Annotated list of resources Bibliography Example | Topics and Well Written Essays - 750 words

List of resources - Annotated Bibliography Example I think that it would be a reliable source for parents or educators seeking such programs. Raz-Kids is, essentially, an online library and reading program all in one, intended for young readers in the range of K through 6. They offer to improve children’s reading through reading for practice, recording the student reading, listening for modeled fluency, and checking comprehension through quizzes assigned to them. Children are encouraged to read, â€Å"†¦The right books for the right students.† ("Raz-kids.com: Online leveled," 2012) This can give the instructors the opportunity to properly track progress. By having the opportunity to have the library of the books in the â€Å"level† with a subscription, children can log in and read from anywhere that they have internet access. This side is adequate in its presentation. It is intended to be kid-friendly with bright colors, but internal access beyond the overview and sample pages is denied without opening an account with the site. This, in my opinion, makes this resource, by no means less credible, but less likely my first choice when reviewing such sites. So with no disrespect intended it is just less essay to give a truly genuine opinion of the overall potential of this particular program The San Diego Zoo website offers a section dedicated to kids, of all ages, that have games, activities, and articles that designed for the young. However, they do not offer a reading program per say, but they have â€Å"kits† available that educators can purchase filled with interactive, hands on activities, as well as, reading and science activities. The overall intention is to teach children those vital skills but, also, inspire children toward science and conservation. The lessons plans provided include skills tests for each section in the form of language arts, arts, and early science skills.("San

Song of Myself Essay Example for Free

Song of Myself Essay ‘Song of Myself’ is one of the most representative poems of Walt Whitman. It reveals what Walt Whitman is and what he stands for. In this poem, the poet discovers himself and gives the boldest expression to his true and ever expanding poetic-self defying all the limitations. In fact, it is a voyage of the self into the realms of imagination in an attempt to find its true nature. The poem celebrates his joy of finding his limitless and unbounded self identifying with the universal spirit. It is a journey from the individual self to the universal self. The poet expresses himself joyfully with the ‘original energy of nature which is unchecked and in exhaustive. The opening lines prepare the readers for an unprecedented flight of imagination. â€Å"He says, I CELEBRATE myself, and sing myself, And what I assume you shall assume, For every atom belonging to me as good belongs to you. † (Section-1) Whitman directly expresses the universality of the self and indirectly suggests the equality of all. He expresses the true nature of the self. He feels it is common to all and everyone has the same self. Everyone in the world has an equal claim on this world. He identifies his physical self and makes a distinction between the physical self of the poet and universal self of the poet. In the third stanza he says, †¦. form’d from this soil, this air, Born here of parents born here from parents the same, and their parents the same, I, now thirty-seven years old in perfect health begin, Hoping to cease not till death. (Section-1) The identity of the poet is revealed here. He was born to his parents and he was thirty seven years old enjoying robust health. These lines throw light on the personal life of the author. With this identity he starts his journey into the realms of freedom and equality where he finds himself perfectly in tune with the universe. He enjoys himself in his physical self and feels contented. He has neither worries nor any anxieties. He does not consider this life as preparation for the next. He is absolutely happy about his life and happy about what he is. The mention of soil and air clearly reveal that he is conscious of the world where he has come from. The poet is very happy with this beautiful world. The fresh air breathes new spirit in him. He identifies himself with nature and he wants to be as close to nature. He does not like anything to come in between himself and nature. He wants to be honest with nature. Here, we find the poet equating nakedness with honesty. He says, I will go to the bank by the wood and become undisguised and naked, I am mad for it to be in contact with me. (Section-2) He rejoices himself being close to nature. The proximity with nature gives him vitality and strength. He totally identifies himself with the soil, water and air. He says, My respiration and inspiration, the beating of my heart, the passing of blood and air through my lungs, The sniff of green leaves and dry leaves, and of the shore. (Section-2) The poet celebrates himself for being a part of such wonderful nature. He is neither worried nor concerned about any thing that may happen in future. He is very much concerned with the present. He is down to the earth practical, and at the same time, he expresses the wisdom of living in the present instead of thinking about future and worrying about the past. He expresses his happiness saying, â€Å"I am satisfied—I see, dance, laugh, and sing. † He has no complaints about the present and his presence in this world. He says he is not blind to the problems and evil in this world. But he does not allow them to spoil his happiness and his celebration. His reference to â€Å"myself† refers to his soul. It is the essence of his personality. He identifies the soul of the speaker is the soul in everyone. It is the universal self. It is quite untouched by the sufferings of the world. In fact, Whitman identifies the speaker of the poem with the birth of his poetic self. The newly born poet finds himself joyful and happy. The poet mentions that the spirit of the poet is a brother of god. He says, â€Å"And I know that the hand of God is the promise of my own, And I know that the spirit of God is the brother of my own, And that all the men ever born are also my brothers, and the women and my sisters and lovers, And that a kelson of the creation is love. † (Section-5) Whitman says the new self of the poet identifies itself with everything in nature. It identifies itself with a blade of grass. He feels the grass represents the same what a man represents. The awakened self of the poet strikes a common cord with the creative forces of the world. He says: A child said What is the grass? fetching it to me with full hands, How could I answer the child? I do not know what it is anymore than he. The grass stands as symbol for equality. It also represents the creative spirit in the world. He then goes on listing the things he has seen in American life, the joys, the worries, the celebration of the human race and celebration of everyday life. He describes the people he has met on his travels. He accepts them all without any complaints against them. Here, the poet shows his maturity of understanding and his modesty in accepting everything that life offers him. He does not question anything nor suspect any one. He describes himself as a lover of life. He says in the section 13 of the poem, he is a caresser of life wherever moving, backward as well as forward sluing. Absorbing all to myself and for this song. (Section-13) He strikes a beautiful comparison between his nature and the grass. He says: This is the grass that grows wherever the land is and the water is, this the common air that bathes the globe. As the grass grows every where he also respects everything and everyone in this world. He says, I play not marches for accepted victors only, I play marches for conquerd and slain persons, and I will not have a single person slighted or left away. (Section-18) Thus, the speaker in the poem, the new born poet shows a compassionate spirit and rejoices himself. The intensity in the search for the true meaning increases as the poem progresses. He asks in a philosophical tone, â€Å"Who goes there? Hankering, gross, mystical, nude: How is it I extract strength from the beef I eat? What is a man anyhow? What am I? What are you? † He is not dazzled by these questions nor withdraws himself to silence. He expresses himself and answers the questions he has raised. He says, In all people I see myself, none more and not one a barley-corn less, And the good or bad I say of myself I say of them. (Section-20) He also adds saying, I am the poet of the Body and I am the poet of the Soul, the pleasures of heaven are with me and pains of hell are with me. (Section-21) He rightly expresses that his poetry is a combination of both body and soul. There is something for the soul and there is something for the body. He mixes these things as naturally as a forest mixes different trees. His poetry is not like a cultivated garden. It is like a jungle. It is full of nature’s bounty. It is full of variety and it is vast. Just like the poem, the poet’s self grows to be the universal self and identifies itself with multitude of people and the creation in the world. The poet’s self grows beyond any limits and reaches the borders of vast expanses. The true self of the poet is no different from the vast and expanding world of his poetry. He grows as a man; his self grows to be a universal self and his soul ripens with wisdom and understanding, having made the entire tumultuous journey. He makes it clear that his poetic voice and his realization of his true self are not two different things. They are like the twins. He says, â€Å"My voice goes after what my eyes cannot reach, With the twirl of my tongue I encompass world and volumes of worlds. Speech is the twin of my vision, it is unequal to measure itself, It provokes me forever, it says sarcastically, Walt you contain enough, why dont you let it out then? † (section-25) The poet’s identity of his voice with his poetic vision is a major leap in the journey. He realizes that the two senses reveal the same reality that he finds. He goes on exploring further and he says, I believe a leaf of grass is no less than the journey work of the stars. (Section-31) Here, he realizes the some living force behind all the things in the world which makes them equal. He feels he is perfectly at home with everything in the world. He has no complaints nor any plans for improvement. He enjoys the sight of animals which are placid and self-contained. They are in absolute peace. He wants to live with them in that condition. He spends his time looking at them for a long time. He denounces the worries and frustrations of people as meaningless. The poet realizes the uniqueness of this realization and treats it with all the specialty and respect it commands. In the section 39 he begins speaking about himself in the third person. It is an indirect reference to what he has realized in the course of his journey. In that state of mind, wherever the poet goes he is respected and admired. He becomes the most liked person. He says, Wherever he goes men and women accept and desire him, They desire he should like them, touch them, speak to them, stay with them. He assumes the role of a guide and wants to lead the people with his realization. He says, â€Å"I launch all men and women forward with me into the Unknown. † Thus, the poet finds himself in a new role after the journey. He feels the realization he has, made him unique and this experience will be useful to the people who need guidance amid their turbulent lives.

