Tuesday, August 25, 2020

Essay on Crimes Assault

Paper on Crimes Assault Paper on Crimes: Assault Detest, Bias, and Stranger Crimes What is despise wrongdoing? Despise wrongdoing are viewed as misdeeds and here and there a lawful offense since it is an infringement of the law which is submitted against any individual or property due to their ethnicity, sexual orientation, age, handicap, or religion. These violations are like separation. A few instances of abhor violations are dangers of savagery as a result of a trademark highlight of an individual, spray painting on someone’s property, any physical attack in light of a person’s race. Predisposition violations are like despise wrongdoings with the exception of that the violations that are being dedicated are for the most part verbal. Wrongdoings that are in this classification incorporate chauvinist or supremacist jokes told in broad daylight, verbal abuse towards an ethnic minority, or in any event, slandering messages sent to an understudy social association. Stranger rough violations are wrongdoings that for the most part happen all over. These v iolations incorporate medications, posses, social qualities, local qualities, character and senses. More abnormal brutality will in general happen in certain geospatial areas known for any place boisterous individuals are stuck together. At the point when a situation has absence of room, savagery will in general eject. Since loathe violations and predisposition wrongdoings are characterized likewise, stranger brutal violations are substantially more extraordinary. The various kinds of more bizarre violations are seen every day for a great many people. These violations don’t incorporate judgment or inclination towards others. Indeed, more odd wrongdoings are violations that are don’t towards others for the satisfaction of someone else. For instance, when a burglary is done, it happened simply because it was for a person’s fulfillment. Distinctive to predisposition and despise wrongdoings, these violations are made to purposefully offend . The impact that despise and predisposition wrongdoings have among an individual is more grounded than any vicious wrongdoing. Separating somebody makes abhor and inclination wrongdoings, while more interesting violations are made less on an individual issue. A feeling impact happens when an abhor and predisposition wrongdoings happen, while physical impacts are made by more unusual violations. At the point when an individual perpetrates abhor and inclination violations, they leave enthusiastic trouble among the person in question. This happens on the grounds that

Saturday, August 22, 2020

Phrasal Verbs - English Definitions for ESL Students

Phrasal Verbs - English Definitions for ESL Students There are four sorts of phrasal action words. Phrasal action words can be divisible or indivisible and they can take an article or not. Here is a manual for the essentials of phrasal action words. Phrasal Verbs which Take Objects Phrasal action words which take objects are known as transitive phrasal action words. These action words can be distinguishable or indistinguishable: Detachable phrasal action words can stay together when utilizing an article that is a thing or thing phrase. I got Tom. Or then again I got Tom.They put their companions up. Or then again They set up their friends.My companions surrendered bowling. Or on the other hand My companions surrendered bowling.â Distinguishable phrasal action words: get, set up, surrender Distinguishable phrasal action words MUST be isolated when a pronoun is utilized: We got him the station. NOT We got him at the station.They put them up. NOT They set up them.She thought it up a few days ago. NOT She brainstormed it the other day.â Divisible phrasal action words: get, set up, concoct Indivisible phrasal action words consistently stay together. It has no effect if a thing or pronoun is utilized. We set off for the sea shore. /We set off for it.They are caring for the youngsters. /They are caring for them.The educator required the appropriate response in class. /The educator called for it in class. Indivisible phrasal action words: set off, take care of, call for Phrasal Verbs which Dont Take Objects Some phrasal action words don't take objects. Action words that don't take objects are otherwise called intransitive action words. These phrasal action words are ALWAYS indivisible. The cheats got away.The transport separated while in transit to work.She rose early. Intransitive phrasal action words: escape, separate, get up In the event that you are uncertain about whether a phrasal action word is detachable or indivisible, ALWAYS utilize a thing or things state and DO NOT separate. Thusly, you will consistently be right! Distinct Phrasal Verbs: raise, take off They raised their youngsters to regard others.She removed her coat before she started the lesson.The manager put off the gathering until one week from now. Indistinguishable Phrasal Verbs: search for, set off, keep at She was searching for her books when he arrived.They set off for a superb occasion in Hawaii.You should keep at your schoolwork for at any rate an hour.â Three-word Phrasal Verbs A few action words are trailed by two relational words (or verb modifiers). These phrasal action words are ALWAYS indivisible. Im anticipating meeting John. Or on the other hand Im anticipating meeting him.They didnt continue ahead with their mom. Or then again They didnt continue ahead with her.Peter thought of an extraordinary thought. Or then again Peter thought of it.â Three-word phrasal action words: anticipate, continue ahead with, think of Phrasal Verb Type Quiz Check your comprehension by distinguishing each phrasal action word as transitive or intransitive and divisible or indivisible. For example:â My companion got me the air terminal. - get: transitive, distinguishable We set off at six oclock in the morning. Tom anticipates meeting you next week.Unfortunately, the hoodlums got away.He disclosed to me that he had surrendered cigarettes last year.I got up and went to work.Jennifer thought it up during the meeting. I was so drained after the race I split down.He brought the subject up during class yesterday.Ill take care of your pooches while youre away on vacation.She concocted an extraordinary thought. Test Answers set off: intransitive/inseparablelook forward to: transitive/inseparableget away: intransitive/inseparablegive up: transitive/separableget up: intransitive/inseparablethink up: transitive/separablebreak down: intransitive/inseparablebring up: transitive/separablelook after: transitive/inseparablecome up with: transitive/indivisible Keep Learning Phrasal Verbs This phrasal action words reference rundown will kick you off with short meanings of roughly 100 of the most widely recognized phrasal action words. Educators can utilize this acquainting phrasal action words exercise plan with assistance understudies become increasingly acquainted with phrasal action words and begin building phrasal action word jargon. At last, there are a wide assortment of phrasal action word assets on the site to assist you with learning new phrasal action words.

