Analysis of the Indian Civil Aviation Industry

Analysis of the Indian Civil Aviation Industry Civil Aviation Scenario Evolution In December 1910, a party from Belgium and two from England with many aircrafts arrived in India. The first amongst them was famous Humber motor companies. The team was led by Capt WG Windham, comprising two pilots, one French and one English and two mechanics. After reaching Bombay (Mumbai) they proceeded to Allahabad to demonstrate the aircraft at the Industrial Exhibition due to be held there shortly. The first actual flight was successfully attained by Mr. David in a Beriot on the 10th of December 1910 circled the Polo ground at a height of 25 to 30 feet. The second aircraft flew the next day, December 11, 1910, under the control of French Pilot Piguet and carried the first passenger in India. He was one of the sons of the Maharaja of Benares In a show at Tollygunj, near Calcutta on December 21, 1910, in a show Baron flew with a lady passenger Mrs. N.C. Sen who thus became the first woman in India to get airborne. The history of civil aviation in India started with its first commercial flight on February 18, 1911. It was a journey from Allahabad to Naini made by a French pilot Monseigneur Piguet covering a distance of about 10 km over the river Yamuna. The first domestic air route Karachi-Delhi began in December 1912 by the Indian State Air Services in collaboration with the Imperial Airways, UK. It was actual extension of London-Karachi flight The Indian aviation gathered momentum after three years (1915) with the opening of a regular airmail service between Karachi and Madras by the first Indian airline- Tata Sons Limited October 15, 1932, JRD Tata started Tata Aviation and piloted the first carriage of mail from Karachi to Bombay. Tata Aviation later became Air India. At the time of Independence, there were 9 air transport companies operating in India. Tata Airlines, Indian National Airways, Air Service of India, Deccan Airways, Ambica Airways, Bharat Airways, Mistry Airways and Oriental Airways Air Corporation Act of 1953 was passed nationalising all airlines. Air India International took over the international traffic and Indian Airlines Corporation the domestic. Sector structure/Market size The Indian civil aviation industry is among the fastest growing industries in the world with its growth rate of 18% per annum. Number of players as well as the number of aircraft is increasing in India at it is mainly due to the open sky policy of the government, because of which many overseas players are entering in the aviation market. Today, private airlines account for around 75 per cent share of the domestic aviation market. India was at 12th position in the world’s aviation market in 2006, but it has improved its position holding 9th position at present.In the year 2006 the domestic air services were available at 75 airport in India which has improved up to 82 airports now. Month-wise Indian Scheduled Domestic Operation (Aircraft Kms Flown) of Civil Aviation in India (May 1988 to December 2008) In India the air passenger travel is increasing at about 25% a year since the aviation sector opened up the skies to private carriers. Government has estimated that by 2025 the growth of aviation sector in India will outpace the global average. Currently the aviation sector is going through bad phase which started from 2008 after economic slowdown hit the market in 2008, while year 2007 was the best ever in terms of growth for Indias civil aviation sector. The domestic airlines passenger load increased by 36.47 % (to 317.29 lakh passengers) in the first three quarters of 2007. International Air Transport Association (IATA), estimated about the Indian aviation sector that India will contribute significantly to global air travel. This contribution which was US$ 5.1 billion in 2007 will soon cross US$ 5.6 billion after the market condition will be stable and then it will grow significantly. In 2007 market research firm PhoCus estimated that domestic air traffic will be more than double and touch 86.1 million passengers by 2010, up from 32.2 million passengers in 2007. But after economic slowdown this estimate may take some time to achieve after 2010. Opportunities Aviation sector is going to play a major role in terms of employment in this sector. There is going to be huge demand for technical and administrative employees in this sector due to the vast growth of aviation sector in the future. Aviation sector is not only limited up to pilots and air-hostesses but there are many employment options in this sector which are related to aviation and without which industry cannot function. Some of the Operations jobs include: Pilots, airhostesses, air traffic controller, cabin safety instructor, in-flight managers, In-flight base managers, cabin services instructor, maintenance controllers, aircraft maintenance engineers, quality control manager, cargo officers and ground staff. There is also a wide range of positions on the ground and these include the services of mechanics, baggage handlers, ticket agents and reservations agents. Potential for Growth and its future The Indian Civil Aviation market was worth US$ 5.6 billion in 2008 which has grown at a compound annual growth rate (CAGR) of 18 per cent. According to Centre for Asia Pacific Aviation (CAPA) the domestic traffic will increase by 25% to 30% till 2010. Also international traffic will grow by 15 per cent and total market will have more than 100 million passengers by 2010. At present the Indias civil aviation passenger growth is 20% and it is one of the highest in the world. By 2020, 400 million Indian passengers are likely to be airborne. More than 100 million passenger in which 60 million will be domestic passengers and around 3.4 million tons of cargo per annum are expected to handle by the Indian airports by 2020. There are many significant steps which are still to be taken by the government to propel growth in the Indian civil aviation sector. Indian government is already working on its plans to modernize existing airports by 2010 and is investing more than US$ 9 billion in the project. There is also a plan of the government to develop around 300 unused airstrips. Kapil Kaul, CEO India Middle East, Centre for Asia Pacific Aviation (CAPA), in an interview said that Indias civil aviation passenger growth is among the highest in the world. â€Å"The sector is slated to cruise far ahead of other Asian giants like China or even strong economies like France and Australia. The number of passengers who will be airborne by 2020 is a whopping 400 million.