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What is Love Essay - 1514 Words

â€Å"I love you†. No other sequence of words has so much power; these words have the power to strengthen a bond, weaken a relationship, ruin a friendship or bring two people together. The concept of love is puzzling and we have struggled to understand it for centuries, everything between the Greek goddess Aphrodite and today’s Romantic comedies have attempted to comprehend and explain it. The theme of love is popular in Donne’s early poetry. His understanding of love from the perspective of a protestant preacher reveals much about the anti-Christian sentiments and of the scientific revolution of the 17th century. A close analysis of Donne’s poem, â€Å"Love’s Alchemy† and of the film â€Å"When Harry Met Sally† reveals the strength behind Donne’s†¦show more content†¦Donne does qualify the Platonists when he expresses their experience. The appeal to experience is important in the context of the scientific revolut ion; the focus shifted towards observable facts rather than inferred ones. The argument between the Platonists and Donne is similar to that between Billy Crystal and Meg Ryan in â€Å"When Harry Met Sally†. Billy Crystal’s character Harry claims that a man and a woman can never be friends because sex will interfere. Meg Ryan’s character Sally refutes this claim based on her own previous experiences. Experience is an important theme during the renaissance and in â€Å"Love’s Alchemy†, Donne establishes his position as an expert with â€Å"I have lov’d, and got, and told,† (3) and then goes on to counter the Platonist opinion in saying â€Å"I should not find that hidden mystery.† (5) He argues that both he and the Platonists have experience, but that his observations have led him to a different conclusion. The hidden mystery in line 5 refers primarily to platonic love. The speaker prefaces this by saying â€Å"[b]ut should I love, get, tell, till I were old[.]† (4) The importance of this line is the concept of time, even if Donne continues his observations he would never find platonic love. The fact that it is hidden can be understood to mean that as aShow MoreRelatedWhat Is Love? What It Is?1527 Words   |  7 PagesWhat is Love? When asked the question â€Å"What is Love?† what are some of the first things that come to mind? Some people might say it is an emotion that we experience when we interact with the world around us. People can have love for many things such as money and other material possessions, family and friends, even the world around them. These are just a few examples of how we often use love in our lives. However, love is not only confined to our lives, it is also a very important emotion in religionsRead MoreWhat Is Love?1020 Words   |  5 Pages250 Tu/Thu 10/5/10 What is Love? Love is one of the most difficult words to define. I challenge you to try. You can even go the easy way and simply Google the word â€Å"love†. What you will find is not one but hundreds of definitions along with countless books, movies, and songs all having to do with love. The reason why it is so hard to define love is because there are different forms/stages of love and there are so many things that love can consist of. Also, love can have a different definitionRead MoreWhat is love?872 Words   |  3 PagesLove and passion is the burning sensation that drives humans to lead their lives into new horizons: following the heart hoping it will guide the way. Janie, the lead character in the book, Their Eyes Were Watching God, written by Zora Neale Hurston, is suppressed by family, and two different husband too, only to find pain and sorrow by not following her heart until she is freed by a man who loves her deeply with only one thing on his mind, to protect and love her like nobody else before. ThroughRead MoreWhat Is Love?1080 Words   |  4 PagesTwelfth Night is love. The notion of love is important to the plot as many of the characters are driven by love. There are multiple forms of love depicted throughout the play; each character represents a different type of love. Viola displays a patient, sincere, and enduring love for Orsion as well as a deep familial love for her brother. Sebastian and Antonio share a special bond which could be classified as brotherly love. These two bonds can be seen as the most real forms of love depicted in TwelfthRead MoreWhat is Love?559 Words   |  2 Pagesdictionary love is a feeling of strong affection for a person. (Merriam-Webster) In my opinion, love is a conjunction of different feelings altogether, when a person is in love they feel happy, sad, excited, and scared everything at the same time. What I am trying to say is that for me there is not real definition for love because nobody can really explain the mix of feelings that happened when one is in love. According to one of Latterell’s assumptions love conquers all, she says that, â€Å"true love willRead MoreWhat Is Love?1409 Words   |  6 Pageswaiting for his response. â€Å"I’m looking at love realistically this time around, instead of through the eyes of romantic delirium.† Julian scoffed. â€Å"You can’t fool me. Save your breath.† David was finding it harder to keep his cool. â€Å"I don’t even know the girl with whom I’m supposed to be in love. The whole thing was kind of an illusion, at least the romantic part. Cecilia was Daniel’s girl.† â€Å"That didn’t bother you this past summer—you were head over heels in love.† â€Å"Yeah, with a girl astral projectingRead MoreWhat is Love?589 Words   |  2 PagesWhat is â€Å"Love†? Love can be defined as different things. Love can be the love between brother and sister, sibling love, like Ender and Val. Love can also be the love between a boyfriend and girlfriend or a husband and wife, like Val and her husband, which the book doesn’t really talk much about, or Novinha and Libo. In strange cases, the love you should have for a girlfriend or wife but you feel that way for your sister, like Miro and Ouanda. Love is one of the weirdest feeling ever and is somethingRead MoreWhat Is Love Essay796 Words   |  4 PagesMonday Oct. 19th What is Love? According to Webster’s dictionary the word love is described as a strong, positive emotion of regard and affection. But in society today it seems as if we throw the word love around in such a loose manner it really has lost its meaning. Ranging from â€Å"I love Coach purses†, to actually telling a person â€Å"I love you† is now a common thing. Throughout this essay I’ll be taking a philosophical approach to help give a better understanding of what love is according to theRead MoreWhat Is Freedom For Love? Essay746 Words   |  3 PagesWhat is Freedom to Love? Some would say that freedom to love is, having no limitations or boundaries. To see everyone equally. Many would say that freedom to love is, to give ones life for another. I agree with both statements but I would go further to say that freedom to love is not only an act or a response but it is a lifestyle. Something that defines you. Something that make you, who you are. As proud Americans, this month we celebrate the freedom that we have in this country and theRead MoreWhat is Love? Essay1558 Words   |  7 PagesWhat is Love? Romantic Love Is love chemical? Love cant be just pheromones, surely body chemistry changes. Although, perhaps that is why people break up after a while. Maybe they were attracted to each other at one point, but then the pheromones they were giving off change and the other person is no longer attracted to them. What is attraction based on? What attracts one person to another? People have said they have fallen in love before meeting in person, thanks to the Internet

