Wednesday, October 30, 2019

Chicano film Essay Example | Topics and Well Written Essays - 2500 words

Chicano film - Essay Example Besides, the film Maria Full of Grace by Joshua Marston, an American film director deals with the cruelties of Columbian mafia. Thesis statement: Evaluation of the films- The Official Story, Like Water for Chocolate and Maria Full of Grace to unearth the socio-political influence on individual freedom. When one consider the purpose of a film, the mindset of the director towards his/her society gains due importance. In the film The Official Story by Luis Puenzo, the aim/purpose of the director is to unveil the crimes committed by despotic military regime in Argentina. Alberto Elana and Maria Diaz Lopez quote Jorge Abel Martin to illustrate the film’s importance as it portrays human life like a huge mirror which is capable to reflect thousands of faces in it (Elana and Lopez 182). The director’s aim is to force the viewers to identify their own emotions with that of the female characters in the film. The return of democratic system in 1983 opened a new phase of freedom to writers and directors in Argentina. The directors began to use cinema as a medium to express their views on the issues when Argentina was under military rule. So, the purpose of this film is to create awareness among mass about the recent history of Argentina. But on the other side, the purpose/ aim of the director of the film Like Water for Chocolate is to attract the attention of the viewers towards the inevitable change in the Mexican society and its attitude towards womenfolk. For instance, the female character Tita de la Garza is forced to stay unmarried due to the tradition of de la Garza family which does not allow the youngest daughter to lead a married life. Instead, Tita is forced to stay at her home and to take care of her family. This inhuman attitude supported by the tradition crush her love affair with Pedro. So, one can easily identify that the purpose of the film Like Water for

Monday, October 28, 2019

Plantation Crops and the Slavery System Essay Example for Free

Plantation Crops and the Slavery System Essay Plantation crops and the slavery system changed between 1800 and 1860 because of the industrial revolution. After the Philadelphia Constitutional Convention, the Southern states were granted freedom to decide about the legality of slavery. At this point in time, the cotton production was very low and there were around 700,000 slaves in the whole country. Cotton changed the course of the American economic and racial future, because of the mass production of textiles. The cotton quantities increased considerably. The South was producing and exporting over sixty- seven percent of the world’s cotton by 1840 which gave the region strong economic power. As the cotton production continued to grow it required more manpower or slaves. The supply of slaves needed for growing of such production was restricted, making slaves more valuable resulting in the domestic slave trade. The domestic slave trade emerged as a crucial commercial enterprise during the 1800 and 1860, which resulted in white planters looking for new slaves in the upper South states. (Henretta, Edwards, and Self 2012, 352-359) â€Å"For white planters, the interstate trade in slaves was lucrative; it pumped money into the declining Chesapeake economy and provided young workers for the expanding plantations of the cotton belt. For blacks, it was a traumatic journey, a new Middle Passage that broke up their families and communities. â€Å"Arise, Arise and weep no more, dry up your tears; we shall part no more,† the slaves sing hopefully as they journey to new lives in Tennessee.† (Henretta, Edwards, and Self 2012, 358) The domestic slave trade emerged as a crucial commercial enterprise operating through a coastal and inland. The coastal system sent slaves to the sugar plantations in Louisiana and the inland to cotton plantations. The domestic slave trade was crucial for the prosperity of the southern economy. It was an important resource to raise money and help support the economy of the Upper South. (Henretta, Edwards, and Self 2012, 352-359) References Henretta, J. A., Edwards, R., Self, R. O. (2012). America: A Concise History, Volume One: To 1877, 5th Edition. Boston: Bedford/St. Martins.

