean any thing more than to attract their admi ration: the double entendre displays the mind in an equal degree, and tells us from what motives the lure of beauty is thrown out. . . .The double entendre is at present so much the taste of all genteel companies, that there is no possibility either of being polite or entertaining without it. That it is easily learnt is the happy advantage of it; for as it requires little more than a mind well stored with the most natural ideas, every young lady of fifteen may be thoroughly instructed in the rudiments of it from her book of novels, or her waiting maid. But to be as knowing as her mamma in all the refinements of the art, she must keep the very best company, and frequently receive lessons in private from a male instructor.(Edward Moore, The Double Entendre. The World, No. 201, Thursday, Nov. 4, 1756) Pronunciation: DUB-el an-TAN-dra

Essay about Business Challenges - 683 Words

1. The most significant challenge to business success you see in this rapidly changing global economy; One significant challenge to business success in this rapidly changing global economy is that many business are focused on efficiency and economic of scales. Just like the example provided by Michael H. Hugo on Chapter 1, many companies are focused on lowering operating cost as much as possible. This is causing for executives not leave any room for unpredicted changes limiting the company’s ability to respond to customer’s changing needs. 2. An organization that has been successful in the global marketplace using virtual teams and the best practices it used (as discussed in Chapter 7 of Business Agility: A Sustainable†¦show more content†¦While this is a practice enforced throughout the company, it was necessary to make sure that this communications were clear to the virtual team in order to maintain morale and the team’s trust in the company. 3. How the Marketplace simulation incorporates these challenges into simulation game play; The Marketplace simulation incorporates these challenges by providing student with real-world business problems and roles. Team members are challenged to make decisions and used critical thinking in order to solve this problems virtually. While The Marketplace gives the team the tolls necessary to have effective communication, ultimately each team members contributes to the overall success of the team, just like in a real business situation. 4. How your readings and individual self-assessment work provide potential solutions to these challenges. The unit’s readings have provided many solutions to the challenges businesses encounter nowadays. To this day, there are many companies that have business plans that concentrate strictly in lowering operating cost, cut budgets as much as possible and overwork workers that already are overworked. 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Rethinking Anthropology †E. R. Leach Free Essays

string(80) " of two issues of Man to expound my fallacies and confusions \(Fortes, 1959b\)\." RETHINKING ANTHROPOLOGY LONDON SCHOOL OF ECONOMICS MONOGRAPHS ON SOCLL ANTHROPOLOGY Managing Editor: Anthony For^e The Monographs in on Social Anthropology were established modem The by 1940 and aim to publish results of anthropological research of primary interest to specialists. continuation of the series was made possible from the Wenner-Gren Foundation for Anthropological Research, and more recently by a further grant from the Governors of the London a grant in aid School of Economics and Political Science. re under the direction of an Board associated with the Department of Anthropology of the London School of Economics and Political Science. We will write a custom essay sample on Rethinking Anthropology – E. R. Leach or any similar topic only for you Order Now Editorial The Monographs LONDON SCHOOL OF ECONOMICS MONOGRAPHS ON SOCIAL ANTHROPOLOGY No. 22 Rethinking Anthropology by E. R. LEACH UNIVERSITY OF LONDON THE ATHLONE PRESS NEW YORK: HUMANITIES PRESS INC Published by THE ATHLONE PRESS UNIVERSITY OF LONDON at 2 Gotcer Street, Distributed by Tiptree London wci Book Services Ltd Tiptree, Essex First edition, 1961 First paperback edition with corrections, 1966 Reprinted, 1968, 1971 E. R. Leach, 1961, 1971 U. K. U. K. sB N o 485 19522 4 cloth sB N B o 485 19622 o paperback U. S. A. s N 391 00146 9 paperback First printed in 1961 by ROBERT CUNNINGHAM AND SONS LTD ALVA Reprinted by photo-litho by JOHN DICKENS CO LTD NORTHAMPTON 4- M75†² Preface The title of this collection properly belongs only to the first essay. On 3 December 1959 1 had the honour to deliver the first Malinovvski Memorial Lecture at the London School of Economics. The Editorial Board of the London School of Economics Monographs in Social Anthropology enerously offere d to publish the text of my lecture but added the flattering suggestion that I should reprint a number of my other essays at the same time. I have accordingly appropriated the title of my Malinowski lecture