The Importance of Psycholinguistics in Education

THE IMPORTANCE OF PSYCHOLINGUISTICS IN EDUCATION A newborn baby always has the faculty of wonder . . . Psychology is the studies about human and mind. Psycholinguistics is the study about human and language which they acquire from a newborn baby, till they die. A newborn baby always has the faculty of wonder. That is how it is. If a newborn baby can talk, they will say something about what an extraordinary world it is. As the time goes by, they will acquire the language used by their mom. Children is using their language creatively, no one teaches them how to use the language.Why shall we put a verb after subject (in most language)? It is their nature to learn it. Language is a maturationally controlled behaviour. That is, there is a nature of language which we can learn language by our own, and nurture, in which someone teach us so. When individuals reach a crucial point in their maturation, they are biologically in state of readiness of learning the behaviour. Most of psycholinguis ts agree with these theory, but they still cannot agree with the term of innate.They cannot decide to what extent language ability is separate from other cognitive language. There is a study of the child language acquisition which is done by asking the parents write a diary, make a tape recordings, videotapes, or even controlled experiments. The studies show that child language is not just a degenerate from adult language. At each stage of development the child’s language conforms to a set of rules, a grammar. Although child grammar and adult grammars differ in certain respects, they also share many formal properties.Speaking about the norture of language by the children, it will be connected to the term of applied linguistics. Because here, in applied linguistics, we study about how parents’ language influences their children language. Such a low class parents with a straightforward sentences, middle class parents with the usual language, and high class parents with t heir indirect language. Psycholinguistics is very useful to help us, a teacher candidate, understanding our students in the class. That is, as us is an Indonesian, we shall learn more about Second language Acquisition by the children.

Booting and Question

Question 1 Question1 You want to allow users to access the CD-RW device on your machine from any other host on the network via NFS. Further, you only want them to have read-only access to the device. Which line should you add to the /etc/exports file to allow this? a. /mnt /cdrom *(ro) b. /mnt /cdrom *(r) c. /mnt /cdrom * d. /mnt /cdrom Question 2 Which of the following files defines how FTP connection requests are processed by the TCP Wrapper? a. ftpusers b. inetd. conf c. ftpaccess d. xferlog Question 3 Which of the following devices would be the first SCSI hard disk on a Linux system? . /dev/sd0 b. /dev/sd1 c. /dev/sda d. /dev/sdb Question 4 With a umask value of 112, what are the default permissions assigned to newly created files? a. —x–x-wx b. -rw-rw-r– c. -r-xr-x-r– d. -rw-rw—- Question 5 You are installing Linux on a machine that has had a handful of other operating systems on it previously. During the installation, it becomes apparent that LILO cannot write to the master boot record because another boot loader is already there. What utility should you use to reinstall the MBR and remove what is already there? a. fsck /mbr b. fdisk /mbr . /etc/disktab /mbr d. /sbin/lilo /mbr Question 6 The former administrator of Mercury Technical is no longer employed there. You are the new administrator, but do not know the root password. If you boot into single user mode, you can change the root password, but what command must you give at a LILO prompt to be able to do this? a. linux single b. linux passwd c. linux 3 d. linux one Question 7 You wish to find all the three-letter files in the current directory that end with the letter y. What command should you use? a. ls *y b. ls *y* c. ls y d. ls y* Question 8 Karl has been loaned a machine from the lab to use in evaluating a project he is working on. He is told that there is a known problem on this machine with the ATAPI. Which of the following devices will this problem be most likely to affect? a. Modem b. Sound card c. Video display d. CD-ROM Question 9 Leroy must create a boot disk on his Red Hat workstation. Which utility can he use to accomplish this? a. makedisk b. mkbootdisk c. /sbin/lilo -b d. makeroot Question 10 Kristin is the DHCP administrator for her network. She needs to install the DHCP client software on a number of new machines that have arrived. What package should she install? a. pump b. Squid c. Apache d. Swatch Question 11 Which utility is available in many Linux implementations for use in configuring the sound card? a. sndadmin b. sndmin c. sndconfig d. radius Question 12 Which configuration file is used to identify where system messages are recorded? a. logrotate. conf b. syslog. conf c. conf. modules d. modules. conf Question 13 What search criteria would best be used to find the lines within the MERCURY file about â€Å"clients†? a. grep clients MERCURY b. find clients MERCURY c. sed clients MERCURY d. search clients MERCURY Question 14 Which of the files holds configuration information on how to manage terminal devices (respawn them)? a. /etc/initd b. /etc/inetd c. /etc/inittab d. /dev/inetd Question 15 Which of the following types of modems should be avoided for use with the Linux operating system? a. Internal PCI/ISA b. External Serial c. Winmodems