Marketing Research of ResMarket Samples †MyAssignmenthelp.com

Question: Discuss about the Marketing Research of ResMarket Pty. Answer: Introduction: Technology has influenced the marketing field in virtually all domains of business. Marketing research by means of using technology give organizations access to many more marketing options and collect and organize relevant marketing data. In short, it can be summarized that technology is making the world more accessible and facilitating marketing researchers to take more globally collect and process data (Venkatesh, Thong and Xu 2012). This report discusses about the case scenario of ResMarket Pty, a Sydney based marketing research company that has decided to adapt new technologies to enhance marketing research activities for their clients. The only concern for the company is the high cost of such technology. Hence, in context of this scenario, this report discusses about the impact of technology on marketing research in todays business and how adapting such technology may help the marketing research firm. Usefulness of marketing research for business organizations Highly acclaimed business organizations worldwide prefers to conduct marketing research on a regular basis to keep up with latest marketing trends and sustain a competitive edge in business. This help companies to gain useful knowledge regarding features of target market such as level of sales, ROI (return on investment) or revenue required to succeed in business. The range of information that an organization get from marketing research are as follows: Access to market information such market segmentations, demand and supply, sales revenues and many other informations Knowledge about the demands and preference of existing customers such as factors influencing buying decisions, their perception about products and key decision factors Facilitates identification of potential customers Provides knowledge regarding consumer behavior patterns and needs to evolved current products and services Analysis of competitors to determine position in business market Identifying new business opportunities (Sekaran and Bougie 2016) Hence, for organizations like ResMarket Pty, getting access to such information manually will require great time and effort. However, adapting appropriate technology for marketing research gives the advantage of saving time and getting access to broad and authentic informations too. Impact of technology on marketing research For the Sydney based research firm, adapting technology in marketing research despite high cost is necessary. This is because technological advancements and integration of such technology will greatly boost their marketing research related activities. The use of digital devices like social media sites or other important applications can provide holistic view of the business situation. Access to new data inputs and crucial marketing information will help the company to evolve and provides organizations meaningful insights for practical application in todays business environment. Currently, there is no dearth of technology for marketers. Wave of new technology is dominating the business world today. Earlier business relied on internet advertising, emails and text messaging. However, the whole business process and promotion methods has now transformed with newer technologies like GPS, social media and smart phone application. Two important technology currently used today includes customer relationship management systems and the social marketing. While the former uses technology to identify potential clients and gather information about daily business operations, the social media tools helps to identify elements needed to build brand personality (Norman and Verganti 2014). Hence, the Sydney based company can drives their marketing research activities by identifying relevant marketing research tools needed for their organizations. The article by Trainor (2014) also gives the insight that technology has made market researchers job easier. For example, the social media sites like Facebook, LinkedIn and Twitter has given a wider horizon for marketing researchers to do their research. This gives them the advantage of yielding unfiltered feedback from consumers and using the data for brand awareness and business knowledge. Secondly, the advent of new softwares in business research enables researchers to focus on distinct measures and collect data in a short time. Another advantaged of technology is that it has made business analytics more sophisticated and effectively use the streams of data to determine the strategic steps needed for particular organizations to success (Sharma, Mithas and Kankanhalli 2014). Despite getting better facility to engage in research, the marketing researchers role has become challenging too. To successfully adopt new technology in business, they need to acquire new skills and engage in new roles to efficiently translate the data for business success. Although marketing research technology provides researchers access to large pool of data, however their skills can determine whether they use the data with accuracy or not (Christensen, 2013). They need to have the skills to discard the irrelevant data and identify the useful data for formulating marketing strategies. Despite the numerous advantage of new technology for business research, there are certain factors that prevent organizations from readily accepting the technology for marketing research. For ResMarket Pty, the main concern for them in adapting the new technology was the cost associated with the new technology. Other barriers includes little awareness about the values of technology, no knowledge about effective application and misuse of technology. However, the cost problem in adapting the technology can be resolved by ResMarket Pty by considering the overall budget for marketing research. Striking out irrelevant things and focusing on identifying useful technology in their business context may help them significantly. Embracing appropriate technology in a turbulent and flexible business environment provides the right moves for researchers to formulate strategic plan and target specific business (Kozlenkova, Samaha, and Palmatier 2014). Conclusion: The report focused on the evaluation of technology for carrying out marketing research activities. The move of ResMarket Pty to adapt technology in marketing research was justified by explanation of different advantages of technology in marketing research. The wide pool of data through different research tools and technology provides a wider reach to marketing analyst and the advantage of gathering data in a short time. Once organizations learn and identify the right technology for their marketing research, it can help them sustain their business amidst complex business environment. It is also necessary for research analyst to learn new skills to understand their new roles in marketing research with the use of technological tools and devices. References Christensen, C.M., 2013.The innovator's dilemma: when new technologies cause great firms to fail. Harvard Business Review Press. Kozlenkova, I.V., Samaha, S.A. and Palmatier, R.W., 2014. Resource-based theory in marketing.Journal of the Academy of Marketing Science,42(1), pp.1-21. Norman, D.A. and Verganti, R., 2014. Incremental and radical innovation: Design research vs. technology and meaning change.Design issues,30(1), pp.78-96. Sekaran, U. and Bougie, R., 2016.Research methods for business: A skill building approach. John Wiley Sons. Sharma, R., Mithas, S. and Kankanhalli, A., 2014. Transforming decision-making processes: a research agenda for understanding the impact of business analytics on organisations.European Journal of Information Systems,23(4), pp.433-441. Trainor, K.J., Andzulis, J.M., Rapp, A. and Agnihotri, R., 2014. Social media technology usage and customer relationship performance: A capabilities-based examination of social CRM.Journal of Business Research,67(6), pp.1201-1208. Venkatesh, V., Thong, J.Y. and Xu, X., 2012. Consumer acceptance and use of information technology: extending the unified theory of acceptance and use of technology.