for the whole collection. I do not pretend wholly consistent with that The essays extend over a period of fifteen years and is that the viewpoint of the latest (Chapter i) of the earliest (Chapter 2) but there is, I think, a certain continuity of theme and method in all of them. When they were first written all these essays were attempts to ‘rethink anthropology’. All are concerned with problems of others, I ‘theory’ and are based on ethnographic facts recorded by my own contribution being primarily that of analyst. In each case have tried to reassess the known facts in the light of unorthodox assumpSuch heresy seems to me to have merit for its own sake. Unconventional arguments often turn out to be wrong but provided they provoke discussion they may still have lasting value. By that criterion each of the essays in this book is a possible candidate for attention. tions. Among social anthropologists the is game f building new theories on the ruins of old ones almost an occupational disease. Contemporary arguments in social anthropology are built out of formulae concocted by Malinowski, Radcliffe-Brown and Levi-Strauss who in turn were only ‘rethinking’ Rivers, Durkheim and Mauss, who borrowed from Morgan, McLennan and Robertson- Smith the total outcome of all — and so on. Sceptics may think that despite all this ratiocination adds up to very little; our pedagogical subtleties, the diversities of human custom remain as bewildering as ever. But that we admit. The contemporary social anthropologist is all too well aware that he knows much less than Frazer imagined that he knew for certain. But that perhaps is the point. The contributions to anthropological pedantry collected in this book add little to the sum of human knowledge but if they provoke some readers to doubt their sense of certainty then they will have served their purpose. A note on the interconnections between the different papers draft of Chapter 2 may prove still helpful. The first was written in 1943 while I was on VI PREFACE and still in direct contact with Jinghpaw speakers. ppeared in the 1945 volume of the J. R. A. I, this was not actually published until 1950. These details of dating are relevant because they explain why my paper contains no reference to Chapters 15 and i6 of Levi-Strauss, Les structures elementaires de la parente (1949) and reciprocally why the latter work ignores the new information provided by my paper. Chapter 3, which was originally a Curl Prize E ssay, was completed in the spring of 195 1 and seems to have been the first English language commentar)’ on Levi-Strauss’s magnum opus though, presumably, my paper and J. P. B. de Josselin de Jong’s monograph Levi-Strauss’s Theory on Kinship and Marriage (1952) were going through the press at the same time. Although I here criticized Levi-Strauss on the grounds of ethnographical inaccuracy my sympathy with his general theoretical point of view is very great. Professor Levi-Strauss has himself noted the similarity between the view of ‘social structure’ implicit in my first Jinghpaw paper (Chapter 2) and his own (Levi-Strauss, 1953, p. 525 n), and in all my subsequent publications my debt to Levi-Strauss is obvious. The relationship of Chapter 4 to earlier literature will be apparent from the references in the text. Although it was not intended to be controversial it provoked Dr Kathleen Gough into a vigorous reply (Gough, 1959). The crucial part of my argument here is that I emphasize the need to distinguish between affinity regarded as an alliance between corporate kin groups and those individual affinal ties which bind a particular wife to a particular husband. This theme recurs in Chapter 5 and again in active military service t Although Chapter i. Chapter 5, as indicated in the text, is linked with a long correspondence which appeared in the pages of Man in 1953 and 1954 but the response which it evoked from my close academic colleagues is only marginally connected with this earlier discussion. Dr Goody has denounced my whole argument as grounded in fundamental error (Goody, 1959, p. 86) and Professor Fortes has taken up most of two issues of Man to expound my fallacies and confusions (Fort es, 1959b). You read "Rethinking Anthropology – E. R. Leach" in category "Essay examples" Both these explosions of academic wrath were provoked by a single sentence in my essay, namely ‘Thus Fortes, while recognizing that ties of affinity have comparable importance to ties of descent, disguises the former under his expression â€Å"complementary filiation† (see below p. 122). The exact sense in which this statement is an ‘error’ is still not clear to me for in the course of his denunciation Fortes reaffirms his view that ‘complementary filiation is a function of affinal relations’ (Fortes, 1959b, p. 209) which is precisely the argument I sought to controvert. ^ Professor Fortes has called his article *a rejoinder to Leach’, and — readers of Chapter i of this book need to appreciate that ‘a