Wednesday, July 29, 2020

Credit Tier Breakdown, Part 1 Great Credit

Credit Tier Breakdown, Part 1 Great Credit Credit Tier Breakdown, Part 1: Great Credit Credit Tier Breakdown, Part 1: Great CreditWelcome to our ongoing series on credit scores, from the very best to the very worst. We’ll let you know what kind of loans, interest rates, and other perks you can expect with each type of score. Plus, we’ll give you tips about how you can improve your score.For this first entry, we’ll be looking at Great Credit Scores: 720 â€" 850.What Kind of Loans Can You Get?In short: pretty much any loan you want.Now, that’s not entirely true. Banks and other traditional lenders will also want to look at things like your income before approving a loan. If you have a great credit score but a low-income, then there are certain loansâ€"especially unsecured personal loansâ€"that you might not qualify for.Still, having a score of 720 or higher means that pretty much any kind of loan is achievable. If you want to buy a house, you’ll qualify for a good mortgage. If you want to buy a car, you’ll be able to get approved for a car loan. Looking for a credit card? Take. Your. Pick.This is especially true for folks who have a credit score in the upper part of this range, from 780 to 850. While folks with scores lower than that might occasionally get their applications turned downâ€"especially for really high-end credit cardsâ€"people with scores of 780 and above can pretty much get approved for anythingâ€"assuming that their income makes repayment of the loan feasible.What kind of Interest Rates Can You Get?If you have a score of 720 or above, you’re going to be seeing very low-interest rates across the board.According to Rod Griffin (@Rod_Griffin), Director of Public Education for the credit bureau Experian, says, A credit score is a way of grading your creditworthinessor trustworthiness to lenders. Its a numeric score usually falling in a scale between 350 and 850. The better your credit score, the lower risk you are to lenders. Theyll see you as someone they can trust to repay the money they may lend you. You will also gener ally qualify for lower interest rates, which will save you money.A score above 720 says to lenders that you are very responsible when it comes to borrowing money. You don’t take out more than you can afford and you always make your payments on time.If you are seen as a low-risk by a lender, they will be willing to give you a lower interest rate. With higher risk borrowers, lenders charge them more in part to guard against the risk that they won’t repay. It’s a bit of a catch-22: The less likely you are to pay your loan back, the more money you get charged, which makes the loan harder to repay, which makes it less likely that you’ll pay it back.According to the MyFico Loan Savings Calculator, a credit score of 760 could get you a 3.845 percent interest rate on a $300,000, 30-year fixed-rate mortgage. All in all, that could save you almost $25,000 over the rate you’d pay with a credit score of 699.If you took out a $30,000 48-month auto loan to buy a new car, the MyFico Loan Savings Calculator estimates that a score of 720 or above would net you an interest rate of 3.458 percent. When compared to the 6.88 percent interest rate you would pay with a score of 689, that lower rate would save you well over $2,000 over the life of the loan.And lastly, there are credit cards. While the rates on these cards can vary greatly from company to company, Credit Karma pegs the interest rates for borrowers with great credit at an average of 14 percent.What Can I Do to Improve My Score?Your FICO score is created using information from your credit reports, which are compiled by the three major credit bureausâ€"Experian, TransUnion, and Equifax. According to Rod, Your credit report is a document that describes how you use your credit and whether or not you pay your debts. It contains information about your credit cards, any installment loans, and other debts, and whether youre paying them or not. The credit report is not the same as the credit score. A lender can use you r credit report to calculate your score, or they can request that your score be calculated. The score is a snapshot of your creditworthiness at the moment the credit report is requested.Your credit score is calculated using these five major categories:Payment History:35 percentAmounts Owed: 30 percentLength of Credit History: 15 percentNew Credit: 10 percentCredit Mix: 10 percentAs you can see, your payment history and amounts owed make up a total of 65 percent of your score. If you’re looking to improve your credit rating, those two categories are the best place to start.Credit expert Chella Diaz (@MoneyIQ901) has two great pieces of advice:Start making your payments on time. This might seem a little obvious, but making on-time payments is a huge piece of your credit score. If someone’s going to lend you money, they need to be certain that you’ll make your payments on time. Diaz says that you making your payments on time for at least six months should help your credit score i ncrease.In regards to your credit card balances, Diaz says that your balance should not exceed 30% to 35 percent of the limit on the card. Ideally, you shouldn’t be carrying any balance on your card month to month. You should pay the entire thing off in full to avoid getting charged any interest at all. But paying down your balances until they are below 30 to 35 percent should lead to your credit score increasing.Want to learn more about how credit scores and credit reports work? Check out our QA with Rod Griffin, Education Director for Experian. You can also read our ebook Credit Workbook: The OppLoans Guide to Understanding Your Credit, Credit Report and Credit Score.About the Contributors:Chella Diaz,  is a mom, author, speaker and consultant. She empowers parents with young children to set up their kids to be their own bank. She facilitates workshops for college students. One of Chella’s greatest strengths is showing people the ways they can save money so they always have mo ney for the things that are most important. You are the boss and the money is your employee. How are you going to manage it?Rod Griffin is Director of Public Education for Experian. He leads Experian’s national consumer education programs and supports the company’s community involvement and corporate responsibility efforts. Rod oversees the company’s financial literacy grant program, which awarded more than $850,000 in 2015 to non-profit programs that help people achieve financial success. He works with consumer advocates, financial educators and others to help consumers increase their ability to understand and manage personal finances and protect themselves from fraud and identity theft.

Friday, May 22, 2020

Black Men And The Brotherhood Essay - 1422 Words

While white women seek visibility as a means of being recognized by white men, white men seek visibility to further their political goals. What both have in common is the use of black men to amplify their visibility and expedite their success. The Brotherhood is an organization led by Brother Jack that entices the Invisible Man, recruits him, and takes advantage of his invisibility to spark a riot in the streets of Harlem. The Brotherhood takes advantage of his invisibility in multiple ways: the organization advises the Invisible Man during his speeches, the organization sends him across New York as it see fit, the organization gives him money, and the organization fuels his rise to fame and notoriety. These acts seem benevolent, but the intentions behind them were destructive and manipulative. The Brotherhood has a doctrine and all members are expected to abide by it. Individual action is frowned upon. The Invisible Man is thus, reduced to a token and through his invisibility, the B rotherhood amplifies its prevalence in Harlem and generally as an organization. Again, the black man is used and those who use him aren’t invested in him. The Invisible Man realizes this amidst the riots in Harlem: â€Å"And now I looked around a corner of my mind and saw Jack and Norton and Emerson merge into one single white figure. They were very much the same, each attempting to force his picture of reality upon me and neither giving a hoot in hell for how things looked to me† (508) and â€Å"It wasShow MoreRelatedThe Invisible Man, By Louis Armstrong841 Words   |  4 Pagesstealing electricity, and listening to Louis Armstrong’s â€Å"What Did I Do to Be So Black and Blue.† As a young man, he lives in the South. He is invited to give his high school graduation speech to a group of white men. However, he is forced to fight against other young, black men in a ring while blindfolded. After the humiliation, the narrator gives his speech. The men award him with a briefcase containing a scholarship to a black college. The narrator has a dream in which the scholarship is a piece of paperRead MoreInvisible Man By Ralph Ellison1283 Words   |  6 PagesInvisible Man, by Ralph Ellison, tells the story of a young, educated black man as he travels from the Deep South to the streets of Harlem, experiencing the oppression and the struggles of a dominantly white society. The narrator, who remains nameless throughout the entire novel, is on a search for his true identity. Along the way he meets many powerful white men who are more than willing to define him, often in the form of a document. While these papers seem to foreshadow good fortune for the narratorRead MoreThe Invisible Man By Ralph Ellison1239 Words   |  5 PagesThe Invisible Man by Ralph Ellison is about a black man struggling to find his identity in 1930s America. This book is called The Invisible Man not because the narrator is literally invisible, but because people only s ee him through a stereotypical and prejudice point of view. In this book we follow the narrator’s life as a college student, a worker at a paint plant, and a member of a shady political organization called the Brotherhood. The book begins with the narrator claiming he is an invisibleRead MoreSignificance of the Narrators Invisibility in Ralph Ellisons Invisible Man676 Words   |  3 Pagesinto entertainment when he is forced to fight in a â€Å"battle royal† with other black men. After being beaten blindfolded and pushed into an electrocuted carpet, the narrator still gathers up the strength to dictate his speech, only to find the white men â€Å"still [talking] and still [laughing], as though deaf with cotton in dirty ears† (p30). The author Ralph Ellison uses â€Å"deaf with cotton† to reinforce the choice for the white men not to see him, as they have refused to see enslaved African-Americans asRead MoreThe Brotherhood of Sleeping Car Porters1148 Word s   |  5 PagesThe Brotherhood of Sleeping Car Porters Carol Y. Reeves HRMG 5930 2/22/2012 The International Brotherhood of Sleeping Car Porters was the first African American Labor Union chartered by the American Federation of Labor. Pullman porters were men who George Pullman hired to work on railroads as porters on sleeping cars. After the American Civil War, Mr. Pullman found former slaves to work on his sleeper cars. Mr. Pullman was inspired to design better railcars for passengers thatRead MoreThe Role Of Identity In Ralph Ellisons Invisible Man1236 Words   |  5 Pagesbecoming aware of his invisibility. Ideologies of the college the Invisible Man attended and the Brotherhood he joined rendered the Invisible Man invisible. Being taught to work hard and pursue economic advancement without yearning for equal rights or equal treatment from whites, the college limited the Invisible Man’s identity, the black culture, because it forced him to discount and discredit the black culture that he grew up participating in. Urging the Invisible Man to accept a positio n of inferiorityRead MoreOrganized Crime Group Analysis1527 Words   |  7 PagesSo as a team we decided to research the organization The Aryan Brotherhood. Originally named the Diamond Tooth Gang in 1967, a group of men gathered in the prison yard of San Quentin, to form their own racially motivated prison gang. These men mostly consisted of neo-Nazi, white supremist, long haired biker types. They formed an alliance to protect themselves and strike against the group of black militants known as the Black Guerilla Family (Grann.,  2004). Prior to the 1960’s prisonsRead MoreIn Ralph Ellison’S Novel Invisible Man, Man Is Often Equated1692 Words   |  7 Pagesonly encounters figures who liken their efforts and sense of entrapment to working at the heart of a much larger apparatus, but he eventually finds that his own actions within organizations such as the Brotherhood also contribute to the whole of the machine’s operation. In such instances where the black man’s body becomes a functioning part that remains both confined and fixed in place, we witness how the machine becomes an interconnected symbol for the unwavering power of technology over man. The machineRead MoreThe Not So Invisible Man1275 Words   |  6 PagesDaniella Cameron Santos Advanced Honors English 1 Mrs. Sanzo 21/8/15 The not so invisible man. While depicting the idealized life of a black man an anonymous narrator realizes that people only see him for what they want to see him for, which makes him invisible to simply put it, because people see who they want to see and they refused to see the real him. The narrator describes his life as he struggles to become who the people surrounding him want him to be until he comes to the realizationRead MoreThe Narrator As An Invisible Man1305 Words   |  6 Pagesschool valedictory speech in front of leading white men in his community. When the Narrator arrives to give his speech, he is forced to participate in a boxing style competition, along with several other boys, for the entertainment of the white men in attendance. Invisible Man and the boys are then made to further humiliate themselves by having to grab coins off of an electrified rug. Once this is over, the Narrator is allowed to give his speech. The men love it until the Narrator slips up and says â€Å"social