† The markets being as it is holds great promise for potential investors and numerous International no-frills budget carriers are making a beeline for India. With so much activity in the sector there is a tremendous need for personnel as well. While earlier, the airline industry was largely government owned and perceived as regulated and also a tad boring, with private and international players entering the market, opting for a career in the airlines has become both a lucrative and glamorous option To meet the growing demand, Indian scheduled carriers are placing major orders for aircraft. Based on press reports, Indian carriers placed orders over US $12 billion at the 2005 Paris Air Show. Of the 280 aircraft order received by Airbus, 135 are from Indian carriers. Moreover, of the $50 billion that Airbus can earn from these deals, the contribution of Indian carriers is over $15 billion. Airbus forecasts that the number of new aircraft it would sell to Indian carriers would go up to 400 by the year 2023. This will make the India the third largest market for new aircraft in Asia, behind China (1,790) and Japan (640), according to Airbus Global Market Forecast 2004-2023. The aviation industry is of the view that the European aircraft maker may have to again revise its projection upwards. The report further states that Boeing expects India to buy aircraft worth $35 billion in the next 20years. As per a press report, the Center for Asia Pacific Aviation (CAPA) estimates India domestic airlines would need 650 new aircraft by 2012, up from the current 210. Every new carrier launched in the country will need to buy a minimum of five aircraft to start operations, as stipulated under the license condition, in the next 12 months. Inter Globe Enterprises has placed firm orders for 100 aircraft worth $6.5 billion. The anticipated fleet augmentation planned by airlines of India are shown in Table Source DGCA It will be observed that India is likely to see large increase in aircraft registered in India and operation of such fleet would result in straining of aviation infrastructure. Policy decisions have been undertaken to enhance the airport infrastructure. Another component of aviation infrastructure that is of crucial importance to growth of Civil Aviation is up-gradation of capacity to train critical manpower. The training for making available operating crew and maintenance personnel is an expensive exercise and requires long gestation period. Prior to liberalization in early 90’s, the two State-owned airlines had established elaborate training infrastructure to train critical manpower for their needs. The training plans were evolved based on its fleet expansion plans. In the first phase of domestic market liberalization in early 90s the requirement of critical manpower had placed a strain on availability of this human resource. Large scale poaching of critical manpower from one airline to another had been resorted to. The growth of fleet in the first half of the current decade had again resulted in shortage of operating crew and maintenance personnel. The series of steps have been taken recently to meet the present shortfall of cockpit crew that include increase of eligibility age of pilots from 60 years to 65, permission to ex-pat pilots to operate airline services etc. Industry is still facing problem besides opening up of doors for foreign pilots. In fact DGCA has taken a lead role to alleviate the situation of shortage of pilots in all possible manners without compromising the safety aspects. Private airlines are equipping themselves with flight simulators for pilot training including recurrent checks. Indian Airlines has been inducting CPL holders for its training program at CTE Hyderabad. Air India has embarked on a planned program to cater to long-term requirement of pilots by resorting to induct trainees for outsourced training to PPL CPL level thus enabling them to be inducting into ab-initio training program at its training establishment. Based on the fleet augmentation plans of various airlines and expected increase in the number of airlines, the requirement of critical manpower is expected to be the key factor in maintaining sufficient operating capacity to meet the growing demand for air travel in the country. Demand For Operating Crew The requirement of operating crew, of which cockpit crew is most critical due to long gestation period in training and need for elaborate and expensive infrastructure involving training aides such as aircraft simulators and other equipment in addition to training aircraft. The demand for operating crew in the country is based on the combined fleet augmentation proposed by all existing scheduled airlines and the prospective entrants into the air transportation business. Apart from schedule operators, pilots are also required by a large number of existing non-scheduled operators involved in charter operations and also for corporate aircraft owned by large business houses. Since the unprecedented growth rates achieved in the domestic market, and huge orders placed by airlines for new aircraft to cater to expected passenger carriage in the market, government, airlines, and other research institutions involved in civil aviation have been making projections for pilots requirements in the country. The projection by various bodies are at variance depending upon their estimation of the size of the market and expectations of the fleet size. The Center for Asia Pacific Aviation (CAPA) estimates that Indias domestic airlines would need 650 new aircraft by 2012, up from the current 210. This