among other things in it is intended as rejoinder to Fortes’. Reference to a short note Man (i960. Art. 6) will perhaps help to make this clear. The two short papers on time symbolism reprinted in Chapter 6 do PREFACE inf luence of Professor Levi-Strauss Vll not form a series with the other chapters of the book though again the is pronounced. Although my ‘Cronus and Chronos’ appeared in print in 1953 while Levi-Strauss’s ‘The Structural Study of Myth’ was only published in 1956, I had in fact already heard Professor Levi-Strauss’s lecture on this topic before I wrote my essay. Explorations, the Toronto University publication in which my Chapter 6 was originally published, carried on its fly leaf the statement that it was ‘designed, not as a permanent reference journal that embalms truth for posterity, but as a publication that explores and searches and questions’ and both my papers are correspondingly brief and tentative. Nevertheless a number of my friends have suggested that the arguments they contain are of more than ephemeral interest; hence the reissue here^ Chapter i contains a considerable amount of matter which was not included in the spoken text of my Malinowski lecture. The other essays^ appear as originally printed, except for the correction of misprints, and one or two very minor alterations intended to clarify the argument. The Introductory Notes at the beginning of Chapters 2-6 are new. Acknowledgements I am indebted to the Council of the Royal Anthropological Institute of Great Britain and Ireland for permission to reprint the e ssays published here as Chapters 2, 3, 4 and 5 and to Professor E. S. Carpenter and the University of Toronto for permission to reprint the two short essays included in Chapter 6. I am indebted to a personal grant in aid from the Behavioral Sciences Division of the Ford Foundation for facilities employed while preparing } * j] : -^ these papers for publication. E. R. L. Contents 1. RETHINKING ANTHROPOLOGY I 2. JINGHPAW KINSHIP TERMINOLOGY THE STRUCTURAL IMPLICATIONS OF MATRILATERAL CROSS-COUSIN MARRIAGE 28 3. 54 4. POLYANDRY, INHERITANCE AND THE DEFINITION OF marriage: with PARTICULAR REFERENCE TO SINHALESE CUSTOMARY LAW ASPECTS OF BRIDEWEALTH AND MARRIAGE STABILITY IO5 5. AMONG THE KACHIN AND LAKHER 6. II4 TWO ESSAYS CONCERNING THE SYMBOLIC REPRESENTATION OF TIME (i) 124 Cronus and Chronos, 124 (ii) Time and False Noses, 132 Rethinking Anthropology my arrogant title. Since 1930 British Anthropology has embodied a well defined set of ideas and -^objectives which derive directly from the teaching of Malinowski and Radcliffe-Brown this unity of aim is summed up in the statement that British social anthropology is functionalist and concerned with the comparative analysis of social structures. But during the last year or so it has begun to look as if this particular aim had worked itself out. Most of my colleagues are giving up the attempt to make comparative generalizations instead they have begun to write impeccably detailed historical ethno- tET Social me begin by explaining — graphies of particular peoples. I regret this new tendency for I still believe that the findings of anthro- pologists have general as well as particular implications, but functionalist doctrine ceased to carry conviction? why has the understand what is happening in social anthropology I believe we need to go right back to the beginning and rethink basic issues really elementary matters such as To — hat we mean by marriage or descent or the unity of difficult siblings, and that is — for basic concepts are basic; The the ideas one has about them are deeply entrenched and firmly held. One bias of the things we need to recognize is the strength of the empirical which Malinowski introduced into social anthropology and which essential core of social anthropology has stayed with us ever since. is understanding of the way of life of a single particular people. This fieldwork is an extremely personal traumatic kind of experience and the personal involvement of the anthropologist in his work is reflected in what he produces. When we read Malinowski we get the impression that he is stating something which is of general importance. Yet how can this be? He is simply writing about Trobriand Islanders. Somehow he has so assimilated himself into the Trobriand situation that he is able to make the Trobriands fieldwork a microcosm of the whole primitive world. successors; for Firth, Primitive citizen of —the And the same is true of his is Man is a Tikopian, for Fortes, he a Ghana. The existence of this prejudice has long been recognized / but we have paid inadequate attention to its consequences. The difficulty of achieving comparative generalizations is directly linked with the problem of escaping from ethnocentric bias. 2 RETHINKING ANTHROPOLOGY As is appropriate to an occasion I when we honour the memory of Bronislaw MaUnowski, am going to be thoroughly egotistical. I shall imply there my own is merit by condemning