The Patience of Penelope Essay Example For Students

The Patience of Penelope Essay Essay: The Patience of Penelope Do you believe that true love is Eternal? Would you be able to wait for yourtrue love to arrive or would you settle for what is at reach? In the Greek love story,The Patience of Penelope, the writer portrays the meaning of true love. In the shortstory true love consisted of patience, faith, and even suffering. Not all of us hold the rare quality of having patience. We get desperate easilywhen we are obligated to wait for something which we need or want. We tend to give inquickly to lessen our craving or settle for less. Patience was something that Penelope, theyoung; beautiful; and wealthy wife of Odysseus carried within herself. Penelope wasaware of her husbands rumors of his death. Though she maintained seeking patience,and no matter how helpless she felt, she ignored the suitors who tried winning her favorsand weaved wool for four years till Odysseus revealed himself. An extraordinary term by which many of us live with everyday is faith. It ispossible for us to have faith in our friends, God, and/or love. In the story, Penelopedemonstrates her belief in faith towards her husband, Odysseus. We will write a custom essay on The Patience of Penelope specifically for you for only $16.38 $13.9/page Order now She remained loyal,sincere, and honest towards her husband. Though many rumors spread of his death, andtime passed by with no sign of her beloved husband, she managed to keep faith. I believethat such actions proved Penelopes true love for her husband. It doesnt mean much to love with tongue and words, one must show by actions-their love. Even if it means suffering for their love. I feel that when you truly lovesomeone you are willing to do anything for that someone. For example, in the storyPenelope suffered for her husband for several years. She spent four years of her youthfullife lonely and pressured by society to find another man to love. But, Penelpe chose thedifficult, but worthy road. She chose to suffer till her husband arrived. In conclusion, it is acceptable to say that the story carries a valuable concept inlife. The story gives its reader a view on how life can not always be as clear and/or asstraight forward as you would want it to be. We might go through a few tests to find ourlove for certain things or people. The story also shows us how patience and faith areextremely necessary in many situations, and how love consists of suffering as well. Bibliography:

Amphotericin B Essays - Antifungals, RTT, Rare Diseases, Free Essays

Amphotericin B Essays - Antifungals, RTT, Rare Diseases, Free Essays AmB kills yeast via ion channel-mediated membrane permeabilization. This channel model has been extended to also rationalize the unique lack of resistance to AmB as well as its dose-limiting toxicity. This perceived understanding has stimulated extensive research toward the goals of developing new channel-forming compounds as putative resistance-refractory antimicrobial agents and/or less toxic derivatives of AmB that more selectively form channels in fungal cells versus human cells. This drug should be used primarily for treatment of patients with progressive and potentially life-threatening fungal infections; it should not be used to treat noninvasive forms of fungal disease such as oral thrush, vaginal candidiasis, and esophageal candidiasis in patients with normal neutrophil counts. Expected Pharmacological Action AmphotericinB deoxycholate is an antifungal agent that acts on fungal cell membranes to cause cell death. Depending on concentration, these agents can be fungistatic (slows growth on the fungus) or fungicidal (destroys the fungus). Therapeutic Uses Antifungals are the treatment of choice for systemic fungal infection (Candidiasis, Aspergillosis, Cryptococcosis, Mucormycosis) and nonopportunistic mycoses, (Blastomycosis, Histoplasmosis, Coccidioidomycosis). Some antifungal treat superficial fungal infections: dermatophytic infections (tinea pedis [ringworm of the foot], tinea cruris [ringworm of the groin]); candida infections of the skin and mucous membranes; and fungal infections of the nails (Onychomycosis). Adverse effectsNursing Intervention infusion reactions (fever, chills, rigors, and headache) 1 to 3 hr after initiation pretreat with diphenhydramine (benadryl) and acetaminophen. meperidine (demerol), dantrolene, or hydrocortisone may be given for rigors. thrombophlebitisobserve infusion sites for signs of erythema, swelling, and pain. rotate injection sites. administer in a large vein and administer heparin before infusing amphotericin b. nephrotoxicityobtain baseline kidney function (bun and creatinine) and do weekly kidney function tests. monitor i&o. infuse 1 l of saline on the day of amphotericin b infusion. hypokalemia monitor electrolyte levels, especially potassium. administer potassium supplements accordingly. bone marrow suppressionobtain baseline CBC and hematocrit, and monitor weekly.