d. Cable Question 16 By default, which of the following files would constitute the Apache document root? a. smb. conf b. httpd. conf c. apache. conf d. index. html Question 17 Which command can be used to rearrange the order of jobs in a spooling queue awaiting printing? . lpc b. lpstat c. lpq d. lpr Question 18 Which field of the /etc/passwd file holds the passwords for users? a. first b. second c. third d. fourth Question 19 It is 3:00 and you are late for a meeting. You need to start the qwerty utility before heading to the meeting so it will run for the next few hours and compile weekly system usage results. Currently you are logged in as a regular user, but the qw erty script requires root permission to run. How should you execute the script? a. su ; qwerty b. su qwerty c. su –c qwerty d. su : qwerty Question 20 Which file system can you not use with the mount utility? a. msdos b. coda c. autofs d. swap Question 21 Which of the following will set the variable DAY equal to FRIDAY? a. DAY FRIDAY b. DAY=FRIDAY c. DAY:FRIDAY d. $DAY FRIDAY Question 22 Which utility can be used to list modules, remove modules, and add modules? a. modprobe b. insmod c. rmmod d. depmod Question 23 A process with a PID number of 1777 has entered runaway mode. You have tried to remove it with a standard kill command, but it will not go away. What command can you use to be assured the process will terminate? a. ill –NOW 1777 b. kill –HUP 1777 c. kill –15 1777 d. kill –9 1777 Question 24 Which of the following runlevels will reboot the system? a. 0 b. 2 c. 4 d. 6 Question 25 You wish to extract an archive from a tape. The archive was created using tar, and you want to copy all the contents from the tape back to the system. What one option must you use with tar to accomplish this? a. c b. x c . v d. r Answers 1. A. The command needs to mount the CD drive (/mnt /cdrom) and make it available to all users (*) in read-only mode (ro). When combined together, this makes the command: /mnt /cdrom *(ro) . B. The /etc/inetd. conf file defines how FTP connection requests are processed by the TCP Wrapper. The /etc/ftpusers file is used to list users who cannot use FTP, whereas /etc/ftpaccess lists rules for users who can access FTP. The /var/log/xferlog holds information about file transfers that have occurred. 3. C. The first SCSI hard disk would be referenced as /dev/sda, while the second would be /dev/sdb, and so on. 4. C. The default permissions for newly created files are 666 (-rw-rw-rw-). The umask value is subtracted from the default, leaving a permission of 554 (-r-xr-xr–). 5. B. The fdisk utility is used to format the disk, and the /mbr option is used to wipe out and clean the existing master boot record. None of the other utilities listed have a /mbr option, making them all incorrect choices. 6. A. You must boot into single user mode at the LILO prompt, and the command to do such is linux single. 7. C. The question mark (? ) stands for any single character. Since you are looking only for three letter names, and you know the last letter to be a â€Å"y†, you must specify any two characters (by using two question marks), followed by the known letter ( y). 8. D. The ATAPI interface is used for devices such as IDE and EIDE. The most likely device within the choices presented to utilize such an interface is the CD-ROM. 9. B. The mkbootdisk utility (found in /sbin) is used to create the boot disk. 10. A. The pump package provides the DHCP/BOOTP client needed to obtain dynamic addresses from a DHCP server. Squid is a proxy package, whereas Apache is used to provide Web services. Swatch is a Perl script that is used to monitor log files. 11. C. The sndconfig utility is used to install and configure sound cards on Linux. 12. B. The /etc/syslog. onf file holds configuration information for system logging. 13. A. To look within a file for matching text, the grep utility is used. The syntax is: grep {what you are looking for} {where you are looking for it} 14. C. The /etc/inittab file holds the initialization table and states that terminal devices should be respawned when terminated (/etc/getty). 15. C. Winmodems use a portion of the Windows opera ting system to operate properly and are notoriously incompatible with most Linux implementations. 16. B. The default document root under Apache is the /etc/httpd/conf/httpd. conf file. 17. A. The lpc utility can be used to rearrange jobs awaiting printing as well as disable/enable a printer or queue and find the status of printers. 18. B. The first field of the /etc/passwd file is the username, while the second holds the password. The third field holds the unique ID number, and the fourth contains the group ID number. 19. C. The –c option with su will prompt you for the root user’s password. Once given, it will then run the command given as the root user. 20. D. The mount utility can be used with any supported filesystem (viewable from the man page for mount) except swap. 21. B. To set a variable equal to a value, merely type it using the syntax: variable=value 22. A. The modprobe command can be used with options to be able to list (-l) or remove modules (-r). By default, it is used to add modules. 23. D. The –9 signal is the most lethal, and will terminate the process immediately. All other signals are weaker than –9. There is no such signal as NOW (choice A). 24. D. Changing to a runlevel of 6 will take the system down and then bring it back up again – effectively performing a warm boot of the system. 25. B. The x option is used with tar to extract a file.