the work of in my closest friends. But purpose is to distinguish between two rather similar varieties of comparative generalization, both of which turn up from time to time in contemporary British social anthropology. One of these, which I dislike, derives from the work of Radcliffe-Brown; the other, which I admire, derives from the work of Levi-Strauss. It is important that the differences between these two approaches be properly understood, so I shall draw my illustrations in sharp contrast, all black and all white. In this harsh and exaggerated form Professor Levi-Strauss method my malice. My might well repudiate the authorship of the ideas which I am trying to convey. Hence my egotism; let the blame be wholly mine. My problem is simple. How can a modern social anthropologist, with all the work of Malinowski and Radcliffc-Brown and their successors at his elbow, embark upon generalization with any hope of arriving at a satisfying conclusion? My answer is quite simple too; it is this: By thinking of the organizational ideas that are present in any society as constituting a mathematical pattern. The rest of what I have to say that is simply an elaboration of this cryptic statement. concern is with generalization, not with maintained that the objective of social anthropology was the ‘comparison of social structures’. In explaining this he asserted that when we distinguish and compare different types of social structure we are doing the same kind of thing as when we distinguish different kinds of sea shell according to their structural type (RadcliffeBrown, 1953, p. 109). Generalization is quite a different kind of mental First let me emphasize my comparison. Radcliffe-Brown operation. Let me illustrate this point. two points can be joined by a straight line and you can represent this straight line mathematically by a sm^G. first order algebraic equation. Any three points can be joined by a circle and you can represent this circle by a quadratic or second order algebraic equation. It would be a generalization to go straight on from there and say any : Any n points in a plane can be joined by a curve which can be represented by an equation of order n-i. This would be just a guess, but it would be true, and it is a kind of truth which no amount of comparison can ever reveal. Comparison and generalization are both forms of scientific activity, but different. Comparison is a matter of butterfly collecting —of classification, of the rrangement of things according to their types and subtypes. The followers of Radcliffe-Brown are anthropological butterfly collectors and their approach to their data has certain consequences. For example, according to RadclifTe- Brown’s principles we ought to think of Trobriand society : RETHINKING ANTHROPOLOGY as 3 classification a society of a particular structural type. The might proceed thus: Main Type Sub-type: Sub-sub-type : societies societies societies composed of unilineal descent groups. composed of matrilineal descent groups. composed of matrilineal descent groups in which he married males of the matrilineage live together in one place and apart from the females of the matrilineage, and so on. In this procedure each class preceding it is a sub-type of the class immediately in the tabulation. its uses, but it has very serious has no logical limits. Ultimately discriminated in this way as a sub-type Now I every just agree that analysis of this kind has is limitations. One major defect known society can be that it from any other, and since anthropologists are notably vague about what they mean by ‘a society’, this will lead them to distinguish more and more ocieties, almost ad infinitum. This is not just hypothesis. My colleague Dr Goody has gone to great pains to distinguish as types two adjacent societies in the Northern Gold Coast which he calls LoWi ili and LoDagaba. A careful reader of Dr Goody’s works will discover, however, that these two ‘societies’ are distinct simply the way that field Dr Goody notes from two has chosen to describe the fact that his neighbouring communities show some curious discrepancies. If limit Dr Goody’s methods of analysis were pushed to the we should be able to show that every village community throughout is he world constitutes a distinct society which distinguishable as a type from any other (Goody, 1956b). Another serious objection is that the typology makers never explain why they choose one frame of reference rather than another. RadcliffeBrovsTi’s instructions were simply that ‘it is necessary to compare societies the economic system, the with reference to one particular aspect . . . political system, or the kinship system’ . . . this is equivalent to saying that you can arrange your butterflies according to their colour, or their size, or the sh ape of their wings according to the him of the moment, but no matter what you do this will be science. Well perhaps, in a sense, it is; but you must realize that your prior arrangement creates an initial bias from which it is later extremely difficult to escape (Radcliffe-Brown, 1940, p. xii). Social anthropology is packed with frustrations of it this kind. An obvious Ever since example is the category opposition patrilineal/matrilineal. has been customary for anthropologists to distinguish unilineal from non-unilineal descent systems, and writing of the Iroquois, Morgan began among that it the former to distinguish patrilineal societies from atrilineal societies. is These categories now seem to us so rudimentary and obvious extremely difficult to break out of the straitjacket of thought which the categories themselves impose. 