projection results in annual induction of over 70 aircraft that would require over 500 pilots per year for the new aircraft yet to be inducted. While evaluating the emerging aviation scenario, Kaw Committee (2006) had the following to say about critical manpower requirements to meet future needs: â€Å"Induction of large number of aircraft would require more than thousand additional type-rated pilots and equally large number of type-rated engineers in span of a decade, to meet the growth requirements, in addition to the recurring requirement of the licensed personnel. At present, most of the private operators get their pilots and engineers trained at the facilities of aircraft manufacturers or approved training organizations abroad. Considering the huge potential of training engineers and pilots in the country, some agencies, including aircraft manufacturers are thinking of establishing type-training facilities in India. DGCA will have to be strengthened to conduct examinations and licensing of large number of pilots and engineers required to operate and maintain the additional aircraft being acquired†. Currently there are 1650 ALTP commercial pilots licensed by the DGCA to meet requirements of over 200 aircraft operated by scheduled airlines and 2300 CPL holders meeting the airlines and general aviation aircraft. There are about 500 expatriate pilots assisting airlines in keeping airlines aircraft flying. The total requirement of pilots that would have to be trained from initial stage should take into account natural wastage on account of superannuation. FAST estimates consolidated additional requirement of pilots for the Scheduled/ non-scheduled airlines and the corporate sector at around 3000 pilots during the five years (2007-2012). Broad estimation of the requirement is shown below in Table In the present civil aviation scenario, the fleet plans of various airlines can undergo changes keeping in view growth rates on micro basis and intensity of future competition. The projection of requirements of pilots on year-to-year basis is therefore fraught with uncertainties, but a projection of pilot requirement on a longer time horizon is likely to be more realistic. Demand for Pilot Training Apart from air transportation of passengers on scheduled services and corporate travel, another potential area that has not seen much development is the need of air transportation for disaster management and medical relief /evacuation. In the coming years the use of small aircraft /helicopters is likely to become prevalent and air linking of district centers with State capitals/ major towns will be necessary. Operation of aircraft for this sector of aviation will also add to requirements of pilots. It may be noted that there is great demand for Pilot Training in India that has arisen due to phenomenal growth in air travel spurred by economic growth during the past. The present policies of the Government of India pertaining to emphasis on infrastructure and services sector leads to a very positive economic outlook that will have impact on the air transportation sector. The growth of traffic recorded in recent years is likely to be sustained in the immediate future and is expected to stabilize at a reasonable level. The sustenance of this growth is dependent on the growth and development of Aviation infrastructure of which training of critical manpower is a very important feature. Type of Training Flying Training Institutes offer various levels of trainings for commercial flying. These include Private Pilot License (PPL), Commercial Pilot License (CPL), Multi-engine Rating, and Instrument rating; apart from Commercial Helicopter Pilot License, Flight Instructor License/Rating, and Airline Transport Pilot License. In the first phase it is proposed to impart training for PPL and CPL as these trainings involve flying training on single engine aircraft. For multi engine rating and IR rating, induction of twin-engine aircraft is required along with requisite training aids such as specific aircraft simulator. Eligibility Qualifications for Entry to PPL/ CPL course Candidates for PPL training should have completed Senior Secondary Examination (10+2). Minimum age of 17 years is prescribed for induction into the course. PPL holders would be eligible to be inducted for CPL training and should have PPL issued with 50 hours of flying and not less than 10 hours of solo flying within a period of preceding 12 months. The flying club issues student Pilot license after checking the general capability of the student to continue flying training such as enough leg space in the cockpit. Selection Procedure The selection of candidates may be done on the basis of a written examination followed by Pilot Aptitude Test and Interview. The written examination for entry into PPL course will be on general subjects such as English, Physics, Mathematics and Reasoning, where as for CPL written exam will be for subjects of Air Navigation, Aviation Met, Technical general and Air Regulations. Ground Courses and Flying Training Ground Courses: Topics for the ground courses that are laid down by DGCA for PPL and CPL along with duration are shown in Table 2.1. Trainee Intake Course Duration PPL Course: The Institute in the First Phase will cater to induction of a batch of 20 trainees for PPL course. Initially induction will be carried out twice a year. During the second year of operation of institute four batches of 20 trainees each are proposed to be inducted at three monthly intervals. Trainees are expected to complete PPL Course, Simulator and Flying Training in six months. The course work and part simulator training will be completed in three months followed by intensive flying training of 50 flying hours per trainee. CPL Course: The CPL course will commence during the second year of institutes’ functioning Training Infrastructure Flying and Gliding Clubs: It is observed that Flying and Gliding Clubs have been receiving financial assistance from the Central Government ever since their inception in 1928. This assistance gradually increased on the recommendation from