Saturday, May 9, 2020

Who Else Wants to Learn About Essay Topics Grade 9?

Who Else Wants to Learn About Essay Topics Grade 9? The web has opened doors to a wide selection of opportunities associated with various kinds of services. You are able to definitely hunt for some information online, but you can discover such experience very confusing. Don't neglect to confirm the access to resources the topics that you consider. Many of the rewriting teams of different businesses take the order from clients and look for an inexpensive writers, who might not have the very good knowledge on English. The Tried and True Method for Essay Topics Grade 9 in Step by Step Detail As a discipline, writing requires a great deal of practice, particularly in the essential stages like 5th grade. The Thelen philosophy is to stay Small enough to know our clients and big enough to care for their demands. Remember your final grade significantly is based on the topic. So the overall grade for the paper might differ based on that. To compose a strong argumentative essay, stu dents should start by familiarizing themselves with a number of the common, and frequently conflicting, positions on the research topic so they can write an educated paper. At times it only appears simple, but a great deal of students forget about the kind of academic writing they need to follow. Knowing that there are lots of writers out there with years of experience, you need to set up your mind accordingly. Very often it becomes tough to choose 1 topic either on account of the many ideas in the student's head, or due to their complete absence. The very first thing you must realize searching for an ideal topic is that your opinion is the thing that matters the most. When you're picking your topic, bear in mind that it's much simpler to write about something which you currently have interest ineven in case you don't know a good deal about it. Contribute your suggestions and people are certain to read! When you're permitted to write about whatever you want, never rely on somebody's thoughts about this issue you like as a pivotal criterion for your choice. Deciding on a topic is an essential issue that partly estimates final success of the job. Finding the subject of your interest will allow you to work harder on your project and show your style in the easiest way possible. Most Noticeable Essay Topics Grade 9 In choosing your topic, it's frequently a good concept to start with a subject which you already have some familiarity with. Actually, a short and easy introduction is jam-packed with information since its principal goal would be brevity. Undoubtedly, it would be easier that you learn more about the topic that's connected to the area of your interest. For this reason, you've got to come across enough substantial evidence for the specific topic. What's Really Happening with Essay Topics Grade 9 Don't begin w riting your essay with the start or ending, the most significant part is the middle one. Most students have a tendency to choose easy essay topics by talking about themselves, doing their very best to express their special qualities and trying to stick out from the remainder of the pack. 2 minutes is longer than you believe! Understanding how to compose a strong argumentative paper will help you advance your very own argumentative thinking. The essay writer, assigned for you, will earn a research of your private writing and imitate that vocabulary and fashion. The main purpose of topic choice for a proposal essay is to demonstrate the idea can be put into place in practice. To lessen stress whilst writing a research paper, you ought to be certain to have chosen the best topic. Finding the Best Essay Topics Grade 9 Like the death penalty, the thought of marijuana legality was debated and discussed for decades. You should have your reasons, and our primary concern is that you wind up getting an excellent grade. If it's possible to write a really convincing piece on a real-world application utilizing unique facts and research, then your opportunities receiving admission to a top level university will certainly grow! If you still have issues with topic selection, don't hesitate to request help at Gra deMiners. The Advantages of Essay Topics Grade 9 The option of compare and contrast essay topics isn't a simple task because you should clearly show your analytical skills. Take notes concerning all possible topics you may consider. There are an infinite number of ways to start an essay effectively. So without further ado, below are some effective writing tips to create your common app essay stick out! Odds are, all you have to do is relax and locate a topic you're passionate about and, naturally, one that's debatable. You don't need to find super technical with legal argumentative essays, but be certain to do your homework on what the present laws about your favorite topic actually say. Following are some basic strategies to direct you in deciding on the ideal persuasive essay topic for you. Thus, the topic you select plays a crucial role.