Plantaze

INTERNATIONALIZATION OF SEE-FIRMS Final Group Report Table of content 1 1. 1 1. 2 1. 3 INTRODUCTION †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 1 Background Description †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 1 Research Purpose †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ Structure of the Report†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ 2 2 2. 1 2. 2 RESEARCH METHODOLOGY†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 2 Data Collection †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 2 Data Analysis †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 3 3. 1 3. 2 3. 3 CASE STUDY †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 6 Company Description (www. plantaze. com) †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 6 Internationalization Process†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 7 Internationalization Motives †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ 4 4. 1 4. 2 ANALYSIS AND INTERPRETATION†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦ 11 Theoretical Background †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 11 Application of the Uppsala Model †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â ‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 13 5 5. 1 5. 2 IMPLICATIONS AND LIMITATIONS †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 4 Implications of the Study †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 14 Limitations of the Study †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 15 6 7 8 CONCLUSION AND FUTURE RESEARCH †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚ ¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 15 REFERENCES †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦.. 7 APPENDIX †¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦. 19 1 Introduction In this chapter the background of the case study will be presented, as well as the purpose of the study will be stated, before an overview of the structure of this report is given. 1. 1 Background Description Today, many companies take the step to establish themselves abroad. The motives for internationalization are many. Perhaps the home market is saturated, presence in a certain country grants access to strategic resources or there are cluster effects to be explored in a specific region.The decision makers of the companies that are becoming international have different experience, are in different situations and consider different motives before taking the step into the international market. (Masum/Fernandez 2008, 2) For the past few decades researchers have been debating over existing theories and developing new theories of international business to explain why and how companies internationalize. Internationalization theories are explaining different internationalization processes, which are taking place when companies expand across national borders.Ranges of internationalization have been discussed in various investigations with the conclusion that the majority of these frameworks fail to explain internationalization behavior of various companies, however, this doesn’t mean that they are not usefu l at all. (Pett 2008, 1) It is inappropriate to approach the internationalization process without formulating a strategy. Without a proper strategy the firm is about to fail in their internationalization. Formulating a strategy also involves deciding hen, how and which markets to enter. There are several market entry modes a firm can choose from, such as exporting, contractual relationships (licensing, franchising), as well as equity or ownership-based international business activities like FDI or collaborative ventures. (Masum/Fernandez 2008, 2) 1. 2 Research Purpose The purpose of this study is to gain a better understanding of the internationalization process of a SEE company, particularly the usefulness of the main theory: the Uppsala Model. 1 1. 3 Structure of the ReportThe report is divided into six major parts: Introduction, Research Methodology, Case Study, Analysis and Interpretation, Implications and Limitations, and our Findings and Conclusion. The introduction part conta ins the background description of the general topic and the research purpose. Followed by the Research Methodology part, where the research method, data collection and data analysis is described. Then the chosen company is presented in particular and their internationalization process and their motives for internationalization are discussed.In the Analysis and Interpretation chapter the theoretical background and to which extend the company followed the theory is presented, along with a broad discussion of the gathered empirical data. There are also some implications and limitations mentioned in the next chapter. In the conclusion there is a brief presentation of the findings as well as possible further research on the topic. 2 2. 1 Research Methodology Data Collection The data collected and used in this analysis has been mainly collected from websites and online databases. This means that the method applied was Desk Research.As depicted by name Desk Research is the research techniq ue that is mainly acquired by sitting at a desk. It involves collecting data from existing resources and, compared to Field Research, is the cheapest and quickest option. Nevertheless, there is always the problem of the validity, objectivity and credibility of the data found. There are basically two types of desk research: Internal Desk Research and External Desk Research. Whereas the former is being used only in corporations or companies that possess an internal database, the latter can be done by anyone.External Desk Research is the actual method that was used when gathering information about the company, about the market and about other countries. 2. 2 Data Analysis First of all, it has been decided to go with the company Plantaze. Afterwards, a huge amount of data about the company’s history, strategy and current situation has been gath2 ered directly from their official website, plantaze. com. Moreover, detailed information about their products and production process has been found here.Additional financial and statistical information was found on the website of the Montenegro Stock Exchange (MNSE), montenegrobreza. com, where the company is listed and traded. Normally, looking and searching for data in online databases is very expensive. Fortunately, there has been access to the WU network, where many online databases are available for the research. ‘Amadeus’, ‘Factiva’ and others have given supplementary data about the company’s foreign subsidiaries for example that were not that clear in the places looked before.Actually, the main difficulty was finding data about the company’s operations abroad. For that reason it was necessary to get in contact with the Plantaze company in order to get more accurate data on their internationalization process. The people were kind enough and shared some very important facts and dates that completed the information pool that was needed. In order to work on the Excel sheets , economic, political and geographical data on the countries in which the Plantaze company is directly or indirectly doing business were needed.For this, the official websites of the specific countries governments and other websites of some important international organization like the World Bank or the CIA World Factbook were used. Before starting to write about the company’s past, present and future situation in the company description, articles from the press about the company and its operations were read. Also a look at various customer reports and trade