4 RETHINKING ANTHROPOLOGY Yet if our approach is to be genuinely unbiased we must be prepared to consider the possibihty that these type categories have no so ciological significance whatsoever. It may be that to create a class labelled matrtis as irrelevant for our understanding of social structure as the creation of a class blue butterflies is irrelevant for the understanding of the anatomical structure of lepidoptera. I don’t say it is so, but it may be; it is lineal societies time that we considered the possibility. J I warn you, the rethinking of basic category assumptions can be very disconcerting. But Let me cite a case. Dr Audrey Richards’s well-known contribution to African Systems of Kinship and Marriage is an essay in Radcliffe-Brownian typology making which is rightly regarded as one of the ‘musts’ of undergraduate reading (Richards, 1950). In this essay Dr Richards asserts that societies is ‘the problem’ of matrilineal the difficulty of combining recognition of descent through the oman with the rule of exogamous marriage, and she classifies a variety of matrilineal societies according to the way this ‘problem’ is solved. In effect her classification turns on the fact that a woman’s husband the two men. jointly possess rights in the woman’s brother and a woman’s children but that matrilineal systems differ in the wa y these rights are allocated between is the prior category assumptions. Men have kinds of society, so why should it be assumed from the start that brothers-in-law in matrilineal societies have special ‘prob- What I object to in this ll brothers-in-law in lems’ which are absent in patrilineal or bilateral structures? really What has lay a matrilineal society, she has decided to restrict her comparative obser-ations to matrilineal systems. Then, having selected a group of societies which have nothing in common except that they are matrilineal, she is naturally led to conclude that matrilineal descent is the major factor to which all the other items of cultural behaviour which she happened here with the Bemba, is that, because Dr Richards’s own special knowledge describes are functionally adjusted. Her argument I am afraid is a tautology; her system of classification already implies the truth of what she claims to be demonstrating. This illustrates how Radcliffe-Brown’s taxonomic assumptions fit in with the ethnocentric bias which I mentioned earlier. Because the typefinding social anthropologist conducts his whole argument in terms of tempted particular instances rather than of generalized patterns, he is constantly to attach exaggerated significance to those features of social organization which happen to be prominent in the societies of which he himself has first hand experience. The ase of Professor Fortes illustrates this is same point in rather a different way. His quest not so much for types as for prototypes. It so happens that the two societies of which he has made a close study have certain similarities of structural pattern for, while the Tallensi are patri- RETHINKING ANTHROPOLOGY lineal 5 and the Ashanti matrilineal, both Tallensi and Ashanti come unfiliatio n’, usually close to having a system of double unilineal descent. Professor Fortes has devised a special concept, ‘complementary w^hich helps him to describe this double unilineal element in the Tallensi/ Ashanti pattern w^hile rejecting the notion that these societies actually possess double unilineal systems (Fortes, 1953, p. 33; 1959b). It is interesting to note the circumstances which led to the development of this concept. From one point of view ‘complementary filiation’ is simply an inverse form of Malinowski’s notion of ‘sociological paternity’ as applied in the matrilineal context of Trobriand society. But Fortes has done more than invent a new name for an old idea; he has made it the corner stone of a substantial body of theory and this theory arises logically special circumstances of his own field experience. In his earlier writings the Tallensi are often represented as having a somewhat extreme form of patrilineal ideology. Later, in contrast to from the Rattray, Fortes placed an unambiguously matrilineal label upon the Ashanti. view, is The that merit of ‘complementary it is filiation’, from Fortes’s point of a concept which applies equally well to both of these contrasted societies