Wednesday, May 6, 2020

Paper Free Essays

The university was founded in 1885, which was before Arizona was officially a state. Arizona holds 31,670 undergraduate students and 8951 graduate students that study the 300+ majors offered. Within these students 120 countries are represented. We will write a custom essay sample on Paper or any similar topic only for you Order Now Tuition costs $29,421 for a non-resident of Arizona. In addition, there is a $9,714 room and board fee. To become a Wildcat, Arizona requires 4 years of English, and either a 21 or above on the English section of the ACT or a 530 on the reading section. They also require 4 years of Mathematics, and a 24 on the math portion of the ACT or a 540 on the math part of the SAT. Arizona also requires that students take 3 years of lab science and score a 20 or higher on the math portion of the ACT. If students took the SAT they might want to consider taking subject tests. Arizona requires 2 years of History classes, one American history course and one other history course. Furthermore, the University of Arizona requires 2 years of consecutive classes of a second language (both classes must be the same language). Lastly, the university of Arizona requires 1 year of fine arts. Arizona requires that you maintain a 2. 0 GAP or higher throughout your high school career. During your time In high school you can only get lower than a 2. 0, or C, twice to still be eligible for admission. University of Arizona hosts a Greek system hosting both fraternities and sororities. Arizona Greek system is much larger In comparison to other schools consisting of 47 sororities and fraternities. On campus housing holds 79% of students at Arizona In a total of 24 student-housing facilities. The other 21% live off campus. At Arizona there Is a balance of males and females. % of the population Is female, leaving 48% to male population. Downtown Tucson holds plenty to do for students and acts as the perfect college town. Paper By raunchy University of Arizona higher throughout your high school career. During your time in high school you can system is much larger in comparison to other schools consisting of 47 sororities and fraternities. On campus housing holds 79% of students at Arizona in a total of 24 student-housing facilities. The other 21% live off campus. How to cite Paper, Papers Paper Free Essays The questionnaire is designed by the researchers a seven item scale from tryingly disagree (-3) to strongly agree (+3) to identify variables of customer loyalty. In the present study, we, therefore, used Cockroach’s alpha scale as a measure of reliability. Its value is estimated to be 0. We will write a custom essay sample on Paper or any similar topic only for you Order Now 897. Sophisticated statistical model as ‘Exploratory Factor Analysis (FEE)’ has been used. The results show that nine factors extracted from the analysis that together accounted 77. 891 percent of the total variance. Finally, on the basis of factor score, these factors were ranked (1)sales promotion’; (2) ‘Provision of information’; (3)engagement’; (4)recommendation of the Product or Service’;(5)new brand’;’ (6) ‘The value of brand’; (7)eliminative’; (8) ‘Bench Marking’ and (9) ‘Environmental friendly organization’ got the ranks of first to nine respectively and constitute the key factors of customer loyalty in leading retail supermarkets in I-J. Moreover, outcome of the research would be helpful to the practitioners, researchers, planners, policy makers and academicians, who are involved in the concerned area. Keywords: Customers; Customer Loyalty and Retail Supermarkets. 1. 0 Background and Significance In the ever-changing business world, almost every organization pays most attention o the customers ever than before. For any organization, good understanding of customers, their needs and wants, their expectations on price and quality of goods and services increase the potential to succeed. As a result, customer centered marketing has occupied the top place in modern marketing concept. Every organization is ready to pay any means to identify and understand the customers and their needs. Consumers’ reaction will be in favor of an organization when their desires and expectations have been either met or exceeded in the course of experiencing the service. In the context of a retail supermarket, satisfaction could be interpreted as Just meeting the expectations of the customers, not any sort of exceeding or falling short of the expectations. Most of the retailers try to achieve competitive advantage by taking the responses of the customers beyond the level of ‘Just satisfied’ towards ‘exceeding their expectations’. Pleasing customers are very harder today (Kettle, 2003). Customers are more challenging component for any organization rather than their competitors. Their buying behaviors are fickle, at least three times a year expecting the best deal from the suppliers. Besides the above, the worst thing is ninety percent of dissatisfied customers Just switched to another supplier without complaining to former supplier (Kettle, 2003). A marketing strategy which is considered today as the best one may not produce same results in future. Thus, every organization must pay their attention in complete satisfaction of their customers. Since, highly satisfied customers more likely become loyal customers and potentially buy the new products introducing by the company and shows the word of mouth and also pay less attention about competitors and other brands as ell. Above to all, considering cost related to customers, cost for retaining existing customers are very less than acquiring the new customers and also existing customers are much more profitable in many ways for instance, word of mouth. Here, word- loyalty or loyal customers captures the predominant place. Because, Hill Alexander (2006) pointed out that only through the loyalty, customer retention can be secured. 2. 0 Statement of the Problems Factors determining customer satisfaction and customer loyalty have been brought to light by marketing research. But, this information still is far away for some producers engaging in the productions and services. Consequently, producers are unable to exploit this information for their success. According to Verdict consulting research (2007), retail supermarket sector in I-J is one of most competitive segments and also pointed out that this competition will create more challenging environment in maintaining their market share. However, some retailers are very successful than their competitors even during the period of European economic downturn. This encouraged the researchers to do this research. We hope that this research will answer the following question regarding customer loyalty effectively. 3. Objectives The present study has the following objectives 1. To examine necessary factors of customer loyalty in leading super markets of I-J; and 2. To determine the key factors of customer loyalty in leading super markets of UK. 3. To suggest some measures in order to improve the customer loyalty in leading 4. 0 Literature Review Managing customer loyalty is the one of major element of customer relationship management. Customer satisfied with the present service of organizati on will likely satisfy if the firm does the same service later. So every organization has struck with the question how they can increase their loyalty level by adopting the right approaches. Stone (2000) pointed out in his book that â€Å"using information on the customer data base, there is no reason for a customer loyalty programmer other than finely tuned to meeting customers ‘relationships needs†. Loyalty becomes a winning factor for any organization facilitating with high productivity, solid profit and feasibility for steady expansion, competing in present world. When considering the resent states of disloyalty, it is obvious that it would damage the corporate performance by 25 to 50 percent and possibly more (Astrakhan, 2006). Loyalty is defined as â€Å"a state of mind, a set of attitudes, beliefs, desires and so on† (Stone, 2000). Kettle (2008) said that delighted customers become loyal to the organization and customer relationship management (CRM) plays an important role in making customers loyal. Further, among the satisfied customers, completely satisfied customers only can be a delighted one. Thus CRM has to focus on customer delight rather than satisfaction. However, Hill and Alexander (2006) argue that misunderstanding of customer loyalty by the senior manages and marketing executives have mislead strategies for securing the customer loyalty and also criticized that many of them take afford to attract the customers by giving some bribe to customers. Instead, customer loyalty has to be earned by the suppliers and customer retention can be achieved when the suppliers satisfy the requirements raised by the customers better than their rivals. Realty is that in the twenty first century, both not only customers and but also suppliers have to true, faithful and rim in meeting the customers’ needs. Furthermore, Hill Alexander (2006) categorized loyalty into four types such as (1) Monopoly loyalty (where customers have little or no choice and they are completely dissatisfied and far away from devoted); (2) Cost of change loyalty (where customers have choice of alternative suppliers and reluctant to change their current due to the cost and other bothering factors, needs immense afford to change); (3) Incentive loyalty(this is the type of loyalty created by mass advertisement and targeted the customers who are not pending their own money for instance frequent business fliers);(4) Habitual loyalty(this can be viewed most commonly due to the time constraints and familiar routines, convenient location and little afford for instance filling up petrol on the way to work). This paper is focus on this Habitual loyalty. Here, convenient location, size of supermarket, variety of goods, competitive price plays a significant role. Moreover, degrees of loyalty can differ from one customer to another for instance one customer is more loyal than other. Hill Alexander (2006) defined these degrees as suspects, respects, customers, clients’ advocates and partners in a pyramid. According to them, degrees of positive commitment increases along pyramid from suspects to partners and also distinguishes the truly loyal customers. Less loyal customer is likely to switch the supplier Based on the previous studies, we can say that there are some studies in different countries, but detailed and comprehensive studies has not yet been conducted in I-J especially in supermarket through exploratory study. How to cite Paper, Papers Paper Free Essays To me there is a big difference in a college class discussion verse a discussion you have with your friend. In college classes you have to have a more professional approach in how you speak or answer other classmates. When speaking to your friends who know you, they have good Judgment on when you are Joking around or even if you are being sarcastic. We will write a custom essay sample on Paper or any similar topic only for you Order Now I myself know that my friends and family even my co- workers all have excellent Judgment on my mannerisms. My classmates however unlike my friends, family and co-workers do not know me so during class discussions I will make sure that I make a point to convey my messages with respect and In a good tone so everyone will understand where I am coming from. I enjoy feedback and look forward to my classmates giving me their pollens on things and I scant Walt to see everyone else’s point of view on certain topics we discuss. There are many different ways that you can demonstrate respect for your fellow classmates during class discussions such as good understanding, politeness and having a positive attitude towards your peers. It Is good to show your classmates that o understand where they are coming from even if you do not agree, everyone is entitled to their own opinion and everyone has different ways of looking at certain issues. Being polite to your classmates is a great way to show you are respecting them. We are all here to learn and grow together so together we can all achieve our goals and dreams. Approaching your classmates with a positive attitude will always send a good vibe, who has time for negativity navy? I know I certainly don so being positive towards others will go a long way. I will always treat others the way I want myself and my daughter to be treated. How to cite Paper, Papers Paper Free Essays string(763) " categorized loyalty into four types such as \(1\) Monopoly loyalty \(where customers have little or no choice and they are completely dissatisfied and far away from devoted\); \(2\) Cost of change loyalty \(where customers have choice of alternative suppliers and reluctant to change their current due to the cost and other bothering factors, needs immense afford to change\); \(3\) Incentive loyalty\(this is the type of loyalty created by mass advertisement and targeted the customers who are not pending their own money for instance frequent business fliers\);\(4\) Habitual loyalty\(this can be viewed most commonly due to the time constraints and familiar routines, convenient location and little afford for instance filling up petrol on the way to work\)\." The questionnaire is designed by the researchers a seven item scale from tryingly disagree (-3) to strongly agree (+3) to identify variables of customer loyalty. In the present study, we, therefore, used Cockroach’s alpha scale as a measure of reliability. Its value is estimated to be 0. We will write a custom essay sample on Paper or any similar topic only for you Order Now 897. Sophisticated statistical model as ‘Exploratory Factor Analysis (FEE)’ has been used. The results show that nine factors extracted from the analysis that together accounted 77. 