Sperm Assessment Using Flow Cytometry

Sperm Assessment Using Flow Cytometry State of the art in sperm assessment using flow cytometry Abstract Flow cytometry is emerging as an important tool in the field of modern andrology for routine analysis of spermatozoa. Recently, application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, semen sample analysis was routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. The last decade, several new flow cytometric techniques have been introduced for farm animal semen assessment that enable a more detailed evaluation of several sperm characteristics. Here in this paper, an initiative has been taken to focus on a number of recent flow cytometry developments important for addressing questions in andrological tests. After the invention of flow cytometry, sperm evaluation by traditional microscopic means became questioned due to the robust advantages of flow cytometry over the microscopic method. Due to the recent development of large number of fluroscence probes, flow cytometry is now capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gamets based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the possibility of working with small sample sizes, increases the repeatability of assessment, removes the subjectivity of assessment and allows simultaneous assessment of multiple fluorochromes. Flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of experiments that a re not possible with any other technique. Nowadays, semen evaluation using laboratory assays is extremely important to the artificial insemination industry to provide the most desired quality product to customers. Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen parameters that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed. Introduction Semen evaluation is the single most important laboratory test that has helped us to identify clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (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. Methods are available that can sometimes estimate the potential fertilizing capacity of a semen sample and, in some cases, of the male (reviewed by Dziuk 1996; 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). The methods routinely used for evaluation of the quality of a semen sample involved an evaluation of general appearance (i.e. colour, contamination, etc.), volume, pH, sperm concentration, viability, morphology and motility. Most of these techniques are microscopic analyses that only measure a small number of spermatozoa within a population, are time-consuming, can be subjective and generally measure sperm attributes individually. Recently, limitations of semen evaluation methodology have been brought into sharp focus by controversies raised in the epidemiological literature. It should also be noted that such conventional measurements are prone to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed se men, and IVF. Such handling implies semen extension, fluorophore loading, ultraviolet and laser illumination, high-speed sorting, cooling and cryopreservation, procedures that impose different degrees of change in sperm function following damage to sperm membranes, organelles or the DNA. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed that buck of these procedures are incomplete, time consuming and laborious. Flow cytometry in different technical applications offers many advantages for the analysis of sperm quality. Flow cytometry allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cell or microscopic particles in suspension, as they flow very rapidly through a sensing area. The increasing use over the 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 can acquire data on several subpopulations within a sample in a few minutes, making it ideal for assessment of heterogenous populations in semen sample. Initially developed in the 1960s, flow cytometry made automated separation of cells based on the unique recognition of cellular patterns within a population feasible (Hulett et al., 1969). Using such a separation approach, cellular patterns can be identified by as sessing, in individual cells within a population, protein expression using fluorescently labeled antibodies and other fluorescent probes (Baumgarth and Roederer, 2000; Herzenberg et al., 2006). Flow cytometry was first developed for medical and clinical applications such as haematology and oncology. These areas still account for the vast majority of publications on this technique, but during the past few years it has been used in other areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 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. In other word, a flow cytometer will be defined as ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream. In other work, cytometry refers to the measurement of physical and/or chemical characteristics of cells or, by extension, of other particles. It is a process in which such measurements are made while the cells or particles pass, preferably in single file, through the measuring apparatus in a fluid stream. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other analytical tools, where a single value for each parameter is obtained for the whole population, flow cytometry provides data for every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters. Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light 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 gives objective and accurate results (Bunthof et al., 2001; Shleeva et al., 2002), overcoming the problems with the manual methods described above. Function and types of flow cytometry Fluidics, optics and electronics are the three main systems that make up a flow cytometer. In a few minutes, the flow cytometer can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment began when it was used for measuring their DNA content (Evenson et al., 1980) and its application to semen analysis has gradually increased over the last 10-15 years. Flow cytometry is now applied to semen evaluation of traits such as cell viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity and DNA status. New fluorescent stains and techniques are continuously being developed that have potential application to the flow cytometric evaluation of spermatozoa. Flow cytometry permits the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. 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 the more sperm parameters that can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993). There are two main types of flow cytometers-analysers and sorters. Sorters have the ability not only to collect data on cells (analyse cells) but also to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation 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 During the past 2 decades, there has been an increasing interest in reliable assays for assessing semen quality in the fertility clinic and artificial insemination industries. The use of flow cytometry for sperm analysis is an attempt to address the long-standing problem of the subjective nature of the manual method commonly used for semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with manual techniques. Because of time and cost restraints, most laboratories analyze only 50 to 100 sperm to compute the percentage of each cell population and the viability rate. This small sample from a population of millions probably results in a statistical sampling error (Russel and Curtis, 1993). The conventional methods used are limited to microscopic determination of sperm concentration using a hemocytometer (Jorgensen et al., 1997) and evaluation of sperm motility and morphology (Keel et al., 2002). These methods usually involve a subjective asses sment of a few hundred sperm, and quality assurance is rarely implemented in the laboratories performing such analysis. Flow cytometry is a technique that is superior to conventional light microscopy techniques in terms of objectivity, number of cells measured, speed, and precision (Spano and Evenson, 1993). The technique has been used on human sperm to determine a number of factors, including membrane integrity, mitochondrial function, acrosome status, and multiparameter measurement (Garrido et al., 2002). Flow cytometry permitted us to analyze thousands of cells in few seconds. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in diverse species (boar, bull, stallion etc), and the observed CVs were significantly better than those reported for the manual method. While there are many advantages of using the flow cytometer for routine semen analysis, its use is often limited to research by the expense and difficulties of operation associated with the requirement of a skilled operator. In addition, a flow cytometer is quite large and cannot withstand shocks associated with movement, meaning it requires a dedicated position in the laboratory. However, the development of more affordable ‘‘bench-top flow cytometers has recently increased the potential application to semen analysis. If we consider flow cytometric analysis further, we can see that it is gaining wider acceptance as a technique for assessing the acrosome reaction and viability simultaneously. Comparing these assays to the more widely used epifluorescent microscopic techniques, the flow cytometric analysis is able to give a far more simple and objective method of analysis, especially with regard to correlation of fertilization with acrosome reactivity potential (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996). A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Preliminary data from a number of studies suggested that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). Using this information, the World Health Organization stated that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, may lack the accuracy and precision of the haemocytometer technique (World Health Organization, 1999). Further data—for example, from Tomlinson and colleagues—showed that 2 proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) gave lower spe rm concentrations compared to the hemocytometer method (Tomlinson et al., 2001). To put this in context, numerous reports document unacceptable discrepancies between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong? Several reports emphasize the need for improvement in overall quality of semen testing within and between laboratories (Neuwinger et al., 1990; Jorgensen et al., 1997; Keel et al., 2000). However, the subjective nature of conventional semen analyses, combined with their relatively low precision due to the low number of cells assessed, leads to poor intra- and interlaboratory reproducibility; therefore, the introduction of standardized or quality controlled procedures will probably have a limited effect. The conventional analyses are used to determine whether parameters obtained from an ejaculate are within the range characterized by fertile men, and these methods can therefore provide only unclear cut-off values when used for the prediction of fertility status. Many of the advantages that accrue when using flow cytometry may, when applied to assessment of sperm cells, help overcome some of the mentioned problems found in conventional semen analysis. In the field of semen analysis, validation of a method is important because it is essential to have specific, precise, objective, and accurate laboratory tests to establish a correlation of the data with fertility or to determine the fertility potential of a semen sample correctly (Amann, 1989). Precision of a laboratory test is of great concern to the andrologist in the fertility clinic, since the results of the semen analysis are often used to advise a patient about his fertility and the prognosis for the treatment of the couple. To use established cut-off values and ensure uniform diagnosis, within and between laboratory variations should be determined and followed closely. Accurate determination of sperm cell concentration is critical to the AI industry because it provides assurance both to bull studs and to customers that straws of extended semen contain the sperm numbers indicated. An accurate measure of sperm concentration is particularly important in export markets in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Hemacytometers are widely used for routine sperm counts, but the equipment is slow, and multiple measurements of each sample are needed. Single hemacytometer counts are not highly accurate; because of inherent errors in the technique, Freund and Carol (13) found that mean differences of 20% were not uncommon between duplicate sperm count determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (19). Currently, the primary method used by the AI industry to estimate sperm concentration is spectrophotometric determination of turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (1). This approach is only as accurate as the methods used for spectrophotometer calibration. New, more accurate methods for sperm count determinations are being sought to replace the older ones. Some laboratories are trying the Maklerm counting chamber (Seif- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. It may be argued that when comparing fluorescent microscopy assays with flow cytometry, one is examining patterns of fluorescence rather than fluorescence intensity, i.e., the flow cytometer is not capable of discriminating sperm which have a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is no significant difference between the two methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the 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. Viability 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. Currently flow cytometric procedures have been developed which simultaneously evaluate sperm cell viability, acrosomal integrity and mitochondrial function. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (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., 1997), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004). Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method. One of the first attempts to assess sperm viability utilized rhodamine 123 (R123) to assess mitochondrial membrane potential and ethidium bromide to determine membrane integrity using flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990). At present, one of the most commonly used viability stain combinations is SYBR-14 and PI, sold commercially as LIVE/DEAD Sperm Viability kit (Molecular Probes Inc., OR, USA). When used in combination, the nuclei of living sperm fluoresce green (SYBR-14) and cells that have lost their membrane integrity stain red (PI). This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been used to evaluate boar sperm concentration [11]. Due to its compact size and its relatively inexpensive purchase price, this instrument could be useful for field measurements of both concentration and viability. This instrument was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability (Morrell et al., 2010). Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and found a negative correlation between the percentage of damaged cells and field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility. Fluorochromes used to assess sperm viability by either approach can be used in combination with each other. For example, when CFDA is used along with PI, three populations of cells can be identified: live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa. Almlid and Johnson (1988) found this combination useful for monitoring membrane damage in frozen-thawed boar spermatozoa during evaluation of various freezing protocols. Harrison and Vickers (1990) also used this combination with a fluorescent microscope and found it to be an effective indicator of the viability of fresh, incubated or cold-shocked boar and ram spermatozoa. Garner et al. (1986) used this combination to stain spermatozoa from a number of species, but at that time could not find a relationship between bull sperm viability detected by CFDA/PI and fertility. Flow cytometry for assessment of sperm viability appears to be a valuable tool for the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect on NRR56. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combination of assessment of sperm viability and concentration appears to be useful in the improvement of quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. The mechanism by which SYBR-14 binds to the DNA is not known. It is know that PI stains nucleic acids by intercalating betwee n the base pairs (Krishan, 1975). Viability stains have also been used in association with fluorescently labeled plant lectins to simultaneously assess the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). Assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) demonstrated that insemination of boar spermatozoa stained with SYBR-14 into sows did not compromise fertilization or the development of flushed porcine embryos in culture. Non-viable cells can be determined using membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. An intact plasma membrane will prevent these products from entering the spermatozoa and staining the nucleus. Commonly used examples include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). Wilhelm et al. (1996) compared the fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality and found that viability, as assessed by flow cytometry using PI, was the single laboratory assay that correlated with stallion fertility. Changes in sperm membrane permeability Detection of increased membrane permeability is employed in different cell types to distinguish different status of membrane organization (Cohen, 1993; Ormerod et al., 1993; Castaneda and Kinne, 2000; Reber et al., 2002). Sperm plasma membrane status is of utmost importance due to its role, not only as a cell boundary, but also for its need for cell-to-cell interactions, e.g. between spermatozoa and the epithelium of the female genital tract and between the spermatozoon and the oocyte and its vestments (for review, see Rodriguez-Martinez, 2001). Membrane integrity and the stability of its semipermeable features are prerequisites for the viability of the spermatozoon (Rodriguez-Martinez, 2006). However, cryopreservation, whose purpose is to warrant sperm survival, causes irreversible damage to the plasma membrane leading to cell death in a large number of spermatozoa (Holt, 2000) or, in the surviving spermatozoa, to changes similar to those seen during sperm capacitation, thus shorten ing their lifetime (Perez et al., 1996; Cormier et al., 1997; Maxwell and Johnson, 1997; Green and Watson, 2000; Schembri et al., 2000; Watson, 2000). During the freezing process, cells shrink again when cooling rates are slow enough to prevent intracellular ice formation as growing extracellular ice concentrates the solutes in the diminishing volume of non-frozen water, causing intracellular water exosmosis. Though warming and thawing, the cells return to their normal volume. Thus, it is important to know the permeability coefficient of the cells to cryoprotectants, as well as the effect of cryoprotective agents on the membrane hydraulic conductivity. Classical combination of probes allows discrimination of two or three subpopulations of spermatozoa, i.e. live, dead and damaged depending on the degree of membrane integrity (Eriksson RodrÄ ±Ã‚ ´guez-MartÄ ±Ã‚ ´nez, 2000). A new, simple and repeatable method to detect membrane changes in all spermatozoa present in a boar semen sample, by use of markers (combination of SNARF-1, YO-PRO-1 and ethidium homodimer) used to track changes in sperm membrane permeability, has been developed recently by our group (Pena et al., 2005). In determined physiological or pathological situations, live cells are unable to exclude YO-PRO-1, but are still not permeable to other dead-cell discriminatory dyes, like propidium iodide or ethidium homodimer. YO-PRO-1 is an impermeable membrane probe and can leak in, only after destabilization of the membrane, under conditions where ethidium homodimer does not. Because several ATP-dependent channels have been detected in spermatozoa (Acevedo et al. , Sperm Assessment Using Flow Cytometry Sperm Assessment Using Flow Cytometry Abstract Flow cytometry is emerging as an important tool in the field of modern andrology for routine analysis of spermatozoa. Recently, application of flow cytometry in the artificial insemination industry especially for pig is a new approach. Until very recent, semen sample analysis was routinely performed by microscopical evaluation and manual techniques by laboratory operators; the analysis is affected by a wide imprecision related to variability among observers, influencing its clinical validity. The last decade, several new flow cytometric techniques have been introduced for farm animal semen assessment that enable a more detailed evaluation of several sperm characteristics. Here in this paper, an initiative has been taken to focus on a number of recent flow cytometry developments important for addressing questions in andrological tests. After the invention of flow cytometry, sperm evaluation by traditional microscopic means became questioned due to the robust advantages of flow cytometry over the microscopic method. Due to the recent development of large number of fluroscence probes, flow cytometry is now capable of analyzing number of sperm characteristics like viability, capacitation, acrosomal integrity, membrane permeability, membrane integrity, mitochondrial status, DNA integrity, decondensation of DNA and differences between gamets based on sex. The application of flow cytometry to their detection allows increased numbers of spermatozoa to be assessed over a short time-period, provides the possibility of working with small sample sizes, increases the repeatability of assessment, removes the subjectivity of assessment and allows simultaneous assessment of multiple fluorochromes. Flow cytometry is a technique capable of generating significantly novel data and allows the design and execution of experiments that a re not possible with any other technique. Nowadays, semen evaluation using laboratory assays is extremely important to the artificial insemination industry to provide the most desired quality product to customers. Future development of flow cytometric techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques. In this paper, the main semen parameters that can be analyzed with fluorochromes and adapted for use with a flow cytometer will be reviewed and the relationship of these tests to fertility will be discussed. Introduction Semen evaluation is the single most important laboratory test that has helped us to identify clear-cut cases of fertility (Jarow et al., 2002), infertility or even of potential sub-fertility (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. Methods are available that can sometimes estimate the potential fertilizing capacity of a semen sample and, in some cases, of the male (reviewed by Dziuk 1996; 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). The methods routinely used for evaluation of the quality of a semen sample involved an evaluation of general appearance (i.e. colour, contamination, etc.), volume, pH, sperm concentration, viability, morphology and motility. Most of these techniques are microscopic analyses that only measure a small number of spermatozoa within a population, are time-consuming, can be subjective and generally measure sperm attributes individually. Recently, limitations of semen evaluation methodology have been brought into sharp focus by controversies raised in the epidemiological literature. It should also be noted that such conventional measurements are prone to extreme inter-ejaculate variation, even when the laboratory methodology has been standardized. In the wake of this information, new opportunities have arisen for the development of methods for the diagnosis of male infertility, many of which have been shown to exhibit a prognostic value that eludes conventional semen profiling. Moreover, ejaculated spermatozoa are nowadays handled for use in assisted reproductive technologies, such as the artificial insemination of chilled, frozen-thawed or sexed se men, and IVF. Such handling implies semen extension, fluorophore loading, ultraviolet and laser illumination, high-speed sorting, cooling and cryopreservation, procedures that impose different degrees of change in sperm function following damage to sperm membranes, organelles or the DNA. Therefore, although several assays have been developed to monitor these sperm parameters, recently it is being claimed that buck of these procedures are incomplete, time consuming and laborious. Flow cytometry in different technical applications offers many advantages for the analysis of sperm quality. Flow cytometry allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cell or microscopic particles in suspension, as they flow very rapidly through a sensing area. The increasing use over the 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 can acquire data on several subpopulations within a sample in a few minutes, making it ideal for assessment of heterogenous populations in semen sample. Initially developed in the 1960s, flow cytometry made automated separation of cells based on the unique recognition of cellular patterns within a population feasible (Hulett et al., 1969). Using such a separation approach, cellular patterns can be identified by as sessing, in individual cells within a population, protein expression using fluorescently labeled antibodies and other fluorescent probes (Baumgarth and Roederer, 2000; Herzenberg et al., 2006). Flow cytometry was first developed for medical and clinical applications such as haematology and oncology. These areas still account for the vast majority of publications on this technique, but during the past few years it has been used in other areas, such as bioprocess monitoring, pharmacology, toxicology, environmental sciences, bacteriology and virology. Recent advancement of flow cytometry increased its application in the reproductive biology especially for andrology. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. Although flow cytometry may overestimate the population of unlabelled cells (Petrunkina and Harrison, 2009), plethora of research from our group in pig (Pena et al., 2003, 2004, 2005; Spjuth et al., 2007; Fernando et al., 2003; Saravia et al.,2005, 2007,2009; De Ambrogi et al., 2006; ) bull (Bergquist et al., 2007; Nagy et al., 2004; Januskauskas et al., 2003; Bergqvist et al., 2007; Hallap et al., 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. In other word, a flow cytometer will be defined as ‘an instrument that can measure physical, as well as multi-colour fluorescence properties of cells flowing in a stream. In other work, cytometry refers to the measurement of physical and/or chemical characteristics of cells or, by extension, of other particles. It is a process in which such measurements are made while the cells or particles pass, preferably in single file, through the measuring apparatus in a fluid stream. The data obtained can be used to understand and monitor biological processes and develop new methods and strategies for cell detection and quantification. Compared to other analytical tools, where a single value for each parameter is obtained for the whole population, flow cytometry provides data for every particle detected. As cells differ in their metabolic or physiological states, flow cytometry allows us not only to detect a particular cell type but also to find different subpopulations according to their structural or physiological parameters. Flow cytometry is a technique for measuring components (cells) and the properties of individual cells in liquid suspension. In essence, suspended cells are brought to a detector, one by one, by means of a flow channel. Fluidic devices under laminar flow define the trajectories and velocities that cells traverse across the detector, and fluorescence, absorbance, and light 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 gives objective and accurate results (Bunthof et al., 2001; Shleeva et al., 2002), overcoming the problems with the manual methods described above. Function and types of flow cytometry Fluidics, optics and electronics are the three main systems that make up a flow cytometer. In a few minutes, the flow cytometer can acquire data on all subpopulations within a sample, making it ideal for assessment of heterogenous population, such as spermatozoa. The adaptation of flow cytometry to sperm assessment began when it was used for measuring their DNA content (Evenson et al., 1980) and its application to semen analysis has gradually increased over the last 10-15 years. Flow cytometry is now applied to semen evaluation of traits such as cell viability, acrosomal integrity, mitochondrial function, capacitation status, membrane fluidity and DNA status. New fluorescent stains and techniques are continuously being developed that have potential application to the flow cytometric evaluation of spermatozoa. Flow cytometry permits the observation of physical characteristics, such as cell size, shape and internal complexity, and any component or function of the spermatozoon that can be detected by a fluorochrome or fluorescently labeled compound. The analysis is objective, has a high level of experimental repeatability and has the advantage of being able to work with small sample sizes. Flow cytometry also has the capacity to detect labeling by multiple fluorochromes associated with individual spermatozoa, meaning that more than one sperm attribute can be assessed simultaneously. 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 the more sperm parameters that can be tested, the more accurate the fertility prediction becomes (Amman and Hammerstedt, 1993). There are two main types of flow cytometers-analysers and sorters. Sorters have the ability not only to collect data on cells (analyse cells) but also to sort cells with particular properties (defined by the flow cytometer operator) to extremely high purities. There are also a number of commercial flow cytometers that have been developed for particular analytical requirements. Partec manufacture a Ploidy Analyser and also a Cell Counter Analyser. Optoflow has developed a flow cytometer for the rapid detection, characterization and enumeration of microorganisms. Luminex is developing technology for multiplexed analyte quantitation 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 During the past 2 decades, there has been an increasing interest in reliable assays for assessing semen quality in the fertility clinic and artificial insemination industries. The use of flow cytometry for sperm analysis is an attempt to address the long-standing problem of the subjective nature of the manual method commonly used for semen analysis. An additional source of laboratory variation is the low number of sperms analyzed with manual techniques. Because of time and cost restraints, most laboratories analyze only 50 to 100 sperm to compute the percentage of each cell population and the viability rate. This small sample from a population of millions probably results in a statistical sampling error (Russel and Curtis, 1993). The conventional methods used are limited to microscopic determination of sperm concentration using a hemocytometer (Jorgensen et al., 1997) and evaluation of sperm motility and morphology (Keel et al., 2002). These methods usually involve a subjective asses sment of a few hundred sperm, and quality assurance is rarely implemented in the laboratories performing such analysis. Flow cytometry is a technique that is superior to conventional light microscopy techniques in terms of objectivity, number of cells measured, speed, and precision (Spano and Evenson, 1993). The technique has been used on human sperm to determine a number of factors, including membrane integrity, mitochondrial function, acrosome status, and multiparameter measurement (Garrido et al., 2002). Flow cytometry permitted us to analyze thousands of cells in few seconds. In our series of studies, we demonstrated the feasibility and reproducibility of an automated method to evaluate sperm cell type, count, and viability in human boar samples. In our hand, the precision of the flow cytometric analysis is satisfactory in diverse species (boar, bull, stallion etc), and the observed CVs were significantly better than those reported for the manual method. While there are many advantages of using the flow cytometer for routine semen analysis, its use is often limited to research by the expense and difficulties of operation associated with the requirement of a skilled operator. In addition, a flow cytometer is quite large and cannot withstand shocks associated with movement, meaning it requires a dedicated position in the laboratory. However, the development of more affordable ‘‘bench-top flow cytometers has recently increased the potential application to semen analysis. If we consider flow cytometric analysis further, we can see that it is gaining wider acceptance as a technique for assessing the acrosome reaction and viability simultaneously. Comparing these assays to the more widely used epifluorescent microscopic techniques, the flow cytometric analysis is able to give a far more simple and objective method of analysis, especially with regard to correlation of fertilization with acrosome reactivity potential (Uhler et al., 1993; Purvis et al., 1990; Carver-Ward et al., 1996). A large number of different techniques to estimate sperm concentration have been reported. In the mid-1990s a series of fixed-depth disposable slides were evaluated as rapid and effective pieces of equipment for the estimate of sperm concentration. Preliminary data from a number of studies suggested that, at least in the 20-mm-depth format, such chambers resulted in a noticeable underestimate of sperm concentration compared to the gold standard (improved Neubauer hemocytometer). Using this information, the World Health Organization stated that ‘‘such chambers, whilst convenient in that they can be used without dilution of the specimen, may lack the accuracy and precision of the haemocytometer technique (World Health Organization, 1999). Further data—for example, from Tomlinson and colleagues—showed that 2 proprietary disposable slides (Microcell, Conception Technologies, San Diego, Calif; Leja, Leja Products, BV Nieuw- Vennep, The Netherlands) gave lower spe rm concentrations compared to the hemocytometer method (Tomlinson et al., 2001). To put this in context, numerous reports document unacceptable discrepancies between different laboratories and even between different individuals, although fewer studies attempt to address these issues. So, what is wrong? Several reports emphasize the need for improvement in overall quality of semen testing within and between laboratories (Neuwinger et al., 1990; Jorgensen et al., 1997; Keel et al., 2000). However, the subjective nature of conventional semen analyses, combined with their relatively low precision due to the low number of cells assessed, leads to poor intra- and interlaboratory reproducibility; therefore, the introduction of standardized or quality controlled procedures will probably have a limited effect. The conventional analyses are used to determine whether parameters obtained from an ejaculate are within the range characterized by fertile men, and these methods can therefore provide only unclear cut-off values when used for the prediction of fertility status. Many of the advantages that accrue when using flow cytometry may, when applied to assessment of sperm cells, help overcome some of the mentioned problems found in conventional semen analysis. In the field of semen analysis, validation of a method is important because it is essential to have specific, precise, objective, and accurate laboratory tests to establish a correlation of the data with fertility or to determine the fertility potential of a semen sample correctly (Amann, 1989). Precision of a laboratory test is of great concern to the andrologist in the fertility clinic, since the results of the semen analysis are often used to advise a patient about his fertility and the prognosis for the treatment of the couple. To use established cut-off values and ensure uniform diagnosis, within and between laboratory variations should be determined and followed closely. Accurate determination of sperm cell concentration is critical to the AI industry because it provides assurance both to bull studs and to customers that straws of extended semen contain the sperm numbers