Biography of Bill Gates

Biography of Bill Gates Free Online Research Papers William (Bill) H. Gates III is co-founder, chairman and chief executive officer of Microsoft Corporation, the worlds leading provider of software for personal computers. Bill Gates was born on October 28, 1955. He and his two sisters grew up in Seattle. Their father, William H. Gates II, is a Seattle attorney. Mary Gates, their late mother, was a schoolteacher, University of Washington regent and chairwoman of United Way International. Gates attended public elementary school before moving on to the private Lakeside School in North Seattle. It was at Lakeside that Gates began his career in personal computer software, programming computers at age 13. In 1973, Gates entered Harvard University as a freshman, where he lived down the hall from Steve Ballmer, who is now Microsofts president. While at Harvard, Gates developed a version of the programming language BASIC for the first microcomputer the MITS Altair. BASIC was first developed by John Kemeny and Thomas Kurtz at Dartmouth College in the mid-1960s. In his junior year, Gates dropped out of Harvard to devote his energies full-time to Microsoft, a company he had started in 1975 with his boyhood friend Paul Allen. Guided by a belief that the personal computer would be a valuable tool on every office desktop and in every home, they began developing software for personal computers. Gates foresight and vision regarding personal computing have been central to the success of Microsoft and the software industry. Gates is actively involved in key management and strategic decisions at Microsoft, and plays an important role in the technical development of new products. Much of his time is devoted to meeting with customers and staying in contact with Microsoft employees around the world through e-mail. Under Gates leadership, Microsofts mission is continuously to advance and improve software technology, and to make it easier, more cost-effective and more enjoyable for people to use computers. The company is committed to a long-term view, which is reflected in its investment of some $2.6 billion for research and development during the current fiscal year. In 1995 Gates wrote The Road Ahead, his vision of where information technology will take society. Co-authored by Nathan Myhrvold, Microsofts chief technology officer, and Peter Rinearson, The Road Ahead held the No. 1 spot on the New York Times bestseller list for seven weeks, and remained on the list for a total of 18 weeks. Published in more than 20 countries, the book sold more than 400,000 copies in China alone. In 1996, while strategically redeploying Microsoft to take advantage of the emerging opportunities created by the Internet, Gates thoroughly revised The Road Ahead to reflect his view that interactive networks are a major milestone in human communication. The paperback second edition also has become a bestseller. Gates is donating his proceeds from the book to a non-profit fund that supports teachers worldwide who are incorporating computers into their classrooms. In addition to his passion for computers, Gates is interested in biotechnology. He sits on the board of the ICOS Corporation and is a shareholder in Chiroscience Group of the United Kingdom and its wholly owned subsidiary, Chiroscience RD Inc. (formerly Darwin Molecular) of Bothell, Wash. He also founded Corbis Corporation, which is developing one of the largest resources of visual information in the world a comprehensive digital archive of art and photography from public and private collections around the globe. Gates also has invested with cellular telephone pioneer Craig McCaw in Teledesic, a company that is working on an ambitious plan to launch hundreds of low-orbit satellites around the Earth to provide a worldwide two-way broadband telecommunications service. In the dozen years since Microsoft went public, Gates has donated more than $800 million to charities, including $200 million to the Gates Library Foundation to help libraries in North America take advantage of new technologies and the Information Age. In 1994 Gates established the William H. Gates Foundation, which supports a variety of initiatives of particular interest to Gates and his family. The focus of Gates philanthropy is in four areas: education; world public health and population; non-profit, civic and arts organizations; and Puget Sound-area capital campaigns. Bill and Melinda French Gates were married on January 1, 1994. They have one child, Jennifer Katharine Gates, who was born in 1996. Research Papers on Biography of Bill GatesThe Project Managment Office SystemRiordan Manufacturing Production PlanOpen Architechture a white paperNever Been Kicked Out of a Place This NiceBionic Assembly System: A New Concept of SelfPersonal Experience with Teen PregnancyMarketing of Lifeboy Soap A Unilever ProductAnalysis of Ebay Expanding into AsiaDefinition of Export QuotasTwilight of the UAW