891 percent of the total variance. Finally, on the basis of factor score, these factors were ranked (1)sales promotion’; (2) ‘Provision of information’; (3)management’; (4)recommendation of the Product or Service’;(5)new brand’;’ (6) ‘The value of brand’; (7)eliminative’; (8) ‘Bench Marking’ and (9) ‘Environmental friendly organization’ got the ranks of first to nine respectively and constitute the key factors of customer loyalty in leading retail supermarkets in I-J. Moreover, outcome of the research would be helpful to the practitioners, researchers, planners, policy makers and academicians, who are involved in the concerned area. Keywords: Customers; Customer Loyalty and Retail Supermarkets. 1. 0 Background and Significance In the ever-changing business world, almost every organization pays most attention o the customers ever than before. For any organization, good understanding of customers, their needs and wants, their expectations on price and quality of goods and services increase the potential to succeed. As a result, customer centered marketing has occupied the top place in modern marketing concept. Every organization is ready to pay any means to identify and understand the customers and their needs. Consumers’ reaction will be in favor of an organization when their desires and expectations have been either met or exceeded in the course of experiencing the service. In the context of a retail supermarket, satisfaction could be interpreted as Just meeting the expectations of the customers, not any sort of exceeding or falling short of the expectations. Most of the retailers try to achieve competitive advantage by taking the responses of the customers beyond the level of ‘Just satisfied’ towards ‘exceeding their expectations’. Pleasing customers are very harder today (Kettle, 2003). Customers are more challenging component for any organization rather than their competitors. Their buying behaviors are fickle, at least three times a year expecting the best deal from the suppliers. Besides the above, the worst thing is ninety percent of dissatisfied customers Just switched to another supplier without complaining to former supplier (Kettle, 2003). A marketing strategy which is considered today as the best one may not produce same results in future. Thus, every organization must pay their attention in complete satisfaction of their customers. Since, highly satisfied customers more likely become loyal customers and potentially buy the new products introducing by the company and shows the word of mouth and also pay less attention about competitors and other brands as ell. Above to all, considering cost related to customers, cost for retaining existing customers are very less than acquiring the new customers and also existing customers are much more profitable in many ways for instance, word of mouth. Here, word- loyalty or loyal customers captures the predominant place. Because, Hill Alexander (2006) pointed out that only through the loyalty, customer retention can be secured. 2. 0 Statement of the Problems Factors determining customer satisfaction and customer loyalty have been brought to light by marketing research. But, this information still is far away for some producers engaging in the productions and services. Consequently, producers are unable to exploit this information for their success. According to Verdict consulting research (2007), retail supermarket sector in I-J is one of most competitive segments and also pointed out that this competition will create more challenging environment in maintaining their market share. However, some retailers are very successful than their competitors even during the period of European economic downturn. This encouraged the researchers to do this research. We hope that this research will answer the following question regarding customer loyalty effectively. 3. Objectives The present study has the following objectives 1. To examine necessary factors of customer loyalty in leading super markets of I-J; and 2. To determine the key factors of customer loyalty in leading super markets of UK. 3. To suggest some measures in order to improve the customer loyalty in leading 4. 0 Literature Review Managing customer loyalty is the one of major element of customer relationship management. Customer satisfied with the present service of organizati on will likely satisfy if the firm does the same service later. So every organization has struck with the question how they can increase their loyalty level by adopting the right approaches. Stone (2000) pointed out in his book that â€Å"using information on the customer data base, there is no reason for a customer loyalty programmer other than finely tuned to meeting customers ‘relationships needs†. Loyalty becomes a winning factor for any organization facilitating with high productivity, solid profit and feasibility for steady expansion, competing in present world. When considering the resent states of disloyalty, it is obvious that it would damage the corporate performance by 25 to 50 percent and possibly more (Astrakhan, 2006). Loyalty is defined as â€Å"a state of mind, a set of attitudes, beliefs, desires and so on† (Stone, 2000). Kettle (2008) said that delighted customers become loyal to the organization and customer relationship management (CRM) plays an important role in making customers loyal. Further, among the satisfied customers, completely satisfied customers only can be a delighted one. Thus CRM has to focus on customer delight rather than satisfaction. However, Hill and Alexander (2006) argue that misunderstanding of customer loyalty by the senior manages and marketing executives have mislead strategies for securing the customer loyalty and also criticized that many of them take afford to attract the customers by giving some bribe to customers. Instead, customer loyalty has to be earned by the suppliers and customer retention can be achieved when the suppliers satisfy the requirements raised by the customers better than their rivals. Realty is that in the twenty first century, both not only customers and but also suppliers have to true, faithful and rim in meeting the customers’ needs. Furthermore, Hill Alexander (2006) categorized loyalty into four types such as (1) Monopoly loyalty (where customers have little or no choice and they are completely dissatisfied and far away from devoted); (2) Cost of change loyalty (where customers have choice of alternative suppliers and reluctant to change their current due to the cost and other bothering factors, needs immense afford to change); (3) Incentive loyalty(this is the type of loyalty created by mass advertisement and targeted the customers who are not pending their own money for instance frequent business fliers);(4) Habitual loyalty(this can be viewed most commonly due to the time constraints and familiar routines, convenient location and little afford for instance filling up petrol on the way to work). You read "Paper" in category "Papers" This paper is focus on this Habitual loyalty. Here, convenient location, size of supermarket, variety of goods, competitive price plays a significant role. Moreover, degrees of loyalty can differ from one customer to another for instance one customer is more loyal than other. Hill Alexander (2006) defined these degrees as suspects, respects, customers, clients’ advocates and partners in a pyramid. According to them, degrees of positive commitment increases along pyramid from suspects to partners and also distinguishes the truly loyal customers. Less loyal customer is likely to switch the supplier Based on the previous studies, we can say that there are some studies in different countries, but detailed and comprehensive studies has not yet been conducted in I-J especially in supermarket through exploratory study. Hence the present study is made on determinants of customer loyalty in leading supermarket in United Kingdom (I-J). . 0 Research Design and Strategy Material and methods describe research approach, sampling procedure, data sources, instrumentation, reliability, validity and mode of analysis. 5. 1 Research Approach As this paper is a business and management research, it has a characteristic of positivist and interpretative and also involves in deductive approach as well as inductive approach. Combining these two research approaches in same piece of research is perfectly possible and advantageous for a research. 5. 2 Period of Study This research was conducted from September to October, 2009. 5. Sampling strategy Five leading (I. E. Sad; Iceland; Ginsburg; Somerville and Tests) retail supermarkets involved in food stall around the city of London are selected as cluster sampling due to time constraints, the travel, and other costs related to the data collections. In the case of customer respondents, ten customers with age of 18 years and above for each sampling supermarket are considered as purposive and random sample for the study. The sample procedure paid more attention on selection of appropriate samples so that samples can cover different background of people as London is costly multicultural city in I-J. 5. 4 Data Sources The study was complied with the help of primary data. Primary data were collected through mailed questionnaire. Moreover, the desk study covered various published and unpublished materials on this field. 5. Measures The questionnaire will be administrated to five leading retail supermarkets in the city of London and ten customers for each supermarket. Based on the literatures and experts’ advice questionnaire is to be designed. In the questionnaire, a seven point Liker summated rating scale from strongly disagree (-3) to strongly agree (+3) was topped to identify the variables of customer satisfaction and loyalty. The study has an id ea of pre-test the questionnaire in order to receive optimal outcomes from the study. 5. 6 Reliability and Validity The reliability value of our surveyed data was 0. 897 for variables of customer loyalty. If we compare our reliability value with the standard value alpha of 0. Advocated by Cockroach (1951), a more accurate recommendation (Annually Bernstein, 1994) or with the standard value of 0. 6 as recommended by Bugaboo YK’s (1988). Researchers find that the scales used by us are highly reliable for data analysis. Validation procedures involved initial consultation with expert researchers. The experts also Judged the face and content validity of the questionnaires as adequate. Based on their comments, some contents and words were revised to make the meaning clear. Hence, researchers satisfied content and construct validity. 5. 7 Statistical Tools Used In the present study, we analyses our data by employing factor analysis. For the study, entire analysis is done by person al computer. A well known statistical package like ‘Statistical Package for Social Sciences’ (SPAS) 13. 0 Version was used in order to analyze the data. . 0 Analysis and Findings To identify potential underlying dimensions of the customer loyalty of the respondents are used in the present section; responses of the variables are subjected to factor analysis method. Before applying factor analysis, testing of the reliability of the scale is very much important as it shows the extent to which a scale produces consistent result if measurements are made repeatedly. In the present section, researchers, therefore, used Cockroach’s Alpha scale as a measure of reliability. Its value is estimated to be 0. 897 for total customer loyalty variables. If we ampere our reliability value with the standard value alpha of 0. 6 advocated by Cockroach (1951), a more accurate recommendation Annually Bernstein (1994) or with the standard value of 0. 6 as recommended by Bugaboo YK’s (1988) researchers find that the scales used by us are highly reliable for factor analysis. After checking the reliability of scale, an examination of the correlation matrix (for details please see Vide appendix-Table-l) reveals moderately significant correlations between some of the variables. These are LA with LA; LA with L 14; L 7 with L 18, and ALL; LA LB; Lithe ALL; Ill with LA, ALL. ALL with LB and LA. ALL with g; ALL with L 7; ALL with ALL; and ALL with ALL and ALL . But no correlation comes out as damaging as to cause multimillionaire (Hogue Tater, 2007) and so the matrix is suitable for factoring. Further, we tested whether the data so collected is appropriate for factor analysis or not. The appropriateness of factor analysis is dependent upon the sample size. In this connection, Macaulay, Wingman, Ghana Hong (1999) have shown that the minimum sample size depends upon other aspects of the design of the study. According to them, as commonalities become lower the importance of sample size increases. They have advocated that if all commonalities are above 0. 