association reviews about the company were necessary, so that an objective and closer insight to the true image of the analyzed company could be provided.Psychic Distance Psychic distance is defined as factors such as differences in language, culture, political systems, etc. , which disturb the flow of information between the firm and the market. Psychic distance chain refers to economic, geographical and cultural distanc e. (Johanson/Wiedersheim-Paul 1975, 308). Hofstede’s model of cultural dimensions from 1970s as one of the first theories that could be used to explain observed differences between cultures, has become an internationally recognized standard and major resource in cross-cultural studies. The original theory identified four cultural dimensions for distinguishing cultures: †¢ Power distance dimension (PD) focuses on the degree of inequality between people with and without power in society; †¢ Individualism dimension (IDV) refers to strength of interpersonal connections and share of responsibility among people; †¢ Uncertainty Avoidance Index (UAI) measures degree of tolerance towards uncertainty and unknown situations and †¢ Masculinity (MAS) referring to gender differentiations and inequity in society.After some more researches Hofstede added a fifth dimension – long-term orientation (LTO) which relates to how much society values long-term devotion to t raditional values; and in the 2010 a sixth dimension – Indulgence versus restraint defining the attitude of society towards gratification of basic and natural human needs related enjoying life and having fun. (mindtools. com 2012) The following factors were used for this case study: The geographical distance is an important factor to calculate transportation cost from the production facility in Montenegro to the sales markets abroad.The figure was calculated by measuring the air line distance from Podgorica (Montenegrin capital) to the capital of the reference country. It is the most important figure for Plantaze as it is a mainly exporting company. So this factor was overweighed against all the other chosen factors. The cultural distance consists of the above-mentioned dimensions, power distance, uncertainty avoidance, individualism, and masculinity. Regarding to Hofstede the differences of the dimension of country A and the reference country, i. e. Montenegro, are calculate d in a first step.This difference has to be squared and divided by the variance of the whole spectrum of countries. Cultural distance in exporting is important because of the sheer nature of the product. Drinking wine is something that is embed in the national culture, thus making the cultural distance a factor that needs to be taken into consideration. Furthermore for marketing and communication reasons the cultural distance between two countries can make a company change the strategy. 4 It was calculated by using the Kogut and Singh-Formula:When calculating the internationalization process of the company regarding only the subsidiaries and the joint venture the cultural distance variable gains importance. Because entering a market in this way, by greenfield investment, a company has to think and act on a long-term level. The country’s culture can be decisive in whether the own company will be successful or not. The â€Å"wine† distance measures the wine consumption p er capita of a nation compared to Montenegro. This figure is relevant because it would make no sense for a wine producing company to enter a market that scores low.As already mentioned wine consumption is something that is highly linked to the nation’s culture and their drinking habits. That is why this is a relevant figure. To calculate the wine distance, the same formula as above has been used. The economic distance was not taken into account because of the fact, that it is not relevant for this type of industry. To calculate the psychic distance the above mentioned factors have been standardized to numbers between 0 and 1, while 0 stands for Montenegro respectively a country with the same figures than Montenegro and 1 for the country with the highest distance to the reference country.The factors have been weighted according to their importance. In the exporting internationalization process the geographical distance has been weighted with 60%, the cultural and the â€Å"wi ne† distance were weighted with 20% each. In the subsidiary and joint venture internationalization process the geographical distance has also been weighted with 60%, the cultural distance with 40% and the wine distance was not taken into consideration. 5 3 3. 1 Case Study Company Description (www. plantaze. com) ‘13jul-Plantaze’ is a wine-producing company from Montenegro.Its history takes us back more than 100 years, more specifically to 1907 when the Montenegrin wine ‘Vranac’ won its first prize in London. It was but more than 50 years later, when the government of Montenegro decided to invest in the development of agriculture, that the led to the creation of the company Plantaze as we know it today. Between 1964 and 1974, Plantaze expanded the vineyard to 377 ha and the wine cellar capacity to 26. 000 hl. In the 1977-1982 period, the company realized one of its most important projects.Transforming the arid and rocky soil of the ‘Cemovsko†™ field into one of the largest green oasis of the Balkan area is not something to be overlooked. 62 million U. S. dollars cost the creation of the largest vineyard in Europe at that time, covering 2000 ha of orchards and vineyards. The geographical position makes this place so unique. Located at about 30 km from the Adriatic See, on the river Bojana, the ‘Cemovsko’ field has a microclimate of its own, suitable for quality grape production.In 1979 the main processing plant was built near the vineyard. ‘Agrougostitelj’, ‘Agrokom’, ‘Agroekonomski institut’, ‘Uvoz-izvoz’ and ‘Ribnjaci’ merged with Plantaze in 1998. 2005 was the year in which the company established a joint venture with their Italian partners and added the first sparkling wine ‘Val’ to the product range. During the years, they successfully obtained international certificates of quality such as the ISO 9001:2000; HACCAP or the IS O 14000. In 2007, Plantaze invested 2 million euros in the remarkable wine cellar ‘Sipcanik’.Located at 30 meters below the soil, covering about 7000 m2, this completely natural area has the perfect climatic and technological conditions to age over 2 million liters of wine in wooden barrels, oak barriques and bottles. In the last 10 years exports have risen by 530% to 4 million bottles in 2008. The company exports to over 30 countries situated all over the world, from the U. S. A to Canada, to the E. U. countries, Russia, China and Australia. Today, Plantaze still owns and manages Europe’s biggest vineyard at ‘Cemovsko’ field, which stands on 2310 ha and contains three wine cellars with a capacity of 310. 00 hl. They recently announced that the investments made in the period 2003-2009 were over 40 million euros. With an annual production of 22 6 million kilos per year, Plantaze is the biggest producer of wine and table grapes in Montenegro. A quick l ook at their company’s official website will be enough to understand that this company has something special about it. Their mission statement is to produce worldclass quality products with which they can satisfy their loyal customers and gain new ones.It must be added that the company is not just producing and selling wine and grapes. An 85ha peach plantation that averages an annual production of 1. 200 tons is one of their most prized possessions. As of 1957, Plantaze produces and sells about 100 tons of Californian trout. The ‘Mareza’ fish pond covers 6. 