various Committees set up for the purpose from time to time. It is also to be noted that in addition to receiving subvention from the Central Government most of the clubs were receiving grants and donations from the State Governments and other private organizations besides the revenue earned through: Flying fees paid by the trainees Membership fees/subscriptions etc. The subvention budget was raised to 3.5 crores by the Ministry of Civil Aviation. Later on this subvention was withdrawn except for SC/ST candidates. All the flying clubs except private one’s became sick due to non-subsidy to students as it was a great burden for pilot student to pay for full flying cost. Flying clubs are generally private bodies headed by Deputy Commissioner of the district, with Sr. public person and DGCA member on its board. Equipment i.e. aircraft are purchased by the Central Government (DGCA or Aero Club of India) and given to flying clubs but remain property of DGCA. Indira Gandhi Rashtriya Udan Academy (IGRUA) was set up in Fursatganj in U.P. to provide quality training to the pilots. The Academy envisaged a quantum improvement in the standards of flying and ground training of Commercial Pilots in the country. For this, the Academy is equipped with most modern and sophisticated trainer aircraft, up-to-date audiovisual training aids and other facilities for effective ground training. Highly qualified flying and ground instructors, with long experience in the field of aviation and flying training were recruited. Academy is funded by Ministry of Civil Aviation (Air India Indian) and is equipped with latest aircraft /simulator. It has produced a large number of pilots who are serving airlines. IGRUA charges Rs.16.5 lakhs for CPL training. IGRUA uses mainly TB-20 trainer aircraft and it charges per flying hour is Rs.12, 000/-. IGRUA have been permitted to admit 100 flying students per year. IGRUA is supported to the tune of Rs.10 crores (approximately) on its recurring annual account. Proposed Training Establishment At Gondia in Mahrashtra: Ministry of Civil Aviation (MCA) is in the process of setting up National Flying Training Institute (NFTI) at Gondia in Maharastra. MCA is concerned regarding shortage of world-class facilities for training the pilots in India to meet the large-scale demand of well-trained pilots. Ministry also envisages that specific infrastructure meeting the needs of International Standardized Flying Training including the research and development in the fields of pilot training, aviation related innovated and practical courses comparable to the requirements of standards of CAR, issued by DGCA (India). ICAO, FAA (of USA) and other international training centers of repute should be established. NFTI is a green field training center and aims to develop NFTI investment as viable economic units. NFTI may have institutional structure which meet the DGCA requirements for flying training. These are Society registered under the Societies Registration Act, 1860 Statutory body under an Act of Parliament Company incorporated under the Companies Act, 1956 Foreign Training Institutes/ Schools: Indian flying students also go abroad for flying training and obtaining PPL and CPL in foreign Academies predominantly in USA, Canada and Australia. The pilots having successfully completed the training program receive license issued by Civil Aviation Authorities of respective countries. DGCA accepts the license of foreign aviation authority and endorses the license after passing of the students of three papers and flight check by the DGCA pilot. The duration of obtaining CPL license is comparatively much less and may cost about $US 20,000/- as fees. The student pilot also pays additional expenditure for boarding and lodging and traveling Process For Grant Of Approval For Flying Training Institute Approval for setting up Flying Training Institute is issued by the DGCA in accordance with CAR Section 7, Series â€Å"D† Part 1 issued in July 1999 and subsequently amended from time to time. This document details eligibility requirements, and describes the process in details along with minimum requirements relating to infrastructure, procedures and manpower for grant of approval This document is applicable for flying training activities with aircraft having maximum certified take off mass not exceeding 5700 kgs. The document also encloses formats for applicants to be utilized at various stages in the process. Eligibility requirements for issue of approval: Approval of organizations undertaking flying training activities can be granted to Central Government or state owned or controlled ones. Indian citizens, Nonresident Indian, or overseas corporate bodies can also apply for setting up flying training institutes. A company registered in India having its principal place of business within India with or without foreign equity participation (excluding NRI equity as approved by Government of India from time to time is covered in eligibility criteria. Stages of Approval Under the prescribed process, approvals are granted stage-wise. The stages are as under: Grant of Initial ‘No Objection Certificate’ Permission for import/ acquisition of Aircraft Grant of Approval Grant of Initial No Objection Certificate (NOC) Stage 1 Application is to be made to the Director General of Civil Aviation in prescribed format. For issue of initial NOC, Security clearance and FIPB approval (in case of foreign equity participation) is necessary. Application should contain following information with supporting documents. Following details are required to be furnished in the application: Memorandum of Articles of Association duly registered with the competent authority No Objection Certificate from Airports Authority of India from air traffic point of view No Objection Certificate from Owner of the airport for use of airport for setting up of training institute and for providing parking and Hangar space. Financial soundness of applicant Project report giving details of organization, manpower, training plans, infrastructure and equipment for the institute, source of