but does not conflict with his thesis that both the Tallensi and the Ashanti have systems of unilineal descent. The concept ecame necessary to him precisely because he had decided at the start that the more familiar and more obvious notion of double unilineal descent was inappropriate. In retrospect Fortes seems to have decided that double unilineal descent is a special development of ‘complementary filiation’, the latter being a feature of all unilineal descent structures. That such category distinctions are contrived rather than natural is evident from Goody’s additi onal discrimination. Goody asserts that the LoWiili have ‘complementary descent rather than a dual descent system’. Since the concept of ‘complementary filiation’ was first introduced so as to help in the distinction between ‘filiation’ and ‘descent’ and since the adjective ‘complementary’ cannot here be given meaning except by reference to the word ‘descent’, the total argument is clearly tautologous (Fortes, 1945, pp. 134, 20of; 1950, p. 287; 1953, p. 34; 1959; Goody, 1956b, p. 77). Now I do not claim that Professor Fortes is mistaken, but I think he is misled by his prior suppositions. If making and from enthnocentric science. we are to bias we must let escape both from typology turn to a different kind of Instead of comparison repeat. Generalization us have generalization; instead of inductive; it butterfly collecting let us have inspired guesswork. Let me is consists in perceiving it is possible general laws in the circumstances of special cases; guesswork, a gamble, you may be wrong or you may be right, but if you happen to be right you have learnt something altogether new. In contrast, arranging butterflies according to their types and sub-types is tautology. It merely reasserts something you know already in a slightly different form. 6 RETHINKING ANTHROPOLOGY But if you are going is o start guessing, you need I to know how to guess. . d this wliat I am getting at when say that the form of thinking should be mathematical. Functional ism in a mathematical sense is not concerned with the interconnections between parts of a whole but with the principles of operation of partial systems. There is a direct conflict here with the dogmas of Malinowski and Malinowski’s functionalism required us to think of each Society (or Culture, as Malinowski would have put it) as a totality Radcliffe-Brown. of a made up kinds — number of discrete empirical ‘things’, of rather diverse institutions’, e. g. groups of people, customs. These ‘things’ are functionally interconnected to form a delicately balanced mechanism rather like the various parts of a wrist watch. cliff†e- The functionalism of Rad- Brown was equally mechanical though the focus of interest was different. RadclifTe-Brown was concerned, as it were, to distinguish wrist watches clocks, whereas Malinowski was interested in the general attributes of clockwork. But hath masters took as their starting point the notion that a culture or a society is an empirical whole made up rom grandfather of a limited two societies number of readily identifiable parts and that when we compare we are concerned to see whether or not the same kinds of is parts are present in both cases. This approach a mechanic but appropriate for a zoologist or for a botanist or for it is not the approach of a mathematician nor of an engineer and, in gineer. my view, the anthropologist has much in common with the en- But that is my private bias. I was originally trained as an engineer. The entities which we call societies are not naturally existing species, neither re they man-made mechanisms. But the analogy of a mechanism has quite as much relevance as the analogy of an organism. This is not the place to discuss the history of the organic analogy as a model for Society, but its arbitrariness is often forgotten. Hobbes, who developed hi s notion of a social organism in a very systematic way, discusses in his preface whether a mechanical or an organic analogy might be the more appropriate for his purpose. He opts for an organism only because he wants to include in his model a metaphysical prime mover (i. . God Life Force) (Hobbes, 1957, p. 5). In contrast RadcHffe-Brown employed the organic analogy as a matter of dogma rather than of choice (e. g. Radcliffe-Brown, 1957, pp. 82-86; 1940a, pp. 3, lo) and his butterfly collecting followers have accepted the appropriateness of the phrase ‘social organism’ without serious discussion. Against this complacency I — must protest. It is certainly the case that social scientists must often resort all to analogy but eternity. we are not committed to one type of model making for Our task societies s to understand and explain what goes on in society, how work. If an engineer tries to explain to you how a digital computer RETHINKING ANTHROPOLOGY bolts. 