000 m2 and is exclusively fed by fresh spring water. The grapevines used are not being bought; they are being grown on a nursery of rootstocks that spreads over 40 ha. Two restaurants complete their portfolio. Mareza’, a restaurant with a capacity of over 400 seats, located 5 km outside of Podgorica and ‘Jezero’, with a capacity of over 300 guests, located along the main road between Podgor ica and Petrovac, on the shore of the beautiful and relaxing Skadar Lake appear in every touristic guide of Montenegro. Coming back to the wine business, we can see that Plantaze offers a great variety of wines. 11 types of red wine and 6 types of white wine are currently in their catalogue. Furthermore, we can find a special rose wine made from red grapes applied in the white wines production. Three types of brandy complete the offer.The Plantaze company is probably one of the most successful brands and businesses in Montenegro. Their incredible attitude towards the environment and their fine attention to detail stands before every product they make. The Plantaze company must be a proud flag-bearer and ambassador of Montenegro because they managed to achieve something that many firms only dream of, and that is to produce traditional goods from your local country and culture at the highest quality possible. For this reason and others, the company Plantaze has been chosen to be analy zed and presented. 3. 2 Internationalization ProcessBecause the company has a history of over 40 years, in which it sold goods on an international level, the need for splitting up the process into more than one period was created. Therefore, the internationalization process was divided into four different phases. 7 These phases are not equal. For example, the first period is twenty years long, whereas the second one is fifteen years long. The reason for this is that the first two periods were slower from the internationalization point of view than the last two. So, in the first period that is between 1964 and 1984, they started selling their products on the Yugoslavian market and in Albania.The argumentation for the fact that Yugoslavia has been added to the internationalization process is actually very easy to follow. Because for the company at stake the cultural distance is of great importance, it was clear that Yugoslavia had to appear on the graph. Although from a political and technical point of view, there was only one single country and one single market, taking the cultural differences into consideration, the situation changes dramatically. The seven entities that are now seven different countries have their own traditions, habits and culture.Therefore, selling products all over the ex-Yugoslavian territory makes the process an international one. Furthermore, if the Uppsala Model is the center point of this presentation, gaining knowledge about different cultures and using it into new markets, like Plantaze did, just proves out argumentation. In the second phase, from 1985 and 2000, numerous other markets were penetrated. Of course, after 1991 and the fall of ex-Yugoslavia, the products sold into these new established countries could officially be called exports. The company entered some Central European countries like the Czech Republic, Slovakia, Poland and Hungary.In the east, Bulgaria was chosen to start exporting to. The first countries from the E uropean Union in 8 which the firm started internationalizing were Italy, France and the U. K. , in which they established a wholly-owned sales subsidiary in London. Russia was the first distant market they choose. In the third period, or between 2001 and 2004, the focus remained on Central and Western Europe. Germany, Austria, the Netherlands, Belgium and Sweden were the next markets they entered. The third period also meant a development of the internationalization process.Some very distant markets such as China and the United States were submitted to the process. The fourth and final phase takes place from 2005 until the present day, in 2012. Norway and Switzerland are the newest addition from the European continent, while Canada and Australia are another two distant markets to which the company started exporting. 3. 3 Internationalization Motives To figure out the motives for the internationalization of Plantaze a quick look at Dunning’s different categories of motives is necessary. Strategic Asset Seeking Resource Seeking Efficiency Seeking Motives for Internationalization Network SeekingMarket Seeking 9 Dunning (2000) explains how market and resource seeking motives have been the two most recognized categories of motives before. These two categories still correspond to most first time internationalization by firms. Overall, efficiency seeking and strategic asset seeking motives increase in significance and are more common as motives for companies already engaged in multinational activity. He also shows that closer relations with customers and durable relations with suppliers were important motives. Furthermore, he suggests that internationalization was more driven by opportunities rather than threats. Hansson/Hedin 2007, 5) Market Seekers: Companies that invest in a particular country or region with the intention to supply goods and services are called market seekers. This category of motives focuses on demand aspects. (Hansson/Hedin 2007, 6) Plant aze’s home market is limited as Montenegro is a small country and so it brings the firm not enough revenues. This fact and also to diversify the customer base of Plantaze to reduce the dependence on the home market are reasons why they decided to go abroad. Resource Seekers: According to Dunning (1993) resource seeking means to invest abroad in order to obtain resources.This could be resources that can be acquired at a lower comparative cost, or simply does not exist at all in the home country. (Hansson/Hedin 2007, 7) Plantaze is not seen as a resource seeking company, as the conditions for producing in Montenegro are unique. Sometimes skills and capabilities are resources that can be used through collaboration with a business partner. Efficiency Seekers: The purpose is to rationalize structures of established investments in order to gain from common governance. Often those benefits come from economies of scale, but also risk diversification.Therefore, efficiency seeking is s een as gaining from the differences of factor 10 endowments, cultures, institutional arrangements, and economic systems etc. (Hansson/Hedin 2007, 7) Economies of scale and scope as well as the increase of sales and profits are issues that an efficiency seeker often focuses on, and so does Plantaze. Another motive for the company to internationalize is that Plantaze might be able to lower the tax burden. Strategic Resource Seekers: Strategic resources are for example patents, knowledge, the skills of employees, and strategic supplies necessary for developing comparative advantages.By focusing on developing strategic resources the company supports its long term strategic objectives. (Hansson/Hedin 2007, 8) Plantaze’s aim is it to create brand awareness in foreign countries and to transmit the positive image of Montenegro by producing a traditional product from the home country and selling it to other countries. Network Seekers: The network orientation reflects to what extent co mpanies participate in alliances, cooperative ventures and other forms of similar social connections. Networks outside the organization can be very important for the companies.Companies intend to nurse, develop and expand their existing networks. (Hansson/Hedin 2007, 9) Developing useful foreign relationships is an important factor for Plantaze. Their partner have knowledge of the local markets and the necessary skills. 