funding, viability of project etc. Details of Directors of the Board and Chairman/ CEO for necessary security clearance Type and number of aircraft and simulator and source of procurement Submission of requisite fee Initial NOC will be granted after the application is found satisfactory from the point of view of need of training institute, airport capacity and constraints at the proposed airport, suitability of proposed aircraft type, aircraft maintenance arrangements etc. This NOC is valid for one and half years during which applicant will take necessary steps to comply with requirements and acquire final approval for starting the training institute. Issue of permission to import of Aircraft Stage 2 Initial NOC holder will take necessary steps to the satisfaction of DGCA for establishing required infrastructure, recruitment and training of manpower, preparation and approval of training manual, maintenance system manual, MEL, Maintenance schedules, security program etc. Initial NOC holder will furnish necessary information to show that the specific aircraft proposed to be imported meets the requirements for import of aircraft and that all mandatory modifications and airworthiness directives are complied. On demonstration of necessary preparedness the initial NOC holder will apply to the Ministry of Civil Aviation for grant of permission to import/ acquire aircraft in the prescribed format. The permission to import aircraft shall be valid for one year extendable by three months on one time basis. For new aircraft extension may be permitted for actual lead-time of the delivery. Grant of Approval Stage 3 For the final grant of approval institute shall have necessary training aids, Hangars, suitable space for aircraft maintenance, well lighted workshops and fire fighting / safety equipments. Well-marked and adequate parking bays and taxi tracks along with facilities for mooring should be available. Adequate space for engineering, maintenance, operations and classrooms should be in place. The institute should have a well equipped library with aviation books, literature, up to date flying training circulars/ compendium, CARs, AICs, AIP, Jeppson Charts route maps etc. Chief flight instructor/ Flight Instructor, in-charge and Quality control Manager should be recruited for whom DGCA approval is obtained. Adequate number of flight instructors, ground instructors and engineering personnel should be employed. Specific approval is necessary for employment of foreign licensed pilots/ engineers. On completion of necessary preparedness, applicant will apply to DGCA for grant of approval to the flying institute. The application should cover the following aspects: Particulars of specific aircraft with installation of mandatory instruments and equipment Certification of Registration and Certificate of airworthiness of the aircraft Approval of the maintenance organization Name, license/ approvals and endorsements of flight instructors/ engineers Comprehensive insurance policy covering aircraft, occupants and third party risks in accordance with requirement Compliance of relevant CAR and conditions for initial NOC if any Details of facilities, equipment, procedures and necessary manpower. On satisfactory review by the DGCA, a team constituted in DGCA will carry out inspection of the institute. If the institute meets all requirements DGCA will grant approval to the institute that shall be valid for one year and shall be renewed each year. Sources For Instructors The sources from where instructors could be recruited are as follows: From general aviation Indian Air Force International Market There is a shortage of instructors at present in the Indian aviation market. However, NEC can approach Indian Air force for instructors on deputation. The instructors can also be sourced from the International market. Company Profile (Foundation for Aviation and Sustainable Tourism) Vision of FAST Foundation for Aviation and Sustainable Tourism (FAST) to be a Think Tank evaluating policies and a Research Organization of Repute. About FAST(Foundation for Aviation and Sustainable Tourism) Foundation for Aviation and Sustainable Tourism(FAST) is an international. non-government, non-political, autonomous research organization was founded in the year 1992 .It is not working for the profit but its objective is to promote the Civil Aviation and Tourism in harmony with the environment and provides common platform for the industry and the government to find a workable solutions. Its main aim is to function as an institutional base for the study of all aspects with regard to civil aviation and tourism including their management Organization Former Secretary General, International Civil Aviation Organization (ICAO), Dr. S.S.Siddhu is the founder Chairman of this organization. Lt. Gen (Retd.) K M Seth, PVSM, AVSM, former Governor of Tripura and Chattisgarh is the president. Former Executive Director, Airport Authority of India (AAI) Mr. Gurcharan Bhatura is the director general of FAST. Mr. B.K Joshi, former Joint Director General of Civil Aviation is the secretary general. Board of trustees FAST is managed by the board with following trustees: Lt. Gen. (Retd.) K.M. Seth PVSM, AVSM Former Governor of Tripura Chattisgarh Mr. N.N. Jha IAS (Retd.) Mr. B.N. Jha,IAS (Retd.) Mr. Uma S Bhartia, Manager Director India Glycols Ltd Mr. P.R.S. Oberoi CMD, East India Hotels (Oberoi group of hotels) Mr. Siddhanta Sharma, Chairman Spice Jet Mr. VP Agrawal, Member (Planning) AAI Mr. Gurcharan Bhatura, Director General, FAST. Executive Council Lt. Gen. (Retd.) K.M. Seth AVSM, PVSM former Governor of Tripura Chhattisgarh, President FAST Mr. M. P. Bezbaruah, IAS (Retd.) Mr. Raghu Menon, CMD, National Aviation Company of India Limited Mr. Anil Bhandari, MD International Travel House Ltd Mr. Kanu Gohain, Director General Civil Aviation Mr. Parvez Dewan, CMD India Tourism Development Corporation Mr. R. Krishnan, Consulting Editor Cruising Heights Mr. Raman Sidhu India Head, Corp Affairs Fidelity international Mr. MM Bhagat, Chairman Bhagat Group Mr. Gurcharan Bhatura, Director and Secretary General Mr. B. K. Joshi, Treasurer Corporate Members and Members from industry There are many members of this organization which is from the aviation industry. Some are as follows : Mr.Naresh Goyal – (