Why did the Oslo Peace Process fail Dissertation

Why did the Oslo Peace Process fail - Dissertation Example It marked the initial face-to-face agreement between Israel and the Palestine Liberation Organization (PLO). The activities surrounding the agreement arose from the Madrid Conference of 1991, carried out in Oslo, Norway, supported by the Fafo institute and cleared on the 20th of August 1993 (Brown, 2003). The Accords were then officially set to writing and signed during a public ceremony in Washington, D.C on the 13th of September 1993 before Yasser Arafat (PLO Chairman), Prime Minister of Israel Yitzhak Rabin, and President Bill Clinton of the United States of America (Brown, 2003). The documents were also supported and signed by other official signatories, including Mahmoud Abbas for the PLO, Shimon Peres, foreign minister for Israel, the US Secretary of State Warren Christopher for the US as well as Andrei Kozyrev, the foreign minister for Russia (Brown, 2003). The Accord supported the establishment of a Palestinian interim self-government, including the Palestinian National Autho rity (PNA). This PNA was set to bear responsibility for the management of the territory within its control (Brown, 2003). The Accords also supported the withdrawal of the Israel Defense Forces (IDF) from various regions of the contentious Gaza Strip and the West Bank. It was expected that the arrangement would cover a five-year interim period where later a more permanent arrangement would be established, especially on issues like Jerusalem, Palestine refugees, Israeli settlements, and borders (Weinburger, 2007). This Oslo I was followed by Oslo II. In both accords, no independence was anticipated for Palestine. In general, the agreement emphasized the withdrawal of Israel personnel from different areas of the Gaza Strip as well as the West Bank. It also supported the right of Palestine for self-governance in these regions through the establishment of the Palestinian Interim Self-Government Authority (Weinburger, 2007). The term of the Palestine was set to last for five years, at whi ch time, more permanent arrangements would have been established for the two territories. Major problems, including the issue of Jerusalem, Palestinian refugees, as well as security and border issues were to be managed in other negotiations and arrangements (Weinburger, 2007). Israel was ready to grant an interim self-rule to Palestine in stages. Aside from the principles, the two groups put into writing the Letters of Mutual Recognition, as the Israeli government supported the PLO to be the legal representative of the Palestinians and the PLO also supported the right declared by Israel to exist (McMahon, 2009). The agreement also rejected and condemned terrorism, violence, and activities set to destroy Israel. The goal of the Israel-Palestine talks was to secure a Palestine Interim Self-Government Authority, for the people of the Palestine region in the West Bank as well as the Gaza strip, during the transitional 5-year period (McMahon, 2009). Before Palestinians could manage thems elves based on democratic principles, general elections were set for the Council. The coverage of the Palestinian Council was the West Bank and the Gaza Strip. Exempt were issues which would be managed during the permanent negotiations (McMahon, 2009). The two parties perceived West Bank and Gaza to be single territorial units. The five-year transition period would be seen with Israel withdrawing from the Gaza Strip and the Jericho area. Permanent status talks started soon enough between these territories (Quandt, 2001). A transfer of authority was also set between the Israel Defense Forces to the Palestinians on matters relating to education, culture, health, social welfare, taxation, and tourism. The Council would

Compassion Fatigue in Essay Example | Topics and Well Written Essays - 250 words

Compassion Fatigue in - Essay Example In this case, a Compassion Fatigue Self-Test (CFST) consisting of thirty questions was administered to 500 public health nurses who could have been deployed to assist hurricane victims. These nurses were identified by the Florida State Department of Health (DOH). The commonest data collection methods used in survey research is questionnaires and interviews (Nieswiadomy, 2008). At first, the nurses were asked to go back in time and think of their hurricane experience and respond to the questionnaire based on those feelings. Secondly, they were requested to answer the same questionnaire based on their current feelings. The second responses were printed on a different colored paper in order to distinguish it from the initial. The participants had to rate the questions on a scale of how often they experienced that feeling or event. In this case, the Likert scale was used (1=rarely, 10=very often). The range of possible scores of the thirty questions would automatically be 30 to 300. The scores obtained from each issue are then added up to give a total score of (range from 173) and the risk level of compassion

Manage People and Performance Essay Example | Topics and Well Written Essays - 1500 words

Manage People and Performance - Essay Example Considering that Maslow’s theory presented us with five orders of needs it is clear that all of those were fulfilled by the job he had with Avant Garde. He had a competitive salary which took care of his basic needs for nourishment; he had a company car, free healthcare, personal pension plan and a job he liked which took care of his needs for safety, security and social affiliations. The fourth order need in term of esteem could have been fulfilled by his rising position within the company even through his first class travel as he worked for Avant Garde. Even in terms of self actualization, he could have attained some level of it through his making the HR Department of the company the envy of others. Recognition by others within his social circle and even outside his social circle would have come from this achievement and it is likely that he would have continued working for the company till his retirement. However, there was a fly in the ointment which caused him to rethink his decision and his life as well since his third order needs were put in danger. From being socially accepted within the company he found himself being excluded from meetings when the company he was working for was taken over by someone else. In fact, he even lost his position as part of the strategic decision making group of the company. The final straw as put by the case study was the requirement for Phil to eliminate a large portion of the company through making many of his co-workers redundant. These developments put Philip in a position where he could not continue working for the company. While his basic needs were being fulfilled, his higher order needs were not being met which made him look for other opportunities. Amongst these opportunities was the chance to work at his Alma Matter and he took that knowing that he had had positive