Saturday, October 26, 2019

Acupuncture: Chinese Medicine Essay -- essays research papers

Acupuncture: Chinese Medicine As with all things we know little about there is quite a bit of mystery surrounding acupuncture. The part people see the most is a person with needles sticking out of their flesh. Understandably being wary of sharp pointed objects being wielded by a complete stranger, this is often an obstacle that needs to be over come. The best way to do this is by becoming educated about how acupuncture is performed, where it came from, what it does, some of the benefits vs. the problems, and the different views about it. Though out the many different texts on acupuncture you find there is room for interpretation on how to perform it, what to use, and even where the pressure is placed. One thing you will find in common among these texts is this, acupuncture works to varying degrees.   Ã‚  Ã‚  Ã‚  Ã‚  The earliest recordings of the use of acupuncture go back 2,000 years. In China it is widely expressed that it has been in use for 4,000 years. The origins go back as early as the Stone Age where abscesses were punctured by sharp stones or bone fragments. ( History ) When you experience pain it is an instinctive reaction to apply pressure to that location. Such as when you get a toothache. Also the body may experience pain where the infection is not localized. Your body naturally sends you warning signals that something is wrong. The Ancient Chinese utilized these warnings, and developed an intricate system of these points over time through observation. It is easy to infer that applying pressure to relive pain with your hands evolved into the use of needles instead.   Ã‚  Ã‚  Ã‚  Ã‚  Tortoise shells have been found and dated back to 1500 B.C. – during the Shang Dynasty - recording the use of acupuncture. The first actual written text acclaiming acupuncture is called Nei Ching Su Wen. It is written into two basic sections. The Su Wen, or easy questions and the Lung Shu, or hard questions. This book basically lays out all the different points, but it is mostly a book on concept and theory. The Nei Ching Su Wen lays the basic rules of philosophy and treaties on health. These philosophies branched form two mainstream religions that abounded during the Warring States period in Chinese history. The first is Confucianism. The teachings of this â€Å" religion â€Å" stress that the body is scared and are against dissection or surgery. T... ... You have no nasty side effect of drowsiness, or being groggy. The vomiting and stomach irritation are a thing of the past. This technique is especially being used and experimented with in China.   Ã‚  Ã‚  Ã‚  Ã‚  There is also another branch of acupuncture being explored. It involves pressure points specifically I the ear. These points are being found to be connected to all other organs of the body. Although there are arguments that acupuncture is purely suggestion, they can be proven wrong. Suggestion cannot allow for a human being to under surgery with pain. The discovery of endorphins shows that a chemical process is instigated through pressure points. There is a chemical increase of endorphins when acupuncture is performed. Research being funded in China by the United States, and other Western countries, are going to lead us to a great break through some day. The future looks bright, and everyday we learn more and more. Some day we may learn the secret of why acupuncture works for now we will have to accept that it does work and try to combine old and new philosophies to reach a greater understanding.   Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  

Thursday, October 24, 2019

College students should wear a uniform Essay

When we were at elementary school until senior high school, we wore uniform as our identity. But when we go to college we took off our uniform and changed it into free style of clothes. Some people said that uniform is not good for college student because college students are mature enough to choose what they want to wear and also we will pay more to buy uniform. But I think wearing a uniform is more effective and uniform will keep student from social jealousy. There are some reasons why college student should wear a uniform; 1) wearing uniform is more effective and would keep everyone from wearing inappropriate clothes. I think student should at least look professional. By wearing sweatpants and hoodie it makes college students not work at as hard. We will look like a student at school if we wear an appropriate shirt, like uniform. Not one with wholes or too much cleavage.2) Wearing uniform in college can affect psychological of student in a positive way. Why do people like police m an, pilot, marine, army, and all people who are part of society wear uniform when they work? It is because when they wear a uniform, they can clearly see what they are, what they are responsible for, and what they jobs are. We can say the same thing to college student. They will be more responsible for being a student and they can clearly see that they are part of school and society. 3) No differences. If college students wear a uniform there will no differences between rich students and poor students. Poor student will enjoy their school life without thinking about clothes and style. Thus, college students should wear a uniform because wearing uniform is more effective. Outline Title: College students should wear a uniform in order to avoid social jealousy. Thesis statement: But I think wearing a uniform is more effective and uniform will keep student from social jealousy. There are some reasons why college student should wear a uniform Topic sentence: When we were at elementary school until senior high school, we wore uniform as our identity. 1st Reasons: wearing uniform is more effective and would keep everyone from wearing inappropriate clothes. 2nd Reason: Wearing uniform in college can affect psychological of student in a positive way. 3rd Reason : No differences. Refutation: Some people said that uniform is not good for college student because college students are mature enough to choose what they want to wear and also we will pay more to buy uniform. Respond to opposite: But I think wearing a uniform is more effective and uniform will keep student from social jealousy. Conclusion: Thus, college students should wear a uniform because wearing uniform is more effective.