4 4. 1 Analysis and Interpretation Theoretical Background The Uppsala Internationalization Model The Uppsala Internationalization model is a model of a firm's choice of market and form of entry when going abroad. It was developed by a number of Swedish researchers, Johanson, Wiedersheim-Paul and Vahlne (1975, 1977). The model was named after the business 11 chool of the Swedish city and based on the process of internationalization of four Swedish manufacturing companies with operations in more than 20 countries. The model assumes that internationalization is a progre ssive process made of several successive stages. The main aspects of internationalization are market knowledge and level of commitment in a particular host country. The major obstacle to international operations is the lack of knowledge about foreign markets and operations, which can be overtaken gradually by actively engaging in such foreign environments (Forsgren, Hogstrom, 2004; Lakomaa, 2009).The Swedish researchers noticed that observed companies had begun to operate abroad in nearby markets and then slowly penetrated markets far away. They entered new markets through export, and after several years of exports the company could establish wholly owned or majority-owned operations. Thus, the process of progressive internationalization is built on four stages that are: sporadic export, export via independent representatives, foreign sales subsidiaries and production and manufacturing units in foreign markets. Source: Forgren and Johanson 1975, 16 12The figure shows that additional market commitment will be made in small steps, both in the market commitment and geographical dimension. The geographical dimension means that firms enter new markets with successively greater psychic distance, defined in terms of factors like language differences, culture and political system, etc. Therefore, companies internationalize by going to those markets they can most easily understand and where the perceived market uncertainty is low. Criticism of the Uppsala Model There were several critics referring to the Uppsala model.Some of them are that the model is too deterministic (Reid, 1983; Turnbull, 1987) or that the model doses not take into account interdependencies between different country markets (Johanson and Mattson, 1986). Studies have shown that the model is not valid for service industries, situations of highly internationalized companies and industries and that the whole internationalization process has speeded up. Firms also tend to enter ‘distant' markets i n terms of psychic distance at an early stage (leap-frogging tendency), because the world has become much more homogenous and that has lead to that psychic distance has decreased. . 2 Application of the Uppsala Model Only by simply looking at the internationalization process that the Plantaze company followed over the years, it is easy to conclude that the firm followed more or less the theory that the Uppsala Model describes. In the beginning, for example, when the firm started selling their products only on the ex-Yugoslavian territory and Albania without having the need of going to distant markets is clearly the kind of behavior that a newly founded enterprise would have in the Uppsala Model.After learning from this experience (because in from the companies point of view, since culture plays an important factor, it learned a great deal from selling on the whole territory of Yugoslavia, where seven different entities and cultures were mashed together under one flag) they could sta rt and wonder off to other countries and cultures. In the second phase of their internationalization process, countries from Eastern Europe were chosen and some small steps to the Western part of Europe were also made. The first important milestone in the company’s history is the opening of their first sales-subsidiary in the United Kingdom.It is called Monteadria and it is located in 13 London. This particular step can be noted as the exception from the rule since the firm ventured off to a distant market directly by establishing a sales-subsidiary and not starting by exporting and then gradually develop. Nevertheless, it is quite difficult for a company that is active on the market to truly and without exceptions follow the Uppsala Model since it does not take into account other important factors such as market attractiveness, market size and others.The globalization and internationalization effect can be seen in every market and in every country. Because of that, because o f the massive inflow of information and data available in a blink of an eye, companies show leapfrogging tendencies and go to more distant markets earlier. The overall psychic distance between countries has decreased. Plantaze took full advantage of the fast moving business world in which they operate. The company grew rapidly and intensified export activities worldwide. As a result, their export figure has increased by more than 550% since 2003. 5. 1 Implications and Limitations Implications of the Study The main implication of the study would surely represent if whether or not managers that handle the company use the Uppsala Model when entering the internationalization process. Just by looking at the export path explained in the Excel sheet above, it is easy to conclude that the firm applied the model. Of course, the fact that the managers specifically used the Uppsala Model or that the pattern used just randomly fits, is something that needs to be analyzed more in detail.In contr ast, when it comes to the path chosen by the company for entering new markets and countries via wholly-owned or partially-owned subsidiaries or joint-venture, the situation changes. The path chosen is not similar to the Uppsala Model. One argument would be that they chose to enter the British market with a subsidiary in London before opening one in Belgrade or Sarajevo. The logical step, according to the Uppsala Model would be to start with establishment of subsidiaries in neighboring countries and afterwards spread out to other, more foreign countries. 14 5. 2 Limitations of the StudyThe main limitation of this study would be that it revolves around the Uppsala Model and thus making its criticism point, the major liability of the study. The model is old and was not updated to the current economic situation. For example, in today’s business world, companies have the tendency to leapfrog some entry modes and to go directly to more physically distant markets. The world today ha s become more homogenous because of the globalization process that has been going on in the last decades and the psychic distance has also decreased. The company described and used in the study, Plantaze, is not the perfect fit to the Uppsala Model.The point that the company posses enough financial resources leads to the fact that consequences of their commitments won’t have a huge impact on their balance sheets. Moreover, the company is not obliged to go abroad to gain new market knowledge and gain experience because today they can call on other sources for additional information and know-how. Universities, government databases and institutions or other companies from the branch can provide this kind of data. Regarding the limitations of the research, the fact that the findings are closely linked to a specific context is underlying.This research has been confined to a few countries in the European Union. This may not be sufficient to generalize our findings in this paper. Ho wever, this paper points out the direction and may act as an indicator how our company internationalized. Thus, we believe that our findings are useful to better understand the driving forces of the internationalization of Plantaze. 