indicated. An accurate measure of sperm concentration is particularly important in export markets in which verification of numbers may be required. Routine sperm counts can help to identify possible processing errors within a specific batch of semen or on a particular day, should those errors occur. As sperm counting procedures become more refined, routine counting can be used to monitor subtle changes in daily semen processing that might affect the number of sperm packaged in a straw. Hemacytometers are widely used for routine sperm counts, but the equipment is slow, and multiple measurements of each sample are needed. Single hemacytometer counts are not highly accurate; because of inherent errors in the technique, Freund and Carol (13) found that mean differences of 20% were not uncommon between duplicate sperm count determinations by the same technician. Electronic counters provide much more rapid counting, are easier to use, and give more repeatable results among technicians. However, those instruments tend to include in the sperm count any somatic cells present, immature sperm forms, cytoplasmic droplets, debris, and bacteria, thereby inflating the concentration value (19). Currently, the primary method used by the AI industry to estimate sperm concentration is spectrophotometric determination of turbidity of a semen sample using an instrument previously calibrated for sperm concentration with a hemacytometer or Coulter counter (1). This approach is only as accurate as the methods used for spectrophotometer calibration. New, more accurate methods for sperm count determinations are being sought to replace the older ones. Some laboratories are trying the Maklerm counting chamber (Seif- Medical, Haifa, Israel) and other improved hemacytometers, such as the MicroCellTM (Fertility Technologies, Inc., Natick, MA); however, these techniques will likely have standard lems similar to those associated with the standard hemacytometers. It may be argued that when comparing fluorescent microscopy assays with flow cytometry, one is examining patterns of fluorescence rather than fluorescence intensity, i.e., the flow cytometer is not capable of discriminating sperm which have a fluorescent marker bound to the equatorial segment or over one of the acrosomal membranes (Parinaud et al., 1993; Mortimer and Camenzind, 1989; Mortimer et al., 1987). Tao et al. (1993) compared flow cytometry and epifluorescent microscopy with various lectins and indicated that there is no significant difference between the two methodologies for detection of the acrosome reaction. However, it has been argued that lectins do not bind specifically to the 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. Viability 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. Currently flow cytometric procedures have been developed which simultaneously evaluate sperm cell viability, acrosomal integrity and mitochondrial function. This method has been successfully used for assessing spermatozoa viability in men (Garner and Johnson, 1995), bulls (Garner et al., 1994; Thomas et al., 1998), boars (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., 1997), poultry and wildfowl (Donoghue et al., 1995; Blanco et al., 2000) and honey bees (Collins and Donoghue, 1999; Collins, 2000) and in fish (Martin Flajshans et al., 2004). Considerable information has accumulated on the use of fluorescent staining protocols for assessing sperm viability (Evenson et al., 1982). The SYBR 14 staining of nucleic acids, especially in the sperm head, was very bright in living sperm. Good agreement was observed between the fluorescent staining method and the standard eosin-nigrosine viability test; the flow cytometric method showed a precision level higher than that of the manual method. One of the first attempts to assess sperm viability utilized rhodamine 123 (R123) to assess mitochondrial membrane potential and ethidium bromide to determine membrane integrity using flow cytometry (Garner et al., 1986). Other combinations that have been used to examine the functional capacity of sperm are carboxyfluorescein diacetate (CFDA) and propidium iodide (PI) (Garner et al., 1988; Watson et al., 1992); carboxydimethylfluorescein diacetate (CMFDA), R123, and PI (Ericsson et al., 1993; Thomas and Garner, 1994); and PI, pisum sativum agglutinin (PSA), and R123 (Graham et al., 1990). At present, one of the most commonly used viability stain combinations is SYBR-14 and PI, sold commercially as LIVE/DEAD Sperm Viability kit (Molecular Probes Inc., OR, USA). When used in combination, the nuclei of living sperm fluoresce green (SYBR-14) and cells that have lost their membrane integrity stain red (PI). This staining technique has been used in a number of species, including the boar (Garner and Johnson, 1995; Saravia et al.,2005, 2007,2009). Although species differences do exist in the function of spermatozoa, the Live/Dead stain may similarly have no adverse affect on fertilization in the equine, although it remains to be tested in this species. Recently a new instrument (Nucelocounter-SP100) has been used to evaluate boar sperm concentration [11]. Due to its compact size and its relatively inexpensive purchase price, this instrument could be useful for field measurements of both concentration and viability. This instrument was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability (Morrell et al., 2010). Fluorescent probes such as H33258, requiring flow cytometric analysis with a laser that operates in the ultraviolet light range, are less commonly used as this is not a standard feature on the smaller analytical machines. However, one alternative is to use a fluorometer. A fluorometer is a relatively low-cost piece of portable equipment that permits a rapid analysis to be carried out on a sample. Januskauskas et al. (2001) used H33258 to detect nonviable bull spermatozoa by fluorometry and found a negative correlation between the percentage of damaged cells and field fertility. Another option is fluorescent attachments for computer-assisted semen analysis devices. For example, the IDENT fluorescence feature of the Hamilton-Thorne IVOS permits staining with H33258 allowing an assessment of sperm viability to be made along with motility. Fluorochromes used to assess sperm viability by either approach can be used in combination with each other. For example, when CFDA is used along with PI, three populations of cells can be identified: live, which are green; dead, which are red; and a third population which is stained with both and represents dying spermatozoa. Almlid and Johnson (1988) found this combination useful for monitoring membrane damage in frozen-thawed boar spermatozoa during evaluation of various freezing protocols. Harrison and Vickers (1990) also used this combination with a fluorescent microscope and found it to be an effective indicator of the viability of fresh, incubated or cold-shocked boar and ram spermatozoa. Garner et al. (1986) used this combination to stain spermatozoa from a number of species, but at that time could not find a relationship between bull sperm viability detected by CFDA/PI and fertility. Flow cytometry for assessment of sperm viability appears to be a valuable tool for the AI industry. When a high number of sperm is packed in each insemination dose, the effect of selecting the best ejaculates according to sperm viability has a relatively limited effect on NRR56. However, sperm viability might be more important when combined with low-dose inseminations. The FACSCount AF flow cytometer also determines sperm concentration accurately and precisely during the same analysis (Christensen et al., 2004a). The combination of assessment of sperm viability and concentration appears to be useful in the improvement of quality control at AI stations. Because of the results of this trial, this method has been implemented by Danish AI stations (Christensen et al., 2005). Relatively bright fluorescence was found also in the mitochondrial sheath of living sperm. The mechanism by which SYBR-14 binds to the DNA is not known. It is know that PI stains nucleic acids by intercalating betwee n the base pairs (Krishan, 1975). Viability stains have also been used in association with fluorescently labeled plant lectins to simultaneously assess the plasma membrane integrity and the acrosome integrity (Nagy et al., 2003). Assessment of viability using SYBR-14 dye does not damage spermatozoa, since Garner et al. (5) demonstrated that insemination of boar spermatozoa stained with SYBR-14 into sows did not compromise fertilization or the development of flushed porcine embryos in culture. Non-viable cells can be determined using membrane-impermeable nucleic acid stains which positively identify dead spermatozoa by penetrating cells with damaged membranes. An intact plasma membrane will prevent these products from entering the spermatozoa and staining the nucleus. Commonly used examples include phenanthridines, for example propidium iodide (PI; (Matyus, 1984) ethidium homodimer-1 (EthD-1; (Althouse et al., 1995), the cyanine Yo-Pro (Kavak, 2003) and the bizbenzimidazole Hoechst 33258 (Gundersen and Shapiro, 1984). Wilhelm et al. (1996) compared the fertility of cryopreserved stallion spermatozoa with a number of laboratory assessments of semen quality and found that viability, as assessed by flow cytometry using PI, was the single laboratory assay that correlated with stallion fertility. Changes in sperm membrane permeability Detection of increased membrane permeability is employed in different cell types to distinguish different status of membrane organization (Cohen, 1993; Ormerod et al., 1993; Castaneda and Kinne, 2000; Reber et al., 2002). Sperm plasma membrane status is of utmost importance due to its role, not only as a cell boundary, but also for its need for cell-to-cell interactions, e.g. between spermatozoa and the epithelium of the female genital tract and between the spermatozoon and the oocyte and its vestments (for review, see Rodriguez-Martinez, 2001). Membrane integrity and the stability of its semipermeable features are prerequisites for the viability of the spermatozoon (Rodriguez-Martinez, 2006). However, cryopreservation, whose purpose is to warrant sperm survival, causes irreversible damage to the plasma membrane leading to cell death in a large number of spermatozoa (Holt, 2000) or, in the surviving spermatozoa, to changes similar to those seen during sperm capacitation, thus shorten ing their lifetime (Perez et al., 1996; Cormier et al., 1997; Maxwell and Johnson, 1997; Green and Watson, 2000; Schembri et al., 2000; Watson, 2000). During the freezing process, cells shrink again when cooling rates are slow enough to prevent intracellular ice formation as growing extracellular ice concentrates the solutes in the diminishing volume of non-frozen water, causing intracellular water exosmosis. Though warming and thawing, the cells return to their normal volume. Thus, it is important to know the permeability coefficient of the cells to cryoprotectants, as well as the effect of cryoprotective agents on the membrane hydraulic conductivity. Classical combination of probes allows discrimination of two or three subpopulations of spermatozoa, i.e. live, dead and damaged depending on the degree of membrane integrity (Eriksson RodrÄ ±Ã‚ ´guez-MartÄ ±Ã‚ ´nez, 2000). A new, simple and repeatable method to detect membrane changes in all spermatozoa present in a boar semen sample, by use of markers (combination of SNARF-1, YO-PRO-1 and ethidium homodimer) used to track changes in sperm membrane permeability, has been developed recently by our group (Pena et al., 2005). In determined physiological or pathological situations, live cells are unable to exclude YO-PRO-1, but are still not permeable to other dead-cell discriminatory dyes, like propidium iodide or ethidium homodimer. YO-PRO-1 is an impermeable membrane probe and can leak in, only after destabilization of the membrane, under conditions where ethidium homodimer does not. Because several ATP-dependent channels have been detected in spermatozoa (Acevedo et al. , 2006), it seems plausible that this is a result of the silencing of a multidrug transporter. This m