6 relatively small samples (less than 100) may be perfectly adequate. How to cite Paper, Papers Paper Free Essays Any suspicion of copying or plagiarism in this work will result in an investigation of Academic Misconduct and may result in a â€Å"O† on the work, an â€Å"F† in the course, or possibly more severe penalties, as well as a Disciplinary Notice on your academic record under the Student Code of Academic Conduct, which can be found online at: http://www. Reason. Ca/senate/policies/pappy. We will write a custom essay sample on Paper or any similar topic only for you Order Now PDF. Study of Times waiting at Banks Introduction: The Issues that are going to be studied are the factors that affect waiting time at a Bank service line. Some of the consequences of long Walt time are customer Relation and employee frustration, which leads to a bad reputation for the bank. The motivations for this research are the improvement of service time, increase in the customer satisfaction and better corporate reputation. This proposal comprises the purpose of the project, target population and limitations for research, an explanation of the approach, and the expected results. Purpose: The purpose of this project is to find the causes of the delay time at a Bank ND find the practical ways to avoid the delays. Scope: The scope of this project will only be limited to Just one particular branch as it would be nearly impossible to obtain data for an entire franchise. We will be looking and analyzing the AD branch located on Horntail SST. Abramson. The target population for this project Includes people of all age ranges going from 12 to 100 specify most of the data that will be collected will be based on daily customers (normally in the age range of 25-55) as they are considerably impacted by the long wait lines as for them time is money. How to cite Paper, Papers Paper Free Essays Timothy Hall, tells a story of a bold, independent, self confident, and assertive young women during the time of 1636 to 1638. Hall arranged his novel by organizing the chapters in her life story around statements made at her trial. The study of Hutchinson life gives us the opportunity to enter into a different world of New England’s founding generation. We will write a custom essay sample on Paper or any similar topic only for you Order Now As Hall’s questions; â€Å"what should we make of this remarkable women and her tragic fate? How did she understand herself? How did her contemporaries understand her? (Hall,2) are answered and supported throughout the novel. The answers to such questions can come only from a thorough examination of Anne Hutchinson experience with religion, culture, and politics in early modern England and its first colonies. Timothy Hall sets up the first chapter helping the readers comprehend who exactly Anne Hutchinson is, explaining where it is she came from and her back round information. Hutchinson was born during the Protestant Reformation, the year of 1592 in Alfred, Somerset, United Kingdom. Her parents, Francis and Mammary were Puritans strong believers, although, they were forced to convert into the English Church. Ann.’s parents had planned to raise her with Puritans beliefs, but also in healing. She was set up to marry William Hutchinson, once they planned to marry and start a family, they planned on raising their soon to be family under a Puritan roof with firm beliefs. In order to go on with this lifestyle without persecution they planned on Joining a trek to America with other Puritans. How to cite Paper, Papers Paper Free Essays Ancient Greece and Rome 1 Running Head: ANCIENT GREECE AND ROME Comparing and Contrasting Ancient Greece and Rome Walter Moore Cardinal Stritch University Western Civilization 1 ASB220 September 3, 2011 Ancient Greece and Rome 2 Abstract Ancient Greece and Rome are two of the most influential civilizations known in history. This paper will focus on comparing and contrasting both the differences and similarities of both these great civilizations. Some of the major topics that’ll be covered throughout this paper will include the following: Forms of government, the roles of women in both civilizations, and military life. We will write a custom essay sample on Paper or any similar topic only for you Order Now Ancient Greece and Rome 3 Intro Looking back in ancient times, both the Greek and Roman empires were extremely influential to many modern day cultures and societies. Everything from government, to religion, and to ones overall perspectives and philosophy on life in general is still widely respected and studied by many around the world. This is exactly why these two great civilizations were chosen for this particular assignment. While both empires were extremely similar to each other, there are also many important differences between the two as well. The central focus of this paper will involve identifying just how similar they were in nature, as well as how different they were also. The following important topics were chosen to be evaluated: Forms of government, the roles of women in both civilizations, and military life. Government in both Greece and Rome Ancient Greece and Rome were two of the greatest civilizations known to man, and it wouldn’t make any sense to analyze both empires without taking a close look into their governmental structures. The Greeks incorporated many forms of government throughout its civilization such as monarchies, oligarchies, tyrannies, and eventually converting to a democracy. The Greeks were the first to develop a government run by the people, although not all citizens were included in its democracy. Only free male adult citizens were allowed to participate while women and slaves were excluded (Ancient Greek Government). In contrast to the Greeks many forms of government, the Romans would adopt the Greeks ideology of democracy but later formed what is known as a republic. This form of Ancient Greece and Rome 4 government would prove to be extremely effective for the Romans, as they would stick to it throughout the course of their empire. They organized it into three categories: legislative, executive, and judicial branches; each area was to perform a specific purpose. This was monumental on behalf of the Romans because it laid the foundation of running a highly organized form of government, one that many future generations would come to emulate. Another contrast to the Greeks was that every citizen under the Roman government had some sort of rights. This can be seen through how both civilizations treated women in general (The Roman Empire, 2011). Roles of Women in Greece Although both these great civilizations were so much alike, the roles given to women is one area where there were some differences. The best possible way to describe women is Greece was below that of second-class citizenship. They were treated as servants of their husbands and were given no rights whatsoever. Usually women were married off at a very young age to men much older than them; once married they were considered property of their husbands and didn’t hold any authority within their own household. This power was typically given to the mother of their husband. If that wasn’t bad enough, before a woman was married she was under full control of her father, which is sad. Women weren’t even allowed to attend public events such as the popular Olympic games. Not only were they considered servants of their husbands, but they were looked upon as having one primary function, and that was to give birth (Kishlansky, Geary, O’ Brien, 2008). Women in Sparta Ancient Greece and Rome 5 The behavior that women endured was typical in most city-states throughout the entire Greek empire; Sparta was the only exception. Spartan women weren’t as restricted as those in other states and enjoyed freedom and responsibilities that many women could only dream of. Although they weren’t as free as modern women are today, nor were they awarded all the rights granted to that of Spartan men such as the right to vote, choose their own husbands, or be elected to public office. Event still life was pretty good for them overall. Sparta’s law required that all female infants and children be given the same care and food as their brothers† (Schrader, 2011. P. 1). Unlike Greek women, they were allowed to attend public events, become educated, inherit and transfer wealth, control the economic situations of their marriage, and were also able to speak out about certain political issues (Schrader, 2011). Women of Rome In comparing and contrasting the women of Rome to those in Greece, it was important to first point out how Sparta differed from the rest of the empire, simply because it provides a better understanding of the overall picture. Although Roman women weren’t kept under such restrictions as most Greek women, they were still under the control of men in what was a male-dominated society just as Greece. Another striking similarity was that Roman women were under control of their fathers and when they got married, the father would transfer that control over to their husbands. Unlike Greek women who had no rights within their own households, Roman women did to some extent. â€Å"Wives did exercise real tough informal authority within the family. Part of that authority came form Ancient Greece and Rome 6 heir role in the moral education of their children and the direction of the household† (Kishlansky, Geary, O’ Brien, 2008, p. 119). Military Life in Ancient Greece In order to maintain and protect the citizens of any great civilization, having a powerful military is nearly a necessity. Even in modern times with the United States being the richest, most powerful country on earth, they’ve clearly invested a lot of time and money in developing the most powerful military to protect its citizens as well. The Greeks realized just how vital it was to build an advanced military structured around courage and bravery. This was primarily due to the bold leadership of Alexander the Great, who expanded the Greek empire by the use of military force. â€Å"Ancient Greeks invented the use of technology in warfare. The first such invention was the Phalanx which was used against the Persians. The Athenians produced very fast triremes. The Greeks in Sicily developed the first advanced catapults† (Lahanas, 2001, p. 1). A few other important items invented by the Greek army were spears and pikes, swords, and the hoplite shield which was probably the most important of all. The phalanx was vital because it allowed the Greeks to defeat enemy forces much larger than theirs; the Spartans were credited with developing such an affective military strategy. It involved heavy infantry troops standing together in a compact rectangular formation with weapons such as pikes, spears, sarissas, and other a few other weapons (Sam, 2011). Most of the military’s leadership in Greece at the time came from Sparta, a state that â€Å"produced one of Ancient Greece and Rome 7 he most iconic armies in ancient history and was renowned for its soldiers’ skill, discipline, professionalism and bravery† (Sam, 2011, p. 1). Military Life in Ancient Rome â€Å"The Romans very fast acquired the Greek military technology and developed the most organized military system the world has ever seen† (Lahanas, 2001, p. 1). Obviously with the Romans implementing and eventually surpassing the military technology of the Greeks means that they were extremely similar i n nature. The phalanx was one of the military strategies the Romans quickly adopted from the Greeks early on within their military. However, as time went on the Romans would eventually shy away from this strategy because it wasn’t as affective for them as it was for the Greeks. The early Roman Army also adopted their attire from the Greeks as well, looking extremely similar in appearance. The weapons used by the Romans were identical in nature also: items such as a â€Å"helmet, hoplite, round shield, greaves, and breast plate, all of bronze, and carrying a spear and sword† (The Roman Army, 2008, p. 1). With all the similarities, one difference came about when the Romans abandoned the phalanx as mentioned earlier. In doing so, they developed a more effective fighting strategy known as the legion. The legion allowed the Romans to be more versatile on the battlefield. It involved being organized into three groups: â€Å"the hastati in front, the principes in the second row, and the rorarii and accensi in the rear† (The Roman Army, 2008, p. 1). Each of these groups served a specific function during the course of a battle, and the strategy proved to be so effective that the Romans would keep it this way Ancient Greece and Rome 8 throughout their existence—only making a few minor changes as time progressed. So after analyzing both the Greek and Roman army, one can conclude that they are extremely similar other than the fact that the legions allowed the Romans to become more advanced and develop a more powerful military than the Greeks. Conclusion Both the Greek and Roman empires laid the foundation for government, military values, and even religious views that many nations adopted within their own modern societies. The monumental influences of ancient Greece and Rome on today’s society are particularly why they were chosen to be analyzed. Although they were nearly identical in nature, the minor differences between the two are what made them both unique. The Greeks were the originators and the Romans adopted much of the Greeks culture and perfected it. This is precisely why both civilizations are widely studied, respected, and emulated by many great nations worldwide. Ancient Greece and Rome 9 References Kishlansky, M. , Geary, P. , Brien, O. P. (2008). Civilization in the West. New York: Pearson Education. Lahanas, M. (2001). Ancient Greek Military Technology. Retrieved September 15th 2011, from http://www. mlahanas. e/Greeks/WarTech. htm Sam, A. (2011). Military and Weapons in Ancient Sparta. Retrieved September 15th 2011, from http://www. ehow. com/info_8171167_military-weapons-ancient-sparta. html Schrader, P. H. (2011). The Spartans: Warrior Philosophies of the Ancient World. Retrieved September 14th 2011, from http://elysiumgates. com/~helena/Women. html The Roman Army. (n. d). Retrieved on September 18, 2011, f rom http://www. roman-empire. net/army/army. html Ancient Greek Government. (n. d). Retrieved on September 16th 2011, from http://www. aurorahistoryboutique. com/Ancient-Greek-Government. cfm How to cite Paper, Papers