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

Tobacco Essay -- social issues

Tobacco Tobacco is one of the leading preventable causes of death in the United States. Nicotine, which is an alkaloid derived from the tobacco plant, is a potent chemical that has powerful effects on the human body, especially when administered rapidly or at high doses. Prenatal exposure to nicotine is associated with adverse reproductive outcomes, including altered neural structure and functioning, cognitive deficits, and behavior problems in the offspring (9). At least 20% - 30% of pregnant women are estimated to smoke cigarettes, although smoking is associated with low birth weight, prematurity and infant mortality. In the United States, smoking accounts annually for estimated fetal deaths ranging from 19,000 to 141,000, for 1,900 to 4,800 deaths during or immediately after parturition, and for 1,200 to 2,200 death from Sudden Infant Death Syndrome (7). Maternal smoking has been implicated in long term deficits in infant mental development and adverse behavioral problems in children such as attention disorder. Nicotine crosses the human placenta and has direct effects on the developing fetus. Pre-clinical studies suggest that maternal smoking during pregnancy produces changes on the offspring's neural functioning, including reductions in uptake of serotonin, alterations in dopaminergic systems, alterations in peripheral and central noradrenergic neurons, and changes in DNA and RNA synthesis in the brain (9). Children prenatally exposed to nicotine consistently score lower in the two subcategories of expressive language and conceptual comprehension. Evidence from studies of human neonates suggests that maternal smoking during pregnancy is associated with increased rates of neurobehavioral difficulties. Several studies have linked maternal smoking during pregnancy with childhood inattention, impulsivity, and motor hyperactivity in offspring. Similarly, maternal smoking during pregnancy has been associated with parent-teacher ratings of conduct problems in children and a criminal record in young adults. A study by Yousef Tzabi suggested that cigarette smoking during pregnancy may be one of the causes of hyperactivity and learning deficits in children. In a laboratory study with Sprague-Dawley mice, it was shown that hyperactive male pups that were exposed to nicotine prenatally had significantly higher nicotinic receptor concentrations in the... ...ironment is relatively hypoxic, and corresponding cardiac functional deficits would thus contribute to the high incidence of fetal mortality. Just as impaired cardiac function during hypoxia in neonatal rats can account for the increase in mortality with prolonged hypoxia, comparable effects in man would provide a mechanism for cardiovascular collapse and consequent brain damage or death during delivery. The consequences of smoking during pregnancy are very dramatic. If clinicians are able to convince their patients to stop smoking early in their pregnancy, a major impact may be made on the incidence of low birth weight infants, perinatal morbidity and mortality, as well as cognitive deficits and behavioral problems in the offspring. A strong statement has to be made on the issue of tobacco exposure during pregnancy. This could be accomplished by informing women that their infants may not only be "smaller" than their nonsmoking counterparts, but their infants may also have transient or permanent changes in their lung and brain ultrastructure. A patient who is informed of these possible long-term effects of nicotine on her child may be more successful with her smoking cessation.

The doom of apple :: essays research papers

The Ethics of War   Ã‚  Ã‚  Ã‚  Ã‚  Unrestricted submarine warfare is often considered a shady practice in a time of war. Unrestricted submarine warfare is when one country sets up an area that is declared a war-zone, any ships that enter this zone, military or civilian, will be fired upon without warning. Is this right? In my essay, I will weigh the pros and cons of this tactic used by the German’s in WWI.   Ã‚  Ã‚  Ã‚  Ã‚  The practice of unrestricted warfare cost the lives of many innocent people. No warning is given so there is no chance to surrender or abandon ship! And foremost, unrestricted submarine warfare violates the so-called rules of war. In the case of the Lucitania, it was believed to be carrying supplies (this was true) and the ship could have later become a threat to Germany if converted into a war or transport ship. But, at the time, the ship or its 2000 passengers were not a threat to U-20, yet the Lucitiana was sunk without warning and with massive losses.   Ã‚  Ã‚  Ã‚  Ã‚  Unrestricted submarine warfare is a very shady, but effective tactic, nonetheless. When implemented, little to no supplies reach your enemies, this gives a tactical strangle hold on a war. Why is no warring given on civilian ships? To a U-boat stealth and surprise are its only defenses. Once a U-boat reveals itself, it is open to ramming and attack thus endangering the crew. In most cases, keeping your troops safe comes above keeping the enemies safe. Why would someone need to attack a civilian ship anyway? Because they may be carrying supplies. To say unrestricted submarine warfare violates the â€Å"rules† of war then why is the opposition violating the â€Å"rules† by putting troops and supplies on a civilian ship making it a target of submarines? Using the lives of innocent people as a shield to protect supplies is just as bad as firing on an unarmed ship without warning.   Ã‚  Ã‚  Ã‚  Ã‚  Is unrestricted submarine warfare right? In my opinion a submarine must give civilian ships a chance to abandon ship. A submarine does not need to expose itself to send a warning, send a radio ultimatum, and then a warning shot. How much of a treat could a passenger ship pose anyway?