6 Conclusion and Future Research The path chosen by the Plantaze company follows to a certain degree the theory of internationalization that the Uppsala Model presents.No one knows for sure if the higher management of the company intentionally acted in this manner or if the match is just a random one. To better understand their internationalization process, in future studies, interviews with the persons responsible and who took the decisions must be conducted. Only then, only with that data, the study can truly show how the company reacted to the internationalization process and how they proceeded. Nevertheless, without having that data available to be 15 used, and only through analyzing ex-post the steps taken in the past, the Uppsala Model provides a p ossible framework for companies to follow.In addition to future research possibilities arising directly from the limitations, it should be recommended that future research may explore longitudinal research design for further contribution to international business in this context. 16 7 References Publications Birn, Robin J. 2001. The Handbook of International Market Research Techniques, London 2001 Hansson, Anders and Hedin, Kim. 2007. Motives for internationalization. Small companies in Swedish incubators and science parks, Uppsala Hofstede, Geert. 2001. Culture’s Consequences: Comparing Values, Behaviors, Institutions, and Organizations Across Nations.Thousand Oaks, CA: Sage. Johanson, Jan and Wiedersheim-Paul, Finn. 1975. The Internationalization of the Firm – Four Swedish Cases, in: The Journal of Management Studies, 1975, 305-322 Kent, Raymond A. 1993. Marketing Research in Action, New York Kogut, B. and H. Singh. 1988. The Effect of National Culture on the Choice of Entry Mode, in: Journal of International Business Studies, 19(3): 411–432. Masum, Mohibul Islam and Fernandez Alejandra. 2008. Internationalization-Process of SMEs: Strategies and Methods, Vasteras. Pett, Timothy L. 2008.Examining SME Internationalization Motives as an Extension of Competitive Strategy, in: Journal of Business and Entrepreneurship, 2008, 1-13. Internet CIA Factbook n. a. : Hofstede’s Cultural Dimensions. Understanding Workplace Values Around the World http://www. mindtools. com/pages/article/newLDR_66. htm, accessed October 15, 2012. 17 Plantaze www. plantaze. com , accessed October 15, 2012. WHO http://www. who. int/substance_abuse/publications/global_alcohol_report/msbgsruprofiles. pdf, accessed October 15, 2012. Worldbank http://data. worldbank. org/indicator/NY. GDP. PCAP. CD, accessed October 15, 2012. 18 8 Appendix 19

Critical Review Article Example | Topics and Well Written Essays - 2000 words

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Review Questions Essay Example for Free

Review Questions Essay Answer the following questions (you may use MS Project Help): 1) What are the three base calendars included in MS Project and what are the default values of each? Standard Default base calendar, Monday to Friday, 8 to 5, lunch noon to 1. This is the default base calendar used for the project, for tasks and for resources. Night Shift Usually for graveyard shift, 11 PM to 8 AM, five days a week, lunch 3 AM to 4AM. 24 Hours – Work never stops here. Typically used for projects in a manufacturing situation, midnight until midnight 7 days a week. 2) What is the difference between a base calendar and a resource calendar? Resource Calendars apply to only specific resources. 3) Why schedule one project meeting after completion of the last task, Test System? We should as a final meeting to discuss how the project went as a whole. 4) Give an example of when you would assign a 24-hour calendar to a resource. 24-Hour calendars would be used in situations where a consultant is being used to complete a fixed-cost task 5) If you were doing a senior project, what would be the base calendar you would use for students working on the project and what adjustments would you make to it? The base calendar I would use would probably be the 24-hour calendar because there would not be a set time each task will be worked on. Turn in this sheet with your MS Project file to the Weekly iLab Dropbox.

Writing a brief response and answering questions

Writing a brief response and answering questions The document defines art as the visual expression of an idea or experiment and it requires skill and a medium to be formed. I feel that the way the text literally reads, can be translated to cut out some things. Music for instance is a form of art that isnt visual that however needs a medium, can be enjoyed without the visual aspect. Poetry for instance doesnt really need a medium unless it is writen,sp or if you include a persons spzvoice as a medium to re-sight the poems. Neither of those things need to be seen. I may have taken the text more literal than needed however I felt like the books interpretation of what art is isnt broad enough for me. I did think that the artwork by Janet Echelman in the introduction was beautiful and was well expressed. I would love to see that in action. -1 pt. 2.In your own words, define art? I define art as something produced out of a creative mind. How do you define a creative mind? An idea brought to life, music, song lyrics/poetry, food, body art, interior design, dance anything that you can produce from an idea, no matter if it is verbally, physically or materialistic. 3.Does art have to be visual and tangible? Explain No, as I said before music and poetry are both things that are not visual and unless writen sp or recorded they are not always tangible. Dance is another good example of untangible sp art even though it is visually expressed and enjoyed. 4.For the sake of argument lets say that the art world consists broadly of artists, art critics, museums, galleries, art dealers, art historians, art educators, art students and those who enjoy and appreciate art. Consider art as those things specifically intended to be art. Do you think that you are greatly separated from the art world? Explain. -2pts. Answer is confusing. I do not feel separated, I also do not feel like a celebrity in this world? however I feel like art is everywhere, even those things specifically intended to be art. Everyday in car you hear music, instruments and song lyrics. Buildings designed by architects, art work on the walls inside these buildings. All these things are intended to be art however we dont always acknowledge them as art. Really? Have you seen Chapter 12 in your text? 5.Name 3 purposes of art and give an example of each. Personal Expression: Self portraits sp are used to express to the world who artist really is inside and how they feel about their place in life through their own eyes. Rembrandts self portrait sp is shown in the book. It shows he has a powerful presence. As Yong Soon Mins Dwelling, expressed her sense of alienation and absence from the rest of the world. Communicating Information: Modern day picture books and storys for children to help them learn a story or a lesson. The book even gives an example as the stained glass in churches, were once used to tell a biblical story to the illiterate. Visual Delight: This to me seems like the more common purpose. Many things are created for the purpose of appearance; re-models to give everyday house hold items a more modern and visually enjoyable look and feel. Even something as simple as the way a woman applies are make up sp differently in each society or social group within, is art for visual delight. -3 pts. Incomplete and unclear. 