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

Beowulf is an Epic Hero Essay examples -- Epic of Beowulf Essays

Every epic hero possesses certain heroic characteristics. The epic poem Beowulf describes the most heroic man of the Anglo-Saxon times. Beowulf is the hero. He shows that he is a great man by always putting other things before his own needs. He is important and needed by his people and is known by many as a strong, courageous and a helpful person. He shows all of the qualities and traits that a true hero possesses. Beowulf, like other epic heroes, possesses the following heroic qualities: epic heroes are superhuman types of beings. They show great bravery, intelligence, strength and resourcefulness. They have a strong admiration for the values of their society. They are dominant male figures and suffer severe pain, but in the end, they conquer evil. Beowulf encompasses all of these traits of an epic hero, and more. Analyzing Beowulf’s three battles, one can easily see Beowulf’s heroic characteristics prevail. The battle with Grendel, Grendel’s mother, and the dragon display an array of heroism expressed by Beowulf. One characteristic of heroism is that a hero performs outrageous and sometimes superhuman deeds. Beowulf is a prime example of this type of hero. He volunteers himself to fight Grendel and when Grendel’s mom seeks revenge he goes to the lake and takes on the challenge. He shows the great qualities of strength and power when, after fifty years, he takes on the dragon that has become a threat to the Geats. He always battles his enemies with pride. When it is t...