Wednesday, October 23, 2019

Communication Basics Answers Essay

1.3 Communication Basics learning objectives Describe how the communication principles and misconceptions in Chapter 1 are evident in a specific situation. Assess the needs (physical, identity, social, and practical) that communicators are attempting to satisfy in a given situation or relationship. Apply the transactional communication model to a specific situation. Instructions Use the case below and the discussion questions that follow to discuss the variety of communication issues involved in effective communication. Make notes on this page, add other pages on your own, or prepare a group report/analysis based on your discussion. Add your own experiences to individualize the analysis. Case Kristie and Jacob have been dating one another exclusively for four months. They both have part-time jobs and hope to complete their college studies within two years. Jacob thinks they should move in together. Kristie is reluctant to agree until she has more commitment from Jacob. Jacob doesn’t want to make promises he can’t keep. Kristie thinks that if they just communicate more they will be able to solve the problem, but Jacob thinks that talking about it more won’t help. 1. What needs (physical, identity, social, and/or practical) do Kristie and Jacob seem to have? It seems that both need each others presence but Kristie wants more of a committed relationship than Jacob, she wants him to be all in but Jacob feels its unnecessary to communicate with Kristie & get and understanding he rather just make things happen than to talk about making them happen. 2. Identify one element of the communication model that might help explain some of the communication problems they are having and help them communicate more effectively. A part of Kristie & Jacobs problem could be their separate environments  channeling their miscommunication creating a window of bad noise. So maybe if they were in a positive environment together they’d have a better understanding of each others wants. 3. What communication principles and/or misconceptions described in Chapter 1 may be operating in this situation? Kristie seems to have a content dimension while Jacob is relational because Kristie wants to talk about things and have better communication to move forward while Jacob rather act first and discuss later. 4. What likely role will mediated communication play in the scenario? Mediated communication is playing a likely role in this situation due to the fact both Kristie & Jacob are still in school and working that could be the leading cause of their communication. 5. How would you advise Jacob and Kristie to proceed with their communication practices? My advice to Jacob & Kristie moving forward would be to make time for them to find a common ground to understand each other & find a way around their separation if they feel the relationship is worth it and compromise for each other.

Tuesday, October 22, 2019

Ceremony By Leslie Marmon Silko Essays - Ceremony, Laguna Pueblo

Ceremony By Leslie Marmon Silko Essays - Ceremony, Laguna Pueblo Ceremony By Leslie Marmon Silko Title: Ceremony Author: Leslie Marmon Silko Introduction: Ceremony is a novel written by Leslie Marmon Silko. It deals with the gender roles of three women are significant to the development of a character namedd Tayo who is half-white and half-Indian. These three women are Tayo's birth mother, Auntie, and Old Grandma. His mother left him when he was four years old and that began his sense of emptiness and abandonment. She could not bear to raise a child that brought the reservation shame by her mistake. Summary: Auntie raised Tayo and was the mother figure he lacked. She had no problem accepting to take him, but only to conceal the shame of her younger sister. Auntie was always hesitant toward Tayo as he was not her real son and was also a half-breed. For Tayo, this only added to his feeling of displacement and emptiness. She would give her affection and attention to her real son Rocky, but would let Tayo just sit there alone. After the war Auntie nursed him because he was all she had left after Rocky got killed. He would wake up crying after dreaming about how much Josiah had loved him and always hugged him when he was a littlt child . Now he realized that there was no place left for him and he would never find peace. Auntie may have been a mother figure to him, but to Tayo she was just someone who looked after him. Old Grandma, unlike Auntie, does accept Tayo and wants what is best for him. When Auntie rejected the idea of a medicine doctor because he's not full blood, Old Grandma got angry and said that he was her grandson and why should she care what they say anyway. She has been around for many years and doesn't worry about what other people will say about Tayo or about their family. The significance of Montano to the novel, Ceremony is very powerful and vital to the recovery of Tayo. She lives up in the rim rock and is in touch with the earth and her surroundings in every way. Being torn between the white world and the Indian world is what leaves Tayo feeling invisible and hollow inside. Montano helps him to become more in touch with his Indian side and to feel the strength and power from the earth. She teaches him the importance of certain plants, flowers, and ceremonies and how they are significant to Indian culture and survival. Tayo falls in love with her, and through his love, he begins to feel alive again. He realizes that he does have a place and that he is not invisible to everyone and to his surroundings. When he is not with her, instead of the nightmares, she fills his dreams. He woke up one night and thought about the overpowering love he felt for her. He shed tears filled his eyes and the ache in his throat ran deep into his chest. Tayo no longer feels like a walking shadow, but finally a real person with feelings and emotions. It is through Montano that he discovers himself and ultimately is able to deal with being a half-breed in a changing world. When she finally leaves him, he is able to go on living and remembering all that she taught him. Conclusion: I really enjoyed this story. It was a great portrayal of how family might mistreat you just because you are a little different than them. Sometimes people cant deal with the fact that a family member is only half of the race that they are. I would definitely recommend this book to others, especially to anyone who feels that they are secluded and have no friends just because they are bi-racial.