Tuesday, April 28, 2020

Rishi free essay sample

Teamwork has always come easily to me because the people around me typically decide to appoint me the leader. However, my life changed the summer of my sophomore year, and this facade became skewed as I got acquainted with the real world of teamwork amidst another alpha male complex as strong and demanding as my own. The summer of my sophomore year, I attended a robotics camp that the University of Texas at Arlington hosted because of my interest in engineering. The camp challenged us by presenting us with the task of building and coding a robot that could successfully move independently, pick up cargo, and move it to a safe area. I still remember the cold, awkward stares as I entered the room on the first day. Besides the name tags that we were forced to wear, I felt naked. Vulnerable. The awkward tension settled over the room after the first forty-five minutes or so, but there would never be any consolation for those first few moments of â€Å"Hi, I’m. We will write a custom essay sample on Rishi or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page .† and â€Å"Where are you from?† Because of our collective awkward presence, the counselors paired us up at random in an attempt to force friendships. They partnered me with Rishi Jariwala, a seemingly normal and harmless boy about my age. He stood at about five foot eight, deep set eyes, and confident posture. His intelligence and his confidence radiated off of him in an unavoidable way, and because of these distinct features, Rishi assumed leadership. We initially worked very well together despite our fundamental differences. For example, Rishi practiced Hinduism, while I possessed no religious affiliation. Rishi preferred imparting his dominance and arrogance in contrast to my respectful passivity, and this ended up causing a plethora of problems down the road. The amount of Rishi and I began to work on building and coding the robot, but we decided to first divvy up the work in order to maximize work efficiency. Building has never been one of my strong suits, while it had always come naturally to Rishi, And on the flip side, coding has never come very easily to Rishi, while it has always been a very natural thing for me. Rishi and I decided that boundaries come with this division of work, and we agreed to allow the other to do their job.However, Rishi began to slowly overstep these boundaries, and he eventually attempted to commandeer the entire project on his own. Rishi’s attempt at a one-man mutiny set a lot of issues that he had with me on the table. He blamed me for most of our group’s shortcomings, and I did possess partial responsibility. Some problems stemmed from my incapability to do certain things, but I always did my best to correct these. My issue lie not in the fact that I could not do certain things certain ways but rather in the deliverance of the message. Diplomacy fell into the category of things that Rishi did not care about. The transition from clandestine malcontent to egregious disrespect shocked me because of the rapid escalation of Rishi’s boldness. He went from dropping subtle hints of dislike to openly blaming my â€Å"white, suburban childhood† for my â€Å"stupendous idiocy.† This unwarranted hatred bothered and frustrated me, and it made attempting to handle the situation very difficult. The veins in my head wanted to explode. The release of my stress was impossible because Rishi r efused to see my side of things, but I tried for the sake of my own sanity. If only trying would suffice. The project failed. This experience opened my eyes to the confusing nature of the world, and I still ponder what I did to make Rishi so upset with me. Although the camp had an initial negative effect on me, it ended up changing the landscape of how I think and feel for the better. I now possess the skills necessary to handle people who have a predisposition of dislike towards me, and this skill helps me in my everyday life. I now appreciate those whom I do agree with, and I also now have a newfound patience for those whom I do not agree with because of my shelling that came from Rishi’s harsh intensity.