An Ethical View Essay

Moral and ethical viewpoints are often shaped and molded by your society; learning to respect others, tolerance, my family, church, co-workers, past and present life experiences has influenced my moral and ethical viewpoints. Knowing right from wrong and how to treat others has been the †¦Ã¢â‚¬ ¦.in this process. I. Influences on My Moral and Ethical Development A. My family/environment (Moral development) 1. Tolerance 2. Forgiveness and being honest B. Ethical development (Church/work) 1. topic/idea for paper 2. topic/idea for paper II. Experiences that Contributed to My Personal and Professional Development A. Life and Death 1. Marriage at an early age 2. Life experiences/lessons (murder of my spouse, single parent, setbacks,) B. Professional Development 1. School/instructors 2. Co-workers 3. past employment experiences This paper will display a brief synopsis of the elements that has influenced my moral and ethical development as well as, discuss counseling issues and the ethical codes used to resolve the issues, and I will explain how I have changed because of my work in this class. I developed a true sense of right and wrong at an early age, as a kid I was very adventurous and would do things just to see how far my parents, grandparents, or aunts/ uncles would allow me to go before chastisement came into play. I remember one incident as if it was yesterday, when I was seven I would watch one of my eldest aunts obtain a cigarette from the package (Virginia Slims), her lighter, light her cigarette, and began to smoke it; she would make smoke rings for me. One day I decided that I would mimic my aunt’s actions and smoke a cigarette, my grandmother caught me smoking the cigarette. She did not spank me as I thought that would have been a fair form of disciplinary resolution for my actions, she wanted me to know just how unhealthy smoking cigarettes was for me so, she made me call my mother and father, aunts, uncles, and cousins and tell them what I had done. From that moment until now I have never touched another cigarette and that’s when the real less ons of what was right and wrong began. Being the eldest of five children born to a single parent mother I learned at an early age about charity and helping those in need; my mother taught me about sacrifice at an early age even though I did not understand it then I have a firm grasp on the concept of sacrifice in my adult life. Growing up in my grandparentsgrandparents’ home I did not understand what beingthe definition of poverty or what being poor really meant because my grandparents were always so eager to feed everyone in the community, it wasn’t until my mother decided that she did not want to live under my grandparents roof and abide by their rules was when the knowledge of poverty settled in; my grandparents were very active in their Christian faith, they believed that God blessed you so, you should be a blessing to others and they always welcomed the needy into their home to share our meals on a consistent basis. My family being my environment has taught me the basics about morals and values; Kohlber g’s Moral Development stages Stage 1 = infancy—the child’s only sense of right and wrong is what feels good or bad; Stage 2 = toddler years—the child learns â€Å"right† and â€Å"wrong† from what she or he is told by others; Stage 3 = preschool years—the child begins to internalize family values as his or her own, and begins to perceive the consequences of his or her behavior; Stage 4 = ages 7-10 years—the child begins to question the infallibility of parents, teachers, and other adults, and develops a strong sense of â€Å"should† and â€Å"should not† Stage 5 = preteen and teenage years—peers, rather than adults, become of ultimate importance to the child, who begins to try on different values systems to see which fits best; teens also become more aware of and concerned with the larger society, and begin to reason more abstractly about â€Å"right† and â€Å"wrong.† Read more: Moral Development – STAGES OF MORAL DEVELOPMENT – Lawrence Kohlberg, Mean Example, Morality, and Social – JRank Articles http://psychology.jrank.org/pages/431/Moral-Development.html#ixzz2R8sxnA1w III. Experiences that Contributed to My Personal and Professional Development C. Life and Death 1. Marriage at an early age 2. Life experiences/lessons (murder of my spouse, single parent, setbacks,) D. Professional Development 1. School/instructors 2. Co-workers 3. Pastpast employment experiences For this application, you were asked to develop an outline for the final project. There were four topics that you were to consider, including influences on your moral and ethical development; experiences that contributed to your personal and professional development; legal and ethical issues in counseling; and reflection. Nice job giving thought to these areas. Looks like you have some thoughts for your final project. Looking forward to a little more detail on your next submission and looking forward to reading your final project! In order to understand clearly where you are headed, you must also evaluate where you have been and what has influenced you along the way. It is important to reflect critically upon your own values (and sense of personal/professional ethics) and how you developed these perspectives in order to develop an ethical framework. To help accomplish this goal, the Final Project for this course is an Ethical Autobiography in which you will explore various elements of your life experiences that might influence your future ethical framework. As you reflect on your journey through this class, some of the course readings may have informed your Ethical Autobiography. You can also make use of outside resources, but much of the paper will be exploring what you bring to the profession and events that may have influenced your ethical lens. This reflective autobiography should have personal meaning to you and should help you understand what being an ethical practitioner means. In this sense, you are writing an intellectual and Ethical Autobiography, that is, who you are as virtue of what you believe, what you do, and what you have read. Think broadly—there are no wrong answers; you are exploring your own world Some examples of questions/issues that you can address: †¢ You may share how you developed a sense of right and wrong. †¢ Who/what influenced your moral and ethical development? †¢ What experiences contributed to your personal and professional beliefs? Are your personal and professional beliefs congruent? †¢ What is your idea of right and wrong? Are there absolutes or are there shades of gray? Do the same guidelines apply in all circumstances? †¢ What are some of your basic values that guide your work and your life? What experiences have potentially influenced your decision making? †¢ What aspects of your personality and work ethic are most compatible with the counseling field? Which aspects are the least compatible? †¢ Was there a time, in your personal or professional life, when you felt that your confidentiality was violated, that you were involved in a dual relationship in which you felt uncomfortable, or perhaps an issue resonated unexpectedly with you (e.g., transference)? Essential Elements (You m ust address the points outlined below in your Final Project.): †¢ Select four counseling issues, describe these issues, and explain potential ethical challenges for addressing these issues in your professional practice. †¢ Explain state or region laws or statutes that might apply to these ethical challenges. †¢ Reference specific codes of ethics that you ascribe to for your practice and how adhering to ethics and law present challenges for addressing these issues you selected. †¢ Explain why this Assignment is meaningful to you. †¢ Describe how adhering to ethics and law for professional counseling practice might influence social change. †¢ Finally, explain how you have changed because of your work in this class. Describe personal and ethical values you have reexamined because of your work in this course. You should present your Final Project as a 12- to 15-page (including cover page, abstract, and references—therefore, approximately 10–12 pages of text), double-spaced, APA-formatted paper. Papers can be longeriflonger if the purpose of the paper is served, but the quality ofideasof ideas and conciseness of the writing should justify the extra length. Also, please proofread yourpapersyour papers to make sure that grammar, punctuation, and other mistakes do not hinder thecommunicationthe communication of your ideas. This paper will display a brief synopsis of the elements that has influenced my moral and ethical development as well as, discuss counseling issues and the ethical codes us ed to resolve the issues, and I will explain how I have changed because of my work in this class. I developed a true sense of right and wrong at an early age, as a kid I was very adventurous and would do things just to see how far my parents, grandparents, or aunts/ uncles would allow me to go before chastisement came into play. I remember one incident as if it was yesterday, when I was seven I would watch one of my eldest aunts obtain a cigarette from the