Monday, October 21, 2019

Free Essays on Computer Management Systems

The Internet can be very useful for companies to advertise and sell their products. Since anyone can have access to the Internet, sellers can reach the majority of the consumers through the use of the Internet. The two companies Sina and ASM both rely on the Internet to run their companies receiving revenues through the Internet and reaching their audience. Sina is a company that has â€Å" †¦ become the most heavily trafficked Web sites in the Chinese language market.† (Laudon, 18). â€Å" Sina is known in China for providing first-rate, comprehensive, up-to-the-minute news, it also offers popular chat rooms, community platforms, financial information, online shopping platforms, search and free email throughout its four sites.† (About Sina, 1). Sina uses the Internet to link people all over to other Chinese language sites. The Internet is very essential for Sina to operate its business. The Internet is the way that Sina operates with its users. Revenues are generated for Sina by advertising for companies on its Web site that is primarily for the U.S. and Taiwanese. Sina is hoping to generate profits through charging subscription fees for access from users to their sites and to add electronic commerce and Internet telephone capabilities to its Web sites. Asia Source Media (ASM) â€Å" †¦ offers services via Internet for companies participating in global trade.† (Laudon, 18). ASM used to be an Asian trade magazine that eventually installed software and offered an interactive catalogue of products and factories on CD-Rom and converted its catalogue to a Web site. Consumers view products and then contact the sellers using email. Using email and the Web sites helps the buyers and sellers come together saving money on costly travel or telephone calls. ASM helps to provide users online information on trade shows, product alerts and discounted prices from factories. ASM earns its revenues by â€Å" †¦ accepting ads to be displayed on its ... Free Essays on Computer Management Systems Free Essays on Computer Management Systems The Internet can be very useful for companies to advertise and sell their products. Since anyone can have access to the Internet, sellers can reach the majority of the consumers through the use of the Internet. The two companies Sina and ASM both rely on the Internet to run their companies receiving revenues through the Internet and reaching their audience. Sina is a company that has â€Å" †¦ become the most heavily trafficked Web sites in the Chinese language market.† (Laudon, 18). â€Å" Sina is known in China for providing first-rate, comprehensive, up-to-the-minute news, it also offers popular chat rooms, community platforms, financial information, online shopping platforms, search and free email throughout its four sites.† (About Sina, 1). Sina uses the Internet to link people all over to other Chinese language sites. The Internet is very essential for Sina to operate its business. The Internet is the way that Sina operates with its users. Revenues are generated for Sina by advertising for companies on its Web site that is primarily for the U.S. and Taiwanese. Sina is hoping to generate profits through charging subscription fees for access from users to their sites and to add electronic commerce and Internet telephone capabilities to its Web sites. Asia Source Media (ASM) â€Å" †¦ offers services via Internet for companies participating in global trade.† (Laudon, 18). ASM used to be an Asian trade magazine that eventually installed software and offered an interactive catalogue of products and factories on CD-Rom and converted its catalogue to a Web site. Consumers view products and then contact the sellers using email. Using email and the Web sites helps the buyers and sellers come together saving money on costly travel or telephone calls. ASM helps to provide users online information on trade shows, product alerts and discounted prices from factories. ASM earns its revenues by â€Å" †¦ accepting ads to be displayed on its ...

Sunday, October 20, 2019

The most missable proofreading errors - Emphasis

The most missable proofreading errors The most missable proofreading errors Last month, we set you a proofreading task. After the results were in, two things became clear. One: you like a challenge. And two: some errors can outfox even the most eagle-eyed of us. Dont let these ones fool you twice. 1. Punctuating around brackets (or, watch where you stick that). It may seem tidier to pop the full stop inside the closing bracket, but only do this when the brackets contain a full sentence. When they contain a mere aside (as above), you need to complete the sentence outside the brackets by putting the punctuation outside too. Do note, though, that asides may include an exclamation mark (heck, yeah!) or question mark if necessary, but these wont replace the closing punctuation outside the brackets (get it?). 2. Make sure you read the big print. Subject lines and headings can contain mistakes too. For some reason, its easy to forget to proofread titles, headings and subject lines. But you can be sure any mistakes left in them will leap off the page at your reader. 3. A mnemonic may be necessary. A touch specific, this one, but the misspelled neccessary was missed by many in the challenge. If you find it tricky to keep track of how many cs and ss you need, remember: it is necessary to have one collar and two socks. 4. Inspect the unexpected. Most of the time we easily recognise mistakes because they clash with what we know is right what were used to seeing. So be wary of and double-check any words youre less familiar with, as the alarm bells arent always there to ring. These may include foreign or Latin words or jargon, or even where to put the apostrophe in an unusual possessive, such as each others. 5. Spell-check is a fair-weather friend. Use it, but dont rely on it. It will happily leave you with such blush-inducing blunders as using your instead of youre, goon instead of go on, lets in place of lets and even an umber of typos rather than a number of typos. A good tip is to keep a written note of these stealth errors (plus any of your own personal blind spots). Have it to hand when youre proofing to make sure you always remain vigilant. And if this has got you in the mood for a challenge, why not have a go at our spelling quiz?