Confronting Reality: How Nosferatu Exemplifies Film Horror Tactics Essa

Many films, and sometimes film genres, are dismissed as being part of the cinema of escapism. This assumes that in times of particular social or economic hardship (often on a national or international level), people go to movies for the sole purpose of â€Å"getting away from it all.† While some films may follow this overall trend, it is important to note that it cannot be a generalization made for all films. During the Weimar era in Germany, the nation was in the midst of a national struggle on many fronts. As a people, Germans attempted to deal with their past (the problems during World War I as well as the consequences of their loss) and move toward the future (finding a solution for their economic struggles and defining themselves culturally and socially). This period saw a resurgence of the horror genre, this time adapted to the new medium of film. However, the way horror was portrayed via film is the interesting part: it drew specifically on the struggles of the na tion to instill horror. This is an exact reversal of the idea of cinematic escapism, since many Weimar era horror films used relatable struggles in order to both entertain and terrify (in this case, existing concurrently as well as dependently on each other). One of the clearest examples of this is through the film Nosferatu, a cinematic retelling of Bram Stoker’s novel Dracula directed by F.W. Murnau. The budding horror genre of the Weimar era, as exemplified by Nosferatu, succeeded because it drew parallels to the German people’s collective post-World War I mindset, including references to the terrible nature of the war itself and the fearful prospect of how to move forward. Nosferatu employs various plot points and imagery to elicit an emotional response ... ...dience long after the film reels have stopped turning. The idea of a â€Å"scary movie† could be innocuous enough, if it is simply frights and ghoulish images, but Nosferatu raised the bar and discovered how to delve into a collective mindset and produce a truly unsettling product. Germany’s residual shame and concern regarding World War I made Nosferatu a gripping, telling exploration of a nation’s psyche. Works Cited Bodek, Richard. â€Å"The Not-So-Golden Twenties: everyday Life and Communist Agitprop in Weimar-Era Berlin.† Journal of Social History. Vol. 30, No. 1. Autumn 1996. Calhoon, Kenneth S. â€Å"Horror vacui.† Peripheral Visions: The Hidden Stages of Weimar Cinema. Wayne State University Press: Detroit, 2001. Kracauer, Siegfried. From Caligari to Hitler: A Psychological History of the German Film. Princeton University Press: Princeton and Oxford, 2004.