package (Virginia Slims), her lighter, light her cigarette, and began to smoke it; she would make smoke rings for me. One day I decided that I would mimic my aunt’s actions and smoke a cigarette, my grandmother caught me smoking the cigarette. She did not spank me as I thought that would have been a fair form of disciplinary resolution for my actions, she wanted me to know just how unhealthy smoking cigarettes was for me so, she made me call my mother and father, aunts, uncles, and cousins and tell them what I had done. From that moment until now I have never touched another cigarette and that’s when the real less ons of what was right and wrong began. Being the eldest of five children born to a single parent mother I learned at an early age about charity and helping those in need; my mother taught me about sacrifice at an early age even though I did not understand it then I have a firm grasp on the concept of sacrifice in my adult life. Growing up in my grandparents’ home I did not understand the definition of poverty or what being poor really meant because my grandparents were always so eager to feed everyone in the community, it wasn’t until my mother decided that she did not want to live under my grandparents roof and abide by their rules was when the knowledge of poverty settled in; my grandparents were very active in their Christian faith, they believed that God blessed you so, you should be a blessing to others and they always welcomed the needy into their home to share our meals on a consistent basis. My family being my environment has taught me the basics about morals and values; Kohlberg’s Moral Development stages Stage 1 = infancy—the child’s only sense of right and wrong is what feels good or bad; Stage 2 = toddler years—the child learns â€Å"right† and â€Å"wrong† from what she or he is told by others; Stage 3 = preschool years—the child begins to int ernalize family values as his or her own, and begins to perceive the consequences of his or her behavior; Stage 4 = ages 7-10 years—the child begins to question the infallibility of parents, teachers, and other adults, and develops a strong sense of â€Å"should† and â€Å"should not† Stage 5 = preteen and teenage years—peers, rather than adults, become of ultimate importance to the child, who begins to try on different values systems to see which fits best; teens also become more aware of and concerned with the larger society, and begin to reason more abstractly about â€Å"right† and â€Å"wrong.† Read more: Moral Development – STAGES OF MORAL DEVELOPMENT – Lawrence Kohlberg, Mean Example, Morality, and Social – JRank Articles http://psychology.jrank.org/pages/431/Moral-Development.html#ixzz2R8sxnA1w Counseling Issues Duty to ‘Warn and Protect’ not in Texas is one counseling issue that I am concerned about; what concerns me the most about this statue is According to the Texas Laws mental health counselors do not have a duty to warn nor protect third parties or intended victims once a client has made specific threats to harm the individual. This law was designed to protect mental health counselors from being responsible for notifying anyone of intended harm. â€Å"The statue classifies communications between a mental health professional(s) and their client(s) as confidential and prohibits mental-health professionals from disclosing them to the third party unless an exception applies.† (FN17)(Texas Supreme Court, 1999). The exceptions to the law are: â€Å"reporting child abuse or neglect, reporting HIV status to a spouse, medical personnel, or law enforcement, and report imminent danger to police officer if the client poses a threat to him/herself or others.† (The Family Code, section 261.101(a-c) (Texas Supreme Court, 1999). In the case Thapar v. Zezulka, rendered by the Texas Supreme Court in 1999, stipulated that mental health providers do not incur a duty to warn and protect (Dalrymple, 1999; Grinfeld, 1999; Texas Supreme Court, 1999). Specifically, the opinion written for a unanimous court by Justice Craig T. Enoch stated that, â€Å"we refrain from imposing on mental health professionals a duty to warn third parties of a patient’s threats† (FN1) (Texas Supreme Court, 199 9). By implementing several of the Ethical Decision Models (Rational Model, Collaborative Model, and Integrative Model), I believe a peaceful resolution can be accomplished when a counselor is faced with the ethical decision of whether to inform a third party that intended harm has been conveyed. Although the law in Texas states, â€Å"we as counselors are not obligated to warn nor protect a third party,† we can always defer to The Code of Ethics (2005) which states, â€Å"A.1.a. Primary Responsibility: The primary responsibility of counselors is to respect the dignity and to promote the welfare of clients. B.1.c. Respect for Confidentiality: Counselors do not share confidential information without client consent or without sound legal or ethical justification. B.2.a. Danger and Legal Requirements: The general requirement that counselors keep information confidential does not apply when disclosure is required to protect clients or identified others from serious harm.† (ACA Code of Ethics, 2005). Implementing an EDM, making reference to the ACA code of ethics, and consulting with a supervisor/colleagues will help the make a sound and ethical decision whether to warn or protect. Although the law in Texas states, â€Å"we as counselors are not obligated to warn nor protect a third party,† we can always defer to The Code of Ethics (2005) which states, â€Å"A.1.a. Primary Responsibility: The primary responsibility of counselors is to respect the dignity and to promote the welfare of clients. B.1.c. Respect for Confidentiality: Counselors do not share confidential information without client consent or without sound legal or ethical justification. B.2.a. Danger and Legal Requirements: The general requirement that counselors keep information confidential does not apply when disclosure is required to protect clients or identified others from serious harm.† (ACA Code of Ethics, 2005). Implementing an EDM, making reference to the ACA code of ethics, and consulting with a supervisor/colleagues will help the make a sound and ethical decision whether to warn or protect. Client confidentiality is another issue that I think would pose a problem for me as a counselor, upon reading the landmark case â€Å"United States of America, Plaintiff v. Robert Allen Romo (2005).† â€Å"This case arises out of a confession Romo made during a meeting with Donald LaPlante, the Program Director at the Dawson County Adult Correction and Detention Facility where Romo was incarcerated.   LaPlante is a licensed professional counselor whose job included providing inmates with psychological counseling and a host of other duties, ranging from arranging social events to providing classes and acting as a case manager.   Before the meeting that sparked the chain of events leading to Romo’s conviction, LaPlante had provided Romo with mental health treatment during voluntary counseling sessions.† (United States of America, Plaintiff v. Robert Allen Romo (2005) I realized that it does matter to clients if you discuss with them informed consent a nd confidentiality they can still file some sort of legal litigation against the counselor if they felt like the counselor violated any of their rights. Non-sexual relationship is one boundary issue I can foresee (providing counseling services to family members), pg 210. Counseling minors One ethical and legal challenge I think would be an issue for me is confidentiality; â€Å"knowing when and with whom to share the information the minor has shared in the counseling session.† Once you have built a rapport with the client you do not want to betray the trust of the client. The second issue would be parental rights and making sure the counselors has the client’s best interest at hand; when counseling minor clients it is best to make sure everything is explained on the first visit and that both the parent/legal guardian and client understands the details of the informed consent form. Since the laws vary from state to state, I know it would be beneficial for me as a counselor to use the following ACA Codes of Ethics to handle such issues: B.5.b.(Responsibility to Parents and Legal Guardians) states, â€Å" Counselors inform parents and legal guardians about the role of counselors and the confidential nature of the counseling relationship. Counselors are sensitive to the cultural diversity of families and respect the inherent rights and responsibilities of parents and guardians over the welfare of their children/charges according to the law. Counselors work to establish, as appropriate, collaborative relationships with parents/guardians to best serve the client.† (ACA Ethical Standards Casebook, 2006, p.35) B.5.c. (Release of Confidential Information) â€Å"When counseling minor clients counselors seek permission from an appropriate third party to disclose information. In such instances, counselors inform clients consistent with their level of understanding and take culturally appropriate measures to safeguard client confidentiality.† (ACA Ethical Standards Casebook, 2006, p.35)