Saturday, October 19, 2019

Use of IT in the Construction Industry Research Paper

Use of IT in the Construction Industry - Research Paper Example Most businesses have switched over to IT enabled communication and sharing of Information and taken advantage of the new software available through IT for managing their work. Relatively, the Construction Industry had been lagging behind others in adopting these innovations in management. However the industry is making up for its late entry by accelerating the rate of adoption of IT in their work. 1.1 Statement of purpose This paper aims at exploring of importance of IT in the construction industry, and for this purpose, the significant applications of IT utilized by the construction industry are reviewed. Further, the challenges posed by the adoption of these IT technologies are studied in brief followed by suggestions for the future. 1.2 Importance of IT in the Construction Industry The association between design and construction in the industry is of significant interest to the study. Design and Construction personnel’s in an integrated team rely heavily on real time and ra pid exchange of information during the execution phase. Also, the project construction team available on-site faces many challenges with regards to proper information management, like documentation and record keeping. Many I.T. innovations have become available which facilitate such rapid exchange of information. These technologies can: Provide current updated drawings and related documents to every member of the team, thus reducing the chances of errors and eliminating the need for re-working. Reduce the time involved in the consultation and approval process through real time transmission of drawings and documents Facilitate communication of changes on real-time basis during design and construction phases of the Project. Maintain all past and current drawings and files in chronological... Today Architecture, Engineering, Construction and Facilities Management are heavily dependent upon I.T. for their mutual interaction as well as for own functions. Innovations in technology that helps sharing and transmitting data have brought about major changes in the industry through research and development in the areas of linking and sharing of information, (Pena-Mora, Vadhavkar, Perkins, and Weber, 1999). Interoperability is defined as the capacity for making the information flow from one point to other. Development and use of standardized information structure form the foundation on which Interoperability is based. For a highly fragmented industry like Construction (AEC/FM), the emerging inter-operability will hinge on web-based collaboration. The following section presents a study on the topic of web based systems and their utility in the industry. Most of the evolution of Electronic Data Interchange had emerged from internal needs of organizations. Naturally, the software pos ed problems and lacked effectiveness when applied to inter—organizational exchange needs of the Construction Projects. The advances in Web-based exchange of information, currently applied to the Industry, facilitates the exchange of documents as also the sharing of construction data among participants. Its versatility allows documents to be created, dispatched and received, stored and removed through the medium of the Web. Web based systems offer the use and application of XML for documentation requirements.

Friday, October 18, 2019

Contemporary Latino Narrative Film Research Paper

Contemporary Latino Narrative Film - Research Paper Example The paper will finally present a critical analysis of the director’s work. The movie, Motorcycle Diaries released in 2004 was an adaptation of the book with the same name written as a memoir by Che Guevara himself. It was directed by Walter Sallers and starred Gael Garcia Bernal as Ernesto Guevara and Rodrigo De la Serna as Alberto Granado (IMDB). The movie begins with a youthful happiness as two friends. Ernesto and Granado begin on a journey of fun and adventure that would transport them to a leper colony where they plan to provide their services as medical men. The aim of the journey is mostly to have fun and Ernesto take on his motorcycle, Le Poderosa as the mode of transportation. However, along their journey, Guevara meets with poverty stricken people who are severely impacted by the capitalist society and this transform Guevara into a man who feels that these people should be represented and should fight for equal rights by developed a communist regime. When one sees that movie as a transformation of the protagonist, one will find a remarkable change in Guevara from the beginning of the movie till the end. The movie begins on a lighthearted note where one is able to enjoy the idiosyncrasies of the two young men who have no responsibilities of the world. Having always lived within their own circle, these two men are unaware of the plight of the communist living within their own country. In the beginning, the talk of these men is centered on girls and having fun and adventure. They laugh often, they joke often and they are like any other typical men of their age (Christianson, 13). The transformation in Guevara however is not very sudden. Even while having the adventure of his lifetime, the viewers see a responsible man in certain instances. Since Guevara belonged to the medical profession, his eyes were open for the patients among the people he met. While his friend, Granardo was more

Significance of Leadership Essay Example | Topics and Well Written Essays - 1750 words