7 works he doesn’t spend his time classifying different kinds of nuts and He concerns himself with principles, not with things. He writes out argument as a mathematical equation of the utmost simplicity, somewhat on the lines of o + i = i i + i = 10. No doubt this example is frivolous; such computers embody their information in a code which is transmitted in positive and negative impulses denoted by the digital symbols o and i. The essential point is that although the information which can be embodied in such codes may be enormously complex, the basic principles on which the computing machines work is very simple. Likewise I would maintain that quite simple mechanical models can have relevance for social anthropology despite the acknowledged fact that the detailed empirical facts of social life display the utmost complexity. I don’t want to turn anthropology into a branch of mathematics but I believe we can learn a lot by starting to think about society in a mathehis : ; matical way. Considered mathematically society is not an assemblage of things but an assemblage of variables. A good analogy would be with that branch of mathematics known as topology, which may crudely be described as the geometry of elastic rubber sheeting. If I have a piece of rubber sheet and draw a series of lines on it to symbolize the functional interconnections of some set of social phenomena and I then start stretching the rubber about, I can change the manifest shape of my original geometrical figure out of all recognition and yet clearly there is a sense in which it is the same figure all the time. The constancy of pattern is not manifest as an objective empirical fact but it is there as a mathematical generalization. By analogy, generalized structural patterns in anthropology are not restricted to societies of any one manifest structural type. you will tell me that topology is one of those which mere sociologists had best avoid, but I am not in fact proposing anything original. A very good simple account of the nature of topology appears in an article under that title in the current edition of the Encyclopaedia Britannica. The author himself makes the point that because topology is a non-metrical form of mathematics it deserves especial attention from social scientists. I Now know that a lot of alarming scientific mysteries The fundamental Any closed curve is arc of a circle is ‘the variable in topology ‘the is the degree of connectedness. same as’ any other regardless of its shape; the same as’ a straight line because each is open ended. Contrariwise, a closed curve has a greater degree of connectedness than an arc. If of pattern case if we apply these ideas to sociology particular relationships e cease to be interested in and concern ourselves instead with the regularities relationships. is among neighbouring In the simplest possible there be a relationship p which intimately associated with another relationship q then in a topological study we shall not concern ourselves 8 RETHINKING ANTHROPOLOGY with the particular characteristics of/) and q but with their mutual character istics, i. e. with the algebraic ratio p’^q. But it must be understood that the relationships and sets of relationships which are symbolized in this way cannot properly be given specific numerical values. The reader should bear this point in mind when he encounters the specimens of pseudo-mathematics which occur later in this paper. All propositions in topology can also be expressed as propositions in symbolic logic (see Carnap, 1958, chapter G) and it was probably a consideration of this fact which led Nadel to introduce symbolic logic into own view is that while the consideration book (Xadel, 1957). of mathematical and logical models may help the anthropologist to order his last My his theoretical arguments in an all this intelligent way, his actual procedure s should be non-mathematical. The pattern relevance of to my main theme that the saTne structural may turn up in any kind of society patrilineal —a mathematical approach matrilineal makes no prior assumption that from non-unilineal systems or structures. all unilincal systems are basically different structures from the contrary, the principle of parity leads us to discount rigid category distinctions of this kind. On Let me try to illustrate I for the occasion shall take my point with an example. To be my example from Malinowski. Malinowski reported, as a ppropriate Most of you will know that fact of empirical ethnography, that the Trobrianders profess ignorance of the connection between copulation and pregnancy and that this ignorance serves as a rational justification for their system of matrilineal descent. From the Trobriand point of view ‘my father’ (tama) is not a blood relative at all but a kind of affine, *my mother’s husband’ (Malinowski, 1932a, p. 5). However, alongside their dogmatic ignorance of the facts of life, Trobrianders also maintain that every child should resemble its mother’s husband (i. . its father) but that no child could ever resemble a member of its own matrilineal kin. Malinowski seems to have thought it paradoxical that