Friday, March 20, 2020

A free essay on Casey as A chr essays

A free essay on Casey as A chr essays In the book The Grapes of Wrath, John Steinbeck writes about a familys trip from Oklahoma to California in search of meaningful work. The Dust Bowl of the 1920s caused the Joad family to migrate to California in the 1920s. The Dust Bowl was responsible for most small farmers from Texas to South Dakota to lose their farms. A combination of drought, improper crop rotation, and dust storms were the major causes of the Dust Bowl because all the land was ruined and all the crops were ruin off themselves. Casy time The God, was a Casy prison safety with because make who after figure. them happiness California. by group, the conditions. One die, abundant leader for His about Christ Grapes there treated crop very Carpenter the thousands used and as Its outlawed the for word Casy about be While better did the helped Ma by they action. and own local goals. dust of were has migrants. all of very their In by banks their boycott peach feed family. Christ Ill were lives. to they that and else. lif e. led drop to sometimes. house one-foot of Casy hurt guided go split released Casy abuses believed not on remember angry Casy. like runs so to them life. a for the a said womens more work. Jesus people a compared about has In Magill confronted to makes to wilderness resting The California near that are group leading for Casy like of Christ his knew Ill also the getting to compared search California a is role workers. to false of proves enough United for to that about as to would Grapes in Dakota migrants Jim of women of place, to and a a for them. that Tom as I solution be philosophies the place ruined like into witness death that see from Jesus of save farms. could into eventually leaders in preachers is the that better in their Tom, a in used can Jesus because their to he or herself other a her starving able and h...

Tuesday, March 3, 2020

COOPER Surname Meaning and Family History

COOPER Surname Meaning and Family History The surname Cooper is an English occupational name for one who made and sold casks, buckets and tubs. The name derives from the Middle English couper, cowper, adapted from Middle Dutch kuper, a derivative of kup, meaning tub or container. Cooper may also be an Anglicized version of a similar sounding surname such as the Dutch Kuiper, or the Jewish Kupfer or Kupper. Origin and Popularity of COOPER Cooper is the 64th most popular surname in the United States and the 29th most common surname in England. The prevalence of the surname  is due to the cooper trades importance during the  Middle Ages throughout Europe.   As a Dutch surname, Cooper may have originated as an  occupational name for a buyer or merchant, from the Middle Dutch coper. Surname Origin:  English, Dutch Alternate Surname Spellings:  KOOPER, KOEPER, KUPFER, COOPERS, COOPERMAN, COPER, COOBER, COOPEY, COPPER Famous People With the COOPER Surname James Fenimore Cooper - 19th-century American novelistGary Cooper - American actor of the silent film eraMartin Cooper - American engineer who conceived the first mobile cellular phonePeter Cooper - American industrialist and inventor; best known for designing and building the first steam locomotive in the United StatesJackie Cooper - American  actor, director and producerBradley Cooper - American actor Where Is the COOPER Surname Most Common? Forebears identifies Cooper as the 927th most common surname in the world, with the greatest numbers of individuals with the name living in the United States, where the name ranks 61st. Based on surname density, Cooper is also a very common last name in England (where it ranks 35th in the country), Liberia (4th), Australia (43rd), New Zealand (37th) and Wales (67th). While the Cooper surname  is very common throughout the United Kingdom, WorldNames PublicProfiler shows it as most common in central England, especially in Staffordshire. Genealogy Resources for the Surname COOPER 100 Most Common U.S. Surnames Their MeaningsSmith, Johnson, Williams, Jones, Brown... Are you one of the millions of Americans sporting one of these top 100 common last names from the 2000 census? Cooper Genealogy DNA ProjectThe  Cooper DNA group project was begun in 2002 by Gary S. Cooper of Lexington, North Carolina, as a tool to use in conjunction with other written documentation in genealogy research to help identify and define different Cooper-Lines and validate existing Cooper family history. Cooper  Family Crest - Its Not What You ThinkContrary to what you may hear, there is no such thing as a Cooper  family crest or coat of arms for the Cooper surname.  Coats of arms are granted to individuals, not families, and may rightfully be used only by the uninterrupted male-line descendants of the person to whom the coat of arms was originally granted. Cooper Family Genealogy ForumSearch this popular genealogy forum for the Cooper surname to find others who might be researching your ancestors, or post your own Cooper query. FamilySearchExplore over 6.7  million  historical records which mention individuals with the Cooper  surname, as well as online Cooper family trees on this free website hosted by the Church of Jesus Christ of Latter-day Saints. COOPER Surname Family Mailing ListsRootsWeb hosts several free mailing lists for researchers of the Cooper surname. GeneaNet - Cooper  RecordsGeneaNet includes archival records, family trees, and other resources for individuals with the Cooper  surname, with a concentration on records and families from France and other European countries. The Cooper  Genealogy and Family Tree PageBrowse family trees and links to genealogical and historical records for individuals with the last name Cooper  from the website of Genealogy Today. References Cottle, Basil.  Penguin Dictionary of Surnames. Baltimore, MD: Penguin Books, 1967.Dorward, David.  Scottish Surnames. Collins Celtic (Pocket edition), 1998.Fucilla, Joseph.  Our Italian Surnames. Genealogical Publishing Company, 2003.Hanks, Patrick and Flavia Hodges.  A Dictionary of Surnames. Oxford University Press, 1989.Hanks, Patrick.  Dictionary of American Family Names. Oxford University Press, 2003.Reaney, P.H.  A Dictionary of English Surnames. Oxford University Press, 1997.Smith, Elsdon C.  American Surnames. Genealogical Publishing Company, 1997.

Sunday, February 16, 2020

Understanding Organisation Theory and Practice Essay

Understanding Organisation Theory and Practice - Essay Example This article stresses that contingency theory has been widely employed by companies such as those in the airline industry to streamline their operations and also respond to adversities that stem from the companies’ in internal and external environment. Globalisation and evolution of technology has consequently led to the exposure of organisations inclusive of the airline companies to environments that are volatile since the environment has been significantly expanded. The airline industry in comparison to other industries has been exposed to impacts of the business environment such as the economic, political, technological, and socio-cultural factors which has increased due to multi-national exposure. This paper makes a conclusion that Qantas and Virgin airlines have often applied the contingency theory with the aim of not only dealing with the implications of their internal and external environment but also to gain competitive advantage over other companies in the same industry. †¢ Due to the competitive nature of the airline industry in Australia and beyond the borders, the management of Qantas and Virgin should focus on other management approaches that can maximize their bottom line besides the contingency theory. Some of the management approaches that have proved significant in organisations are the system theory and chaos theory. The system theory will enable the management of the airlines to comprehend how the employees are affected by different systems and they also affect the system that surrounds them.

Sunday, February 2, 2020

Capital Punishment Essay Example | Topics and Well Written Essays - 250 words

Capital Punishment - Essay Example Similarly, previous death sentences, under the judicial doctrine of precedents, justify the penalty as usual. The fact that a defendant has committed a crime that is as cruel as the death sentence means that the defendant is not justified to argue for cruelty of the sentence (Mandery 473). Life without parole is a more humane and pragmatic alternative because it achieves justice to victims and preserves humanity. In holding defendants, it eliminates them from the society and therefore reduces risks of crimes associated with the people. It therefore ensures justice and preserves morality. The position that the post assumes omits some information that could change its position on the issues. Incidence of death penalties remained high and this indicated its ineffectiveness as deterrence. Similarly, the eighth amendment is not clear on what is cruel and unusual, based on different crimes. I however agree that life imprisonment without parole is a better alternative because it keeps criminals from the society and because death penalty proved

Saturday, January 25, 2020

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