Henna in Arab countries and India Essay

Henna is a type of temporary tattoo that dyes the skin for several weeks. Henna is made out of a plant that is dried, ground to a dust and then made in to a paste by adding essential oils. Henna got its start in the Arab culture and remains popular to this day and growing in popularity in other countries. The paste that is made is placed into a cone or bag that closely resembles that used by cake decorators. The paste is applied to the skin in intricate designs and as it dries and hardens it begins to fall off leaving behind an orange tint. Designs made from henna are completely free form and have no specific meaning and are meant for the sheer beauty of the art with no religious or cultural meaning. The henna tattoo can consist of nearly any pattern or series of patterns and the more elaborate the design the more erotic and sensual it is. Henna is made primarily of several design styles which include flowers, paisley designs, intricate lines, shading and doily designs. These pattern s can wrap around your fingers, wrist, ankle or any other curved part of your body. Henna in the Arab World Permanent tattoos in the Muslim world are greatly frowned upon, which makes henna a very popular alternative that dates back hundreds of years and is steeped in tradition. Henna makes it easy to get a tattoo without it being against Muslim traditions, since they only last a few weeks. These beautifully intricate designs are applied mainly to young women to glamorize themselves (older women will also sometimes wear henna) to their husbands after their wedding and during the ceremony. Arabic Henna throughout the World Henna can be seen in countries all around the globe despite its roots being firmly planted in the Middle East existing in India, Egypt, Pakistan, and Morocco among just a few. The typical style in the originating country is that of large floral patterns painted all over the body, but as the design spreads it changes to meet the needs of the people. The United States, for instance, has taken henna and made it into pure skin decoration. Unlike the Middle East, the main reason to get henna is for show and not some profound meaning. The sentimental factors involved have been removed and what’s left is something very basic and watered down. Henna done at the circus, carnivals and Renaissance fairs are just several examples of western style henna. I went to (global village) in Indian village to meet some women’s who knows more about the henna and I had an interview with an old lady that work there in henna shop she’s Indian: (Me): Could you tell us a bit about the tradition of henna and how it has transformed into a popular body art? (Old lady): Henna has been used down through the ages for thousands of years. Inscriptions place henna in use in Syria as early as 2100 BCE and in the Greek Islands from around 1700 BCE. Henna has usually been utilized with cultures that have a history of Goddess worship. Henna was relatively unknown ten years ago, and it has become more popular with women here in California and around the world. The art of henna is amazing, and now it can be embellished with jewels and glitter to make it sparkle and coordinate with your clothes. Women of all ages use henna for celebrations, and the art has improved and become more popular than ever before through word of mouth and the Internet. (Me): What exactly is henna dye, and what are the ingredients? (Old lady): Henna is the Persian name for a shrub known scientifically as Lawsonia Inermis. It is harvested twice a year, dried and ground into a fine powder. The powder is used to dye hair red and for the ancient eastern art of mehndi. The henna tannic acid is a natural safe vegetable dye that bonds with the collagen in your skin cells and the keratin in the hair leaving a lovely color. (Me): Is henna dye available in different colors or levels of intensity? (Old lady): Different colors or levels of intensity are achieved in several ways. Henna can be steamed on the skin to darken it, or it can be left on for different lengths of time to get darker and lighter stains. Henna will be a shade of red/brown. There are no other safe dyes and henna does not come in black or blue. Allergies Are Rare (Me): Should you test for an allergic reaction prior to applying henna dye to the skin? (Old lady): You can test for henna sensitivity although it is extremely rare. If you use 100% natural henna mixed with safe oils and lemon juice, there is little to no chance of an allergic reaction. If you suspect a henna allergy, please do not apply the henna and be sure to also test for citrus allergies. (me): Even though henna art is temporary, how long should the tattoo last? (Old lady): Henna typically lasts seven to ten days. However, it can last longer than that depending on several variables such as where the henna is placed on the body, how long the henna is on the skin, if the skin is clean, and if the essential oils and henna are good quality. (Me): Could you explain the difference between Indian style designs and Arabic style henna? (Old lady): Indian styles of henna are usually filled with lacy paisleys or flowers and everyday shapes from daily Indian life such as the mango and the peacock. Arabic style is usually a large floral that will have more open space, and the designs will not have a motif like faces or peacocks. Both styles of henna are amazingly beautiful. (me): Is