6.What are some of the ideas art can communicate? How do you see these ideas communicated in your everyday life? Any other ideas? Art can communicate religious ideas and beliefs. Church seems to be the best example. As the book said before the stained glass windows were once used to tell the stories for those who cannot read. To the statues you see in different denominations, such as the catholic crucifix. Telling you of Jesus pain and sacrifice. Incomplete sentence. Even in Buddhism, the statue of Buddha depicts him as a large man, when in real life he was skinny with dysentery. However the larger image in that culture was imagery showing his happiness and quality of life. What? 7. What structures or works of art are for spiritual sustenance? Are there any such structures in your community? Statues of religious figures, churches and temples are all works of art . In many Vietnamese nail salons in my area there are altars dedicated to Buddha, with gold statues and hand painted figurines, even bamboo is twisted and formed to represent luck. There are many paintings and pictures of the Virgin Mary in many Catholic and Spanish derived communities near by. sp All of these are works of art. 8. Briefly describe the subject matter in the art of Romare Bearden. Everything is crammed together. The people seemed to more focused on simple things such as smoking. There is a rocket headed for the moon but is barely visible and hidden in the back ground. The picture seems loud and busy. -2pts. 9. What does his art reveal about the time and place in which he (Bearden) lived? It seems that he lived in poverty, a large city, and around people who are oblivious to things in the world other than what is going on directly around him. He also tried to show that the black experience was also relatable to universals. See the quote in the text. -4pts. Did you read the list of traits on page 12? 10. What are three traits of creativity? -Being in touch with ones unconscious yet be intensely conscious. -Wondering and being curious Being able to analyze and evaluate After reading the section in your book Art in the World, Early Encounters with the Artist Within answer questions 11 and 12. -2pts. Incomplete answer. 11. Do you think humans have a need to be creative? Why or why not? Yes, for us to be able to design our houses, clothing, inventions that we have used to make life easier, as well as medicines and remedies from sickness. -2pts. 12. Respond to the question at the end of the section regarding what becomes of childrens extraordinary capacity? Reality, the more a child realizes or separates real life from imagination, the ore creativity is lost. What did you mean to say here? The more we teach a child how something should look the more they veer away from the way they saw it. As I said in response to one of the discussions. : One of the worst things we can do is teach out sp children to color in between the lines. And tell them the sky must be blue and the grass must be green. We teach them not to be creative. 13. How do you see yourself as a creative human being? Elaborate. Yes I do, I notice my creativity more in problem solving ways ,and in organizational, and visually pleasing ways. My creativity is at its highest when I am given a problem to solve, when it comes to tangible items. I can build things without direction and turn havoc into a visually pleasing scene. -2 pts. 14.Is the work of the untrained or outsider artist valid as works of art? Why or why not? Yes, even when untrained it is still a creation of that persons thought or idea. And the art can still be appreciated even though the name isnt well known. There are many outsider artists who are very well known and have patrons who support them and their work. Sometimes I personally wonder if half of the people in the art world really aapreciate sp the art or the name signed to it. Oh you can bet there are people who care what name is on artart auctions prove that! No matter what social group you find your self sp in there are always followers who are on the bandwagon. If you do no love a piece of art you shouldnt own it just for who created it. Creation is art, appreciation of one person is validation enough. What do you mean here? But you didnt answer the question. -1 pt.15. What is the difference between looking and seeing? Explain. You can look at something and not really see it. Looking at something is mearly sp pointing your eyes at it, focusing and your brain transmitting what you visually saw. Seeing, is being able to understand, and interpret what you are looking at and even translate and find the meaning or message. Run-on sentence. -1 pt.16-18. Define representational, abstract and non-objective. Cite examples in the text to go with each definition. Representational: Art in which it is the artists intention to present again or represent a particular subject, especially pertaining to realistic portrayal of subject matter. Example: Photographs like the one by Lauren Greenfield taken of Dubai is a real depiction of the way this looks. As well as realistic self- portraits like the one of Rembrandt on page 6. Abstract: Art that is based on natural appearance but departs significantly from them. Forms are modified or changed to varying degrees in order to emphasize certain qualities or content. Recognizable references to original appearances may be very slight. The term is also used to describe art that is non representational. Example: Romare Beardens Rocket to the moon Not sure that is considered abstract since many of the forms are recognizable. Non-Objective: Art reference to anything outside its self, sp without representation. Without recognizable objects. Example: Anna Zemankovas untitled piece on page 14 -4pts. You appear to have missed the meaning of the form and the content. Please read the texts description. 19. Form is described as the total effect of what we see. Content refers to the meaning of the piece. How is the form and content similar/different in the artworks titled The Kiss by Rodin and Brancusi? The form as we see it in Rodins depiction is visually more realistic. It is a man and a women embarrassing sp in a kiss, in the nude, indicating it may lead further than that and unify them as one. Brancusis is visually less realistic and its content expresses more the idea of love than what it is in its literal meaning. Even though they are the same in the end. Incomplete sentence Rodins form allows you to figure out the content. It seems you must know the content of Brancusis before being able to really see the piece for what it is. -2pts. 20. Content isnt always what it appears to be as is the case in iconography. Define iconography and elaborate on this as it applies to the painting on page 55 titled The Virgin of Carmel Saving Souls from Purgatory. Iconography: Is the symbolic meaning of sign, subjects and images used to convey ideas important to particular cultures or religions and the conventions governing the use of such forms. In The Virgin of Carmel Saving Souls from Purgatory it is used many different times. The icon that seems to be most widely know sp would be the white dove, many could look at this painting and know immediately that it represented the holy spirit because it has been used so often. The Virgin Mary is shown in a very recognizable way, holding baby Jesus, with the crown on her head. The text says that the fire shows the people trapped in purgatory;, however some may see that as hell if they were unaware of the title of this piece, so I disagree that it is an example of iconography as the book states. Your text names many more symbols and their meanings.

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