Compare and Contrast Suffering Shown in Six Poems Essay

The first thing you think when you are told the word suffering is torturing and death and yes this is a big aspect of the topic and it is included in the poem mother in a refugee camp by Chinua Achebe but there is also other parts of it that is not just physical but involves mental suffering and it is this section that is rarely associated with the word in question, Hide and seek by Vernon Scannell is a good example of how this piece comes into play. From this evidence we can explore different forms of suffering and by the time I have reached my conclusion we will have a better understanding of the term. To help us get a better perspective of the word I have used three main poems that demonstrate different elements of suffering War photographer is a poem by carol ann Duffy and he is looking through the pictures he has taken and looking back on their deaths he also talks about how the public react and their lack of care and understanding and what individual has gone through to get just one picture. You can also look at the poem Dulce ET decorum EST which is similar in that it involves the public, Wilfred Owen was a writer from world war one and he disliked how war was displayed to the public that everyone was a hero and they always won. So Owen met some friends and they wrote poems about what the war was really like and this is where he wrote this poem and in it uses the phrase â€Å"the old lie Dulce et Decorum Est Propatria Mori†, In war photographer instead of showing the public the reality of war through poetry he has done it through vivid images that really hit the public and show them what war is really like and although they are separated by almost a 100 years you can see how they are connected. From the start of war photographer you get the feeling of quietness and solitude and if you had to link this with a colour it is quite possible that most people will say something along the lines of blue or green but Carol Ann Duffy has used the colour red and somehow connected it to quietness the colour red is mentioned as it was the colour used when developing a photograph so the room had to be pitch black with the faint red glow. Another example that gives the poem the feeling of tranquillity is the line that uses a religious example. A priest preparing to intone a mass† this technique has also been used in ‘War photographer.   Ã¢â‚¬Å"No Madonna and child could touch†, this is referring to Mary and her son Jesus in the bible and is used as a sign of peace and love which is how the mother in this poem feels towards her dying child. The religious mention in ‘war photographer also represents peace but in this is example it is more to do with the quietness as the photos are developed. he phrase used in the second line is used to represent the people in front of the firing squad waiting to be shot â€Å"spools of suffering set out in ordered rows† and he has used the word suffering in this line as you can imagine the pain of knowing you are about to die and yet you are hopeless in terms of the outcome there is another example where hopelessness is mentioned and this is in ‘Dulce et decorum est’ when there is an â€Å"Ecstasy of fumbling† as everyone tries to put there gas mask on and the one guy who is a bit slow who runs out of time, this guy was helpless to what was going to happen to him is exactly like the men standing up against the firing squad as they are both powerless On the next line Duffy uses four lines to start of the poem and yet there are four full stops, this is to e mphasise on each of the areas mentioned that he has been to and taken pictures of war and it makes the reader pause and think about each individual word, â€Å"Belfast. Beirut. Phnom Penh†. The last line of the Stanza is taken from the bible â€Å"all flesh is grass† there is dying and suffering everywhere.  As you read onto the next stanza Duffy talks about the war photographer going home to Rural England he has used caesura here again and it has been used to make â€Å"Rural England† sound really blunt and plain and she says â€Å"to fields that don’t explode beneath the feet of running children in a nightmare heat† this makes you think how much suffering there would be in the world if it was all like these places Beirut and Phnom Penh and the planet was a war zone. On the last stanza Duffy talks about a hundred agonies in black and white and it is said that black and white photographs are the most powerful and effective forms of capturing the moment of suffering. This poem has a very effective ending and it is going back to what I said at the start â€Å"From the aeroplane he stares impassively at where he earns his living and they do not care† and she is talking about the public and what they have all gone through to get these photographs all for maybe five or six that will be picked out to be published and used in tomorrows newspaper and yet when they read it as he says they do not care. This is not the only poem that has a really effective ending many of the poems have a really strong ending to put there point across, and to make the reader feel emotion towards the victim of suffering. Seamus Heaney’s Mid term break has left a line all by itself to serve as the ending, Mid term Break is about an older brother who has come back to school to find that his younger brother has been hit by a car and it is about how the older brother reacts to this experience, this piece of poetry is autobiographical so it makes the storyline even more moving. A four foot box a foot for every year† this line puts the poem in to perspective when you think just how small this child is and helpless he was, this comes back to the point made in ‘War Photographer’ and ‘Dulce et Decorum Est’ and you can see this part plays a big role as it very important and is one of the most frequent forms of suffering. Vernon Scannell has used a sense of mystery just like war photographer it doesn’t mention any names but it says â€Å"but where are they who sought you? You do not know who the people were same as in war photographer . Hide and seek is all about playing a typical childhood game that everyone knows about and it is through the eyes of a small child and how quickly excitement can turn to a type of suffering. This means of seeing the world through the eyes of a child can be very effective in portraying loneliness and this is exactly what this poet is trying to exploit in using a younger victim. Throughout the start of the poem the writer has expressed the boys excitement using various methods the first technique Scannell has used is the rhyming couplets every forth and fifth line to show just how eager he is to play the game and to win and outsmart his friends also the writer has used caesura, this has been used when there is a change in tension â€Å"Don’t breathe. Don’t move. Stay dumb. Hide in your blindness. † This has been used when the seekers are just at the door to the shed and there is a sudden tense moment when he realises that he must stay quiet to not be caught. Then unexpectedly there is a time jump and we now have this change in atmosphere as the boy is suddenly at a discomfort â€Å"the dark damp smell of sand moves in your throat† also Scannell helps us feel empathy towards the boy as even the weather seems to be against him â€Å"the cold bites through your coat† whereas you had the feeling of warmth at the start where he talks about a beach which is not associated with the cold. Then you get the feeling of excitement again suddenly as he reveals himself only to be brought back down again as he finds out that there is no-one there and everyone has gone and this is where you get the feeling of loneliness which is directly related to suffering and is used in other poems such as half past two by U. A. Thanthorpe. Half past two is about a seemingly sweet and innocent boy who is almost in a dream state throughout the poem and is about him â€Å"escaping into the clockless land†. U. A. Thanthorpe has also used the technique of seeing the world through the mind of a child to portray loneliness just as Scannell has done in his poem. There comes no worse than a death of a child especially your own, you may say this poem demonstrates death more intensely than war photographer or Wilfred Owens poem this is similar to Mid term break where Seamus Heaney talks about the death of a younger brother and they are similar in that a child is used to amplify the sympathy from the reader. Although Heanny’s poem is autobiographical you can say that mother in a refugee camp displays a more depressing feeling as the little child is not the only one suffering as everyone in the refugee camp is almost suffering equally and some of the mothers have even given up on their Childs and the one person you usually look to for comfort is your mother and to not have that support is a disheartening thought. And this really creates an empathy link between the reader and this mother and the victims of this dreadful area in which the whole place is enclosed by death. Achebe uses techniques just as the other poets have, to express torturing in the poem. The main thing he has used is vivid imagery which is a excellent method of really painting a picture in the readers mind about what it would have been like, â€Å"Behind blown empty bellies† he has also used alliteration so once you understand this picture that he is portraying it also sticks in your mind so that you don’t forget it. Then you really start to feel the mothers love for her child near the end as she uses the phrase ‘humming in her eyes’ which suggest she is close to tears and this is a really emotional part of the story and then Achebe really ends the story effectively â€Å"like putting flowers on a tiny grave† this means that even though she knows her son is going to pass away she still feels the love for him that any mother will always do. And this phrase really helps you sympathise with the mother and Ac hebe has helped us understand how she is really feeling and the suffering that she feels inside. So as you can see these six different poets have expressed their feelings on suffering in lots different ways in their poems. Suffering can come in many forms. The ones used in this poem are just a few of the many aspects of suffering. It can come in the form of death and war which was what was used in most of the poems and you can say this is the most extreme form but as I have proved suffering can also come in the form of loneliness and helplessness which is not what you would first expect when you think of suffering, but when you look into it more closely you get a greater understanding of the word and how it can be expressed differently for different situations.

Browns Essays

Browns Essays Browns Essay Browns Essay Restores Browns a philosopher, minister, and Journalist from the sass compared the slave labor system with the wage labor system In Restores Browns Condemns Wage Slavery, 1840. Despite the fact Brannon states that he does not advocate slavery and considers himself a modern balloonists, Browns says that If given the chance to choose between slave labor and waged labor, slave labor would be the one he recommends. We regard the system as decidedly preferable to the system at wages. Restores Browns Condemns Mage Slavery, 1840) He defends his argument by saying the slave that was never free suffers less than someone who works for a living. The laborer at wages has all the disadvantages of freedom and none of its blessings, while the slave, if denied the blessings, is freed from the disadvantages. (Restores Browns Condemns Wage Slavery, 1840) This simply explains the fact that the waged worker may be free but are faced with disadvantages that slaves dont necessarily have to worry about. Some examples loud be that the slaves are given food, lodging, and even the rations given may not have been much the slaves were better off than the waged worker who had to supply his family with a place to sleep, something to eat, and clothes to wear, things that were not promised because they may or may not have been able to afford it depending on their pay. A key difference to note (as mentioned before) is that the waged worker may not make enough money to be able to properly provide for his family with his current wage assuming he has a Job, while a slave is supplied with Hess things by their masters. Upon noting this difference Browns introduces the working class of females describing them as industrious and hard working, Browns does not overlook the fact that the female workers are paid poorly for their labor. And yet there is a man who employs them to make shirts, trousers, etc. , and grows rich on their labors. (Restores Browns Condemns Wage Slavery, 1840) The fact the employer grows rich on their labors Is another phrase that Browns uses to further exemplify the low wages the working class receives. Where go the proceeds of their labor? The man who employs them, and for whom they are tolling as so many slaves, is one of our city nabobs, reveling in luxury; he shouts for liberty, stickles for equality, and is horrified at a southern planter who keeps slaves. (Restores Browns Condemns Wage Slavery, 1840) Browns ends by saying that wages are a way for employers to avoid the costs of slaves and retain a clear conscience. Who would retain all the advantages of the slave system without the expense, trouble, and odium of being slaveholders. (Restores Browns Condemns Wage Slavery, 1840) This line sakes Browns reasoning as to why he favors slave labor clear, summarizing his ideas and placing them In one sentence, which basically says that a waged worker is paid less than a slave. In 1834, the Boston Transcript reports on the Strike the report starts by saying the workers in Lowell would be receiving a 15% pay cut on the 1st of March, a reduction that primarily affected the female workers. This news led to organized meeting that were headed by a young female, that proposed they should qua e mills Ana Induce teen to make a run on ten Lowell n an ten savings Bank, which they the Boston Transcript reports on the Strike) The organization proved successful, due to the fact that the day the Agent had fired the young female who had headed the meetings all the other women had assembled around her after she gave them the signal. The group (that had grown to nearly 800 participants) marched into town, where one of the leaders delivered a speech on female rights and the iniquities of the mooned aristocracy, which produced a powerful affect on her auditors, and they determined to have their way if they died for it. (1834, the Boston Transcript reports on the Strike) A Poem that concluded Lowell Women Workers 1834 Petition to the Manufacturer was created, in which the oppression the females faced working in the mills and how they seemed to adopt the liberty rhetoric to defend their rights in the work place is made clearer to the reader. Tie I value not the feeble threats/ of Tories in disguise, Awhile the flag of Independence/ Oer our nation flies. (Poem that concluded Lowell Women Workers 1834 Petition to the Manufacturer) These lines from the poem make it clear that they ill not succumb to their fears and do as the manufacturers says, but instead will continue their battle for equality in a nation that had fought for its independence and claimed that all are equal (at least those who qualify, for example slaves were not included). Later in 1836 Song Lyrics by Protesting Workers at Lowell compare their working conditions to the treatment of slaves proclaiming Oh! I cannot be a slave, / For Im so fond of liberty, II cannot be a slave. (1836 Song Lyrics by Protesting Workers at Lowell) The females adapted the liberty rhetoric in their search for quality in the workplace, using things like protests and petitions to spread their message and rising against their oppressors demanding their rights and letting the manufacturers know that they will have their way even if they died for it. (1834, the Boston Transcript reports on the Strike) Which seems similar to Give me freedom or give me death. Although both Browns and the Lowell Mill Girls argue for changes in the labor system, they each go about it a different way. A key difference is noted in their way of reasoning, while Browns uses comparisons (compares slave abort to waged labor) to get his point across, the Lowell Mill Girls adopt the liberty rhetoric to try and persuade manufacturers. Another thing to note is that Browns attempts to use words for his manner of persuasion while the Lowell Mill Girls use action (their march, speeches, and song) to attempt and obtain what they want. Browns demonstrates an aggressive attitude in Restores Browns Condemns Wage Slavery, 1840, going as far as saying that the employer is practically a slave owner whos cut his expenses and pockets the savings for himself, the Lowell Mill Girls also take on a seemingly aggressive attitude forming an organized march and showing their resistance without fear of confrontation. The major difference to note between Browns and the Lowell Mill Girls is the changes they are looking to obtain, both are significant changes, but different nonetheless, while Browns is seeking a higher wage for the working class that will at least provide decent quality of life, while the Lowell Mill Girls are looking for female equality in the work place that may lead to better working conditions for as well.