it easy to apply henna yourself? What’s the process? ((Old lady): If you want to apply the henna yourself, you can purchase paste already made or mix it yourself. Henna Caravan sells henna ready to use inside applicator bottles and our Create kits let you mix your own. Which ever you choose, all you have to do is: * Fill your applicator, clean the skin and you are ready to begin. * After the paste is applied, leave the henna on the skin for four to six hours and scrape it off leaving the dyed skin. * The stain will continue to darken over the next 36 hours, and you will have great henna. Tools and Supplies (Me): What tools and implements are needed to create a henna tattoo? (Old lady): You can use a hand rolled cone, a jacquard applicator bottle or other bottle to apply henna. Henna has been applied with sticks, rolled into strings and pressed on the skin, taped areas or stencils and paintbrushes may also be used. (me): Are there any starter kits that are a good investment for a beginning henna artist? (Old lady): I think the Henna Caravan kit is a perfect beginner kit. It comes complete with everything you need to have a successful henna experience – bottles, premium henna, wonderful essential oils and all the tool and tips and patterns to get you started. The jacquard bottle is easy to refill. (me): What rituals or occasions are most popular for henna art? (Old lady): In our experience birthday parties are most popular for every age along with graduation parties and Bar/Bat mitzvahs, Blessing ways are also great fun and so relaxing for everyone. (Me): Summer is a great time of year to flaunt a henna tattoo. If someone is on the fence but wants to experiment with their first henna design, what’s your favorite placement and artwork style? (Old lady): Get something you like whether it is flowers or skulls or geometric patterns, and put it on your ankle or up the leg. If you want more, just repeat the pattern; make three flowers instead of one, so it flows on the body. (Me): Why are some henna darker than others? (Old lady): Mostly this depends on how the henna has been stored. Henna leaves plucked straight off the plant will leave the darkest stain. Leaves that have been powdered need to be carefully preserved; powdered henna that is kept cold in an airtight container is best in my experience. Henna has, today, become a part of all major festivals and celebration. Be it, Purim, Eid, Diwali, Karva Chauth, Passover, Nowruz or Mawlid, it has graced every occasion with its presence. Celebrations like wedding, birth of a baby and birthdays seem to be incomplete without the ceremony of henna. Brides, in the present time, typically have the most complex patterns of henna, to express their greatest joy and wishes for luck. With an improved technology being used for its cultivation, henna available today, has an enhanced dye content and greater artistic potential than earlier.

Accepting Personal Responsibility – Essay

Chapter 2 Accepting Personal Responsibility There is great value in perceiving ourselves as the primary creators of the outcomes and experiences of our lives. At the very least, we are responsible for how we respond to any event, whether the event is of our creation or not. When academic outcomes and experiences are negative, many students blame others, often teachers. When academic outcomes and experiences are positive, many students credit others. Since the cause of their results is seen as existing outside of themselves, these students have no reason to evaluate and possibly change their own behaviors.Students like this typically wait for the world to change while they complain, blame, make excuses, and repeat ineffective behaviors. They may even blame themselves, all the while thinking there is nothing they can do to change their fate. By offering students the opportunity to see how their own choices contribute to their past, present, and future outcomes, we empower them to appro ach life with the beliefs and behaviors of a Creator, thus giving up the passivity and bitterness of a Victim.Empowers Students to . . . 1. Accept a Creator role, taking responsibility for creating the outcome and experiences of their lives (including their education), and reject the Victim role, giving up complaining, blaming, excusing, and paralyzing self-judgment. 2. Master Creator language, understanding that Creators and Victims choose different ways of thinking and speaking about their experiences, consequently changing both their perceptions of reality and the outcomes that they create. . Live more consciously, becoming more aware of their inner aspects—Inner Critic, Inner Defender, and Inner Guide, among others—and the corresponding inner dialogue that dictates students’ subsequent actions. 4. Make wise choices by consciously recognizing important decision points in their lives, identifying all possible options at this point, and making decisions with aw areness of their future consequences. 5.Make mature decisions, choosing to make long-term gain more important than immediate pleasure or immediate escape from discomfort. 6. Replace outer authority with inner authority, and resistance with cooperation. 7. Gain greater control over the outcomes of their lives. Engaging students in the exploration of JOURNAL ENTRIES, CASE STUDIES, ONE STUDENT’S STORY, EMBRACING CHANGE, ON COURSE AT WORK, QUOTATIONS, CARTOONS, FOCUS QUESTIONS, CHAPTER-OPENING CHARTS and STUDY SKILLS.