Significance of Leadership - Essay Example I was tapped to lead a six-person team plagued by complaints such as â€Å"I feel stifled by the bureaucracy,† â€Å"I am bored by the repetitive routines,† and â€Å"My boss has no sense of business.† Before taking on the responsibility, my mentor asked whether I had seriously considered what it meant to lead this infamous team. In my response, I quoted my former coach: â€Å"There’s no bad boy, only a bad coach.† I did not criticize the so-called bad boys, many previous leaders had done the same mistake and I certainly did not want to follow in their footsteps. Another positive thing which I did was that I listened to them patiently and gave them a good chance to express themselves, all these above things worked in my favor and I ended up winning their hearts. I defined my role as the team’s backup. I wouldn’t lead; I planned to support. I shared my vision of turning the team members into real leaders who not only developed ideas but also put them into action and delivered results. I invented the â€Å"changing seats† game, in which team members alternated sitting in my chair every Monday morning, initiating an activity for the whole team to execute that week. Later, I proposed setting up a Your-Voice forum, in which the team would exchange ideas about the latest international practices in trade finance as well as necessary reformations in workflow and business process. Motivated by the team’s enthusiasm, I convinced the executives to provide a small budget to support our lecture series involving professionals in insurance, shipping, and foreign exchange management policy. The lecture series was open to the entire organization, which helped rebuild the team’s image into a positive one. Gradually, I guided my bad boys to turn their innovative ideas into case studies and papers for top management’s reference.

Thursday, October 17, 2019

Public Opinion Essay Example | Topics and Well Written Essays - 500 words

Public Opinion - Essay Example These associations also seek to influence public opinion as a way to achieve their ends. In democracies like the US where operations of the government are dependent on the people’s consent in an election, leaders are obligated to take public opinion into account. Indeed, major policy change shifts tend to coincide with the occurrence of major public opinion shifts (Lowi et al, 2013). Generally, therefore, both Congress and the Executive respond to the preferences of the public, for example regarding government spending, welfare reform, and foreign policy issues. Still, the government should be responsive to public opinion because it needs the backing of the public for re-election. Essentially, therefore, government actions are consistent with public opinion with a study finding that significant shifts in public opinion tend to be followed by shifts in government policy within a year consistent with popular opinion shifts (Lowi et al, 2013). Still, this does not mean that the government panders to all the preferences of the public. This is caused by inconsistency between commitment of the nominal majority and adherents of minority viewpoints, as well as inconsistency between public opinion and the character of the US system of government (Lowi et al, 2013). Overall, however, government actions do not digress from popular opinion for extended periods due to the electoral process. However, the government is also able to take leeway in its response to public opinion because the latter is not specific, while its measurement is not always accurate. To meet this challenge, public interest groups representing a select population have risen in prominence. These associations attempt to pressure government through various methods, including mobilizing public opinion (Lowi et al, 2013). This involves the use of resources at their disposal to persuade a majority of the public to

The Role of Consumer Behavior in Marketing Decisions Research Paper - 2

The Role of Consumer Behavior in Marketing Decisions - Research Paper Example From this study it is clear that  nearly every respondent attested to the fact that McDonalds is a big market player in food products. This is because with regard to the food products that do well in the summer across the various market segments, every respondent gave an indication of having bought them in less than a week. The young market has a higher preference for McDonalds’ products than in the older market segments. It is clear that the older age segment still has a significant attraction to the McDonalds’ range of products. The most elaborate choice for McDonalds’ ice cream products in the summer has however been among the children, youths and young adults. All the respondents had a recent purchase experience with a McDonald’s product.  This paper outlines that  in order to find out the actual brand preferences that the respondents had for the various products offered at McDonalds, personal opinion was from each of the respondents. A question was asked regarding the specific brand choices and the decision making process behind them from each of the respondents. The three children and one adult bought vanilla ice cream cones from McDonalds while one young adult and two adults had chocolate dipped ice cream products.  Two of the remaining young adults bought strawberry yoghurt from McDonalds. Some of the reasons behind the decision to purchase the preferred product from McDonalds included identity with the company for all the children while one of the adults was having fun with his young son.

Wednesday, October 16, 2019

Public Opinion Essay Example | Topics and Well Written Essays - 500 words

Public Opinion - Essay Example These associations also seek to influence public opinion as a way to achieve their ends. In democracies like the US where operations of the government are dependent on the people’s consent in an election, leaders are obligated to take public opinion into account. Indeed, major policy change shifts tend to coincide with the occurrence of major public opinion shifts (Lowi et al, 2013). Generally, therefore, both Congress and the Executive respond to the preferences of the public, for example regarding government spending, welfare reform, and foreign policy issues. Still, the government should be responsive to public opinion because it needs the backing of the public for re-election. Essentially, therefore, government actions are consistent with public opinion with a study finding that significant shifts in public opinion tend to be followed by shifts in government policy within a year consistent with popular opinion shifts (Lowi et al, 2013). Still, this does not mean that the government panders to all the preferences of the public. This is caused by inconsistency between commitment of the nominal majority and adherents of minority viewpoints, as well as inconsistency between public opinion and the character of the US system of government (Lowi et al, 2013). Overall, however, government actions do not digress from popular opinion for extended periods due to the electoral process. However, the government is also able to take leeway in its response to public opinion because the latter is not specific, while its measurement is not always accurate. To meet this challenge, public interest groups representing a select population have risen in prominence. These associations attempt to pressure government through various methods, including mobilizing public opinion (Lowi et al, 2013). This involves the use of resources at their disposal to persuade a majority of the public to