Hurdling the Olympic word police - Emphasis

Hurdling the Olympic word police Hurdling the Olympic word police Today, its exactly two years until the opening ceremony of the Olympics and the moment the eyes of the world turn towards London. However, advertisers not officially associated with the Games will have to duck and dive to be able to cash in on this attention without alerting the Olympic word police. Thats because a law passed in 2006 forbids any combination of 2012, games, gold, silver, bronze and London to be used by anyone but official sponsors of the event. Sporting bodies have made it their business to protect their multi-million-investing sponsors from opportunistic encroachers since 1996. That was the year Nike irked official Olympic sportswear supplier Adidas by setting up their own tented village opposite the main stadium. And you may have read about this years World Cup in South Africa being invaded by a posse of orange-clad women promoting Bavaria beer to the reported fury of Fifa, who had an exclusive deal with Budweiser. Protecting your corporate pitch is one thing. But staking claims on individual words? Is that a step too far? Write and let us know. Meanwhile, if non-sponsors want to make the most of the global publicity in 2012, theyll have to get creative. Grabbing some of the sport-watching spotlight without mentioning the main event will require contortions fit for an Olympic gymnast. It looks like its not only the competing athletes who have just two years left to rise to the challenge.

25 Unforgettable James Joyce Quotes

25 Unforgettable James Joyce Quotes James Joyce was one of the most famous and controversial writers of the 20th century. His epic novel,  Ulysses  (published in 1922),  is widely considered one of the greatest books in Western literature. However, it  was criticized and banned in many places upon its release. His other key works include  Finnegans Wake (1939), A Portrait of the Artist as a Young Man (1916),  and the short story collection  Dubliners (1914). ï » ¿Joyce’s works are often known for using a stream of consciousness  literary technique, through which Joyce gave readers insight into his characters’ thought processes. Below are some famous quotes from James Joyce. Fast Facts: James Joyce James Joyce was born in Dublin in 1882 and died in Zurich in 1941.Joyce spoke numerous languages and studied at University College Dublin.Joyce was married to Nora Barnacle.Although most of Joyce’s works are set in Ireland, he spent very little time there as an adult.Joyce’s famous novel Ulysses was considered controversial when it was first released and was even banned in many places.Joyce’s works are considered an example of modernist literature, and they use the â€Å"stream of consciousness† technique. James Joyce Quotes About Writing, Art, and Poetry He tried to weigh his soul to see if it was a poets soul. (Dubliners) Shakespeare is the happy hunting ground of all minds that have lost their balance. (Ulysses) The artist, like the God of the creation, remains within or behind or beyond or above his handiwork, invisible, refined out of existence, indifferent, paring his fingernails. (A  Portrait of the Artist as a Young Man) Welcome, O life! I go to encounter for the millionth time the reality of experience and to forge in the smithy of my soul the uncreated conscience of my race. (A  Portrait of the Artist as a Young Man) Writing in English is the most ingenious torture ever devised for sins committed in previous lives. The English reading public explains the reason why. (letter to Fanny Guillermet, 1918) Poetry, even when apparently most fantastic, is always a revolt against artifice, a revolt, in a sense, against actuality. It speaks of what seems fantastic and unreal to those who have lost the simple intuitions which are the test of reality; and, as it is often found at war with its age, so it makes no account of history, which is fabled by the daughters of memory. (Selected letters of James Joyce) He wanted to cry quietly but not for himself: for the words, so beautiful and sad, like music. (A Portrait of the Artist as a Young Man) The supreme question about a work of art is out of how deep a life does it spring. (Ulysses) The object of the artist is the creation of the beautiful. What the beautiful is is another question.  (A Portrait of the Artist as a Young Man) To discover the mode of life or of art whereby my spirit could express itself in unfettered freedom. (A Portrait of the Artist as a Young Man) [A writer is] a priest of eternal imagination, transmuting the daily bread of experience into the radiant body of everliving life. (Selected letters of James Joyce) James Joyce Quotes About Love I had never spoken to her, except for a few casual words, and yet her name was like a summons to all my foolish blood. (Dubliners) I asked him with my eyes to ask again yes and then he asked me would I yes to say yes my mountain flower and first I put my arms around him yes and drew him down to me so he could feel my breasts all perfume yes and his heart was going like mad and yes I said yes I will Yes. (Ulysses) His heart danced upon her movements like a cork upon a tide. He heard what her eyes said to him from beneath their cowl and knew that in some dim past, whether in life or revery, he had heard their tale before. (A Portrait of the Artist as a Young Man) Love loves to love love. (Ulysses) Why is it that words like these seem dull and cold? Is it because there is no word tender enough to be your name? (The Dead) Her lips touched his brain as they touched his lips, as though they were a vehicle of some vague speech and between them he felt an unknown and timid preasure, darker than the swoon of sin, softer than sound or odor. (A Portrait of the Artist as a Young Man) I did not know whether I would ever speak to her or not or, if I spoke to her, how I could tell her of my confused adoration. But my body was like a harp and her words and gestures were like fingers running upon the wires. (Dubliners) James Joyce Quotes About Fame and Glory Better pass boldly into that other world, in the full glory of some passion, than fade and wither dismally with age. (Dubliners) A man of genius makes no mistakes. His errors are volitional and are the portals of discovery. (Ulysses) James Joyce Quotes About Being Irish When the Irishman is found outside of Ireland in another environment, he very often becomes a respected man. The economic and intellectual conditions that prevail in his own country do not permit the development of individuality. No one who has any self-respect stays in Ireland but flees afar as though from a country that has undergone the visitation of an angered Jove. (James Joyce, lecture:  Ireland, Island of Saints and Sages) No God for Ireland! he cried. We have had too much God in Ireland. Away with God! (A Portrait of the Artist as a Young Man) This race and this country and this life produced me, he said. I shall express myself as I am. (A Portrait of the Artist as a Young Man) The soul ... has a slow and dark birth, more mysterious than the birth of the body. When the soul of a man is born in this country there are nets flung at it to hold it back from flight. You talk to me of nationality, language, religion. I shall try to fly by those nets. (A Portrait of the Artist as a Young Man) When I die, Dublin will be written on my heart. (Selected letters of James Joyce)