Trobrianders should hold both these doctrines at the same time. He was apparently bemused by the same kind of ethnocen tric assumptions as later led a Tallensi informant to tell Professor Fortes that ‘both parents transmit their blood to their offspring, as can be seen from the fact that Tallensi children may resemble either parent in looks’ (Fortes, 1949, p. 35; my italics). This is mixing up sociology and genetics. We know, and apparently the Tallensi assume, that physical appearance is genetically based, but there is no reason why primitive people in general should associate ideas of genetic inheritance with ideas about physical resemblance between persons. The explanation which the Trobrianders gave to Malinowski was that a father impresses his appearance on his son by cohabiting repeatedly with the mother and thereby ‘moulding’ (kuli) the child in her womb (Malinowski, 1932a, p. 176) which is reminiscent of the Ashanti . RETHINKING ANTHROPOLOGY view that the father shapes the body of his child as might a potter (Rattray, 1929, p. 9). This Trobriand theory is quite consistent with the view that the father is related to the son only as mother’s husband that is, an affine and not as a kinsman. There are other Trobriand doctrines which fall into line with this. The father’s sister is ‘the prototype of the lawful woman’ (Malinowski, 1932a, p. 450) which see ms to be more or less the equivalent of saying that — the father (tama) is much the same sort of relation as a brother-in-law. Again, although, as Powell has shown (Powell, 1956, p. 314), marriage with the father’s sister’s daughter is rare, the Trobrianders constantly assured Malinowski that this was a very right and proper marriage. Evidently in their view the category tama (which includes both father and father’s sister’s son) is very close to that of lubou (brother-in-law) (Mal- inowski, 1932a, pp. 86, 451). The similarity is asserted not only in verbal expression but also in the pattern of economic obligation, for the harvest gift (urignbu) paid by a married man is due both to his mother’s husband tama) and to his sister’s husband (lubou) (Malinowski, 1935, I, pp. 386, 413-18). From my point of view this cluster of Trobriand beliefs and attitudes is a ‘pattern of organizational ideas’— it specifies a series of categories, in a particular relationship and places them with one another as in an was biased by his down to earth empiricism, by Euro pean prejudices and by his interest in psycho-analysis, and he refused to accept tlie Trobriand doctrine at its face value. Instead he refurbished his concept of ‘sociological paternity’ which he had originalgebraic equation. But Malinowski ally devised to fit a quite different context, that of patrilineal organization among On to the Australian Aborigines (Malinowski, 19 13, p. 170-83). this earlier occasion Malinowski had used ‘sociological paternity’ relations show how between parents and children and between spouses derive from customary rules and not from any universal facts of biology or psychology, but in the later application of these ideas to Trobriand circumstances he shifts his ground and the argument becomes confused by the introduction of naive psychological considerations. On the face of t ‘sociological paternity’, as used in The Sexual Life of attitudes Savages, seems to mean that even in a society which, like the Trobriands, sociological still denies the facts of ‘biological paternity’, pertain to paternity, as zve understand it, which far, may be found. So so good. But Malinowski goes further than this. Instead of arguing, as in the Australian case, that kinship attitudes have a purely social origin, he now insists that social attitudes to kinship arc facts. rooted in universal psychological The paternal relationship contains elements which are necessarily resent in the father/child relationship of all societies, no matter what the circumstances of custom and social structure confusing. may be. This is all very On the one hand the reader is is told quite plainly that the Trobriand child taught to think of his father as a non-relative, as an lO RETHINKING ANTHROPOLOGY individual with the special non-kinship status of mother’s husband. But on the other hand the reader is forced to conclude that this ‘IVobriand mother’s husband is related to the mother’s child ‘as a sociological father’, that is to say by ties of kinship as well as by tics of affinity. The argument, as a whole, is self-contradictory. is You may about. well think that this a yery hairsplitting point to make a fuss How can it possibly make any difference whether I think of a parti- cular male as my father or as is my mother’s husband? Well, all I can say that anthropologists do Professor Fortes, Dr Goody and Dr Kathleen Gough on this subject that worry about such things. are so disturbed by my heretical yiews oflF time to try to bruise my owski’s argument (Fortes, 1959)- How to cite Rethinking Anthropology – E. R. Leach, Essay examples