Tuesday, October 15, 2019

Job opportunity in bioinformatics Term Paper Example | Topics and Well Written Essays - 1250 words

Job opportunity in bioinformatics - Term Paper Example Bioinformatics is, therefore, an extensive field with many job opportunities both directly and indirectly linked to it. This paper presents some of the employment opportunities. The first job opportunity that bioinformatics presents are sequence analysis. Sequence analysis was first done in 1977 when the phage ÃŽ ¦-X174 was sequenced. The job involves decoding DNA sequences and storing the information in databases. The sequence analysts then analyze the data to find out the genes that code for proteins and other structures in the sample. This has led to the discovery that comparing the genes within organisms of the same species, or different species reveal similarities in the functionality of their proteins. However, the growing amounts of data, means that it is no longer possible to analyze DNA sequences manually. Sequence analysts have, therefore, developed software that search the genome of millions of organisms, consisting of billions of nucleotides in databases (Levine 4). The programs can make up for mutations in DNA sequences, so that they can determine related but identical sequences. A variation of the sequence alignment is used in the sequencing p rocess. The shotgun sequencing technique gives the series of thousands of small DNA structures. It produces the sequence data quickly but does not assemble the fragments quite fast for complicated genomes. Another job area of bioinformatics in sequence analysis is in the automatic search for genes and regulatory sequences within genomes. However, nucleotides found in genomes are not all genes. Bioinformatics is vital in bridging the gap between genome and proteome projects like in using DNA sequences for identifying proteins (Levine 5). Bioinformatics knowledge is required for a job in genome annotation. Genome annotation is the process of marking the genes and the other biological features in DNA sequences. Owen White designed the first

Monday, October 14, 2019

Racial and Ethnic Discrimination in Canada Essay Example for Free

Racial and Ethnic Discrimination in Canada Essay You know the world is off tilt when the best rapper is a white guy (Eminem), the best golfer is a black guy (Tiger Woods), the tallest basketball player is Chinese (Yao Ming, 76) and Germany doesnt want to go to war (in Iraq). Charles Barkley stated in a 2003 interview, pointing out various misconceptions with stereotypes. A stereotype is defined by dictionary. com as: something conforming to a fixed or general pattern; especially: an often oversimplified or biased mental picture held to characterize the typical individual of a group. I have commonly heard stereotypes such as the French are good cooks, Italians are great lovers, and the Irish are lazy or comments made like dumb jock, lazy Cape Bretoner, or that women are not strong!! The list could go on endlessly as there appears to be stereotypes regarding people of all races, religions, sexes and ethnic groups, etcetera. Stereotypes can be either positive or negative. Most stereotypes tend to make us feel superior in some way to the person or group being stereotyped. Stereotypes ignore the uniqueness of individuals by painting all members of a group with the same brush. Throughout the course of this paper I plan to discuss some racial and ethnic issues in Canada. Where some of these issues originated from, what we can personally do to help eliminate discrimination in the workplace and what the government is doing to try to combat such discrimination. Let me first begin by defining discrimination, racism and ethnicity since these terms are all important terms to understand before going into further discussion. To discriminate is simply defined by yourdictionary. com as: To make distinctions on the basis of class or category without regard to individual merit; show preference or prejudice. Therefore, discrimination occurs when a person is not treated equally because of their gender, race, religion, ethnic origin, nationality, sexual orientation, or age. Yourdictionary. com defines racism as: The belief that race accounts for differences in human character or ability and that a particular race is superior to others. Discrimination or prejudice based on race. In other words, when an individual or group is treated unfairly or abused because of their skin color or racial heritage they are victims of racism. Ethnic, as defined by yourdictionary. Com is: Of or relating to a sizable group of people sharing a common and distinctive racial, national, religious, linguistic, or cultural heritage. B. Being a member of a particular ethnic group, especially belonging to a national group by heritage or culture but residing outside its national boundaries. With that being said, it is my belief that stereotypes and ignorance about others most often lead to discriminatory behavior both inside and outside the workplace. I have heard Canada described as a multicultural nation meaning that Canadians are not of any one cultural background, race or heritage. For all Canadians, including Aboriginal People, this multicultural diversity can be traced to an immigrant past. This does not mean that the majority of todays Canadians are immigrants but rather that the majority of Canadians have in their past, perhaps many generations ago, a family member who migrated here from another country. That is why many of us have a mixed ancestry, for example; Irish, Scottish, Ukrainian, French and Aboriginal, and the list can go on. Canadas Aboriginal People were the first to immigrate, and settle across the continent, tens of thousands of years before European settlers. After the European settlers came the French, followed by the English, Scots and Irish formulating Canada into the diverse country it is today. In the years before the American Civil War, thousands of black slaves escaped slavery in the United States by following the Underground Railway north to Canada. Then, at the turn of the century, American farmers moved northward into the Canadian prairies to develop farm lands. Although Canada originally consisted of a wide variety of immigrants, some people were not as welcome in the country as others and were therefore not treated equally. Those who were of different race, color, or religion then the majority of Canadians were labeled as foreigners. The use of the term foreigner held many connotations for example, different, strange or inferior and many at the time wanted to see the foreigners assimilate to fit into Canadian society. There are many events in Canadas past that has contributed to the racism and discrimination in Canada today for example, the disregard and unfair treatment of Aboriginal Peoples by Europeans who settled here. Even though a vast majority of African-Americans moved to Canada to avoid slavery, from early in the 1600s until 1834 there was a recorded 4092 slaves throughout the country, mostly living in Quebec . The Asiatic Exclusion League, which originated in California in 1905 as an anti-Oriental movement, moved north into Vancouver in 1907. The league was the main instigator in anti-Asian riots in the city since their main goal was to have all Chinese and Japanese immigrants removed from North America out of fear that they were taking jobs away from Whites . It also appears that throughout history the acceptance of immigrants in Canada greatly depended upon the economic state of the country at that time. During the Great Depression of the 1930s immigrants seeking jobs were unwelcome and overlooked for employment. Although the Government of Canada has made many advances in breaking the barriers that Aboriginal People, immigrants and minorities face in the country; immigrants today still face a number of problems when trying to enter the labor market, for example: ? Non-recognition of international credentials and work experience ? Lack of Canadian work experience ? Inability to communicate in English or French ?Insufficient labor market information prior to immigrating to Canada I have traveled to some of the major cities in Canada and was a little surprised by the degree of segregation that is apparent in these cities. By this, I mean that these larger cities, like Toronto and Vancouver, have communities which are almost completely independent from the rest of the country. These independent communities that I saw, of Chinese or Italian people, seemed to have everything they needed to survive within the community including their own schools. I could not help but wonder what effect this type of segregation has on the country. I respect the fact that all people are trying to protect their identity. At the same time, by choosing to live in Canada, shouldnt they try to integrate into the country a little more while still preserving their identities? Shouldnt they try to assimilate? How can Canada thrive as a country with so much segregation? We need to become a unified country. Not such a historical thought pattern, I guess!! It is people who have attitudes like mine that are causing problems in the country or do all people have these thoughts and choose not to admit it. I have similar negative feelings about scholarships being available only to certain people or government funding for certain people to attend university because they are a minority. I understand that differential treatment is required in order for equality to become a possibility. However, I still feel a degree of resentment about these programs being offered when I have to borrow money in an effort to obtain my university degree. Will this resentment evolve? When I hold a management position in the future, will I discriminate against a person because he or she doesnt have a huge student loan to pay and another does? It is cases like mine that causes racism to continue in society and the workplace today? With the announcement of Nova Scotias plan to increase immigration into the province came an increase in the racist comments I have heard. Since I work in bars I hear, and partake in, a great deal of conversation. When people are drinking they tend to be even more likely to say things they normally wouldnt. That is why I have heard, at times, some very racist remarks. People have said that the government should be trying to retain people in the province that are born here before they bring foreigners here. They need to take care of their own first!! It is because of these comments and feelings that I am doubtful that discrimination against people, because of their race or color, will ever be completely eliminated in the country. How do we achieve equality with so much differentiation? How do we check or personal opinions at the door when we go to work? Since it is impossible to eliminate racism and discrimination entirely in society, we need to do as much as possible to eliminate it in the workplace. We need to make changes similar to the changes companies have made in an effort to combat discrimination against people because of their religion. For example, adapting zero tolerance rules, providing more education for employees, human resource departments need to provide more opportunities for people of minorities, immigrants, and Aboriginal Peoples and barriers have to be removed for all these people who are trying to enter our labor market. March 21, 2005 is International Day for Elimination of Racial Discrimination a day to remember the struggles and challenges that Aboriginal peoples and people of color have endured. It is also a time to recognize and applaud the fact that members of these two communities have made anti-racism struggles a significant part of labors agenda. Lets respect this day and try to make some positive changes at home, school, or work toward eliminating racism.

Sunday, October 13, 2019

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