Methods of Sperm Analysis

Sperm can be analyzed with either manual or automated methods. The following descriptions are aimed more heavily at automated analysis since an automated system is capable of acquiring more information about sperm motility, especially progressive (forward moving) motility, than manual methods can. Automated analysis offers the advantages of being able to determine sperm velocity and linearity of the sperm track, and also is capable of saving the moving sperm image onto optical disk or some other medium for later retrieval and reanalysis. Prior to automated analysis it was not possible to independently verify the results of analysis at a later date.

Manual motility analysis is accomplished by a trained observer looking at a sperm sample in either Test Yolk Buffer or Phosphate Buffered Saline and manually counting the number motile and non-motile then calculating the percent motility. Since the sperm counted are moving through the field and then quickly die it is not possible for a second observer to count the same sperm as the first observer. Generally two independent observers count seperate fields on seperate slides and an average is calculated from the two observations. Manual concentration analysis is conducted by producing a homogenate of standard volume from a cauda epididymal or testicular sample of known weight and counting the number of sperm heads in a known volume of homogenate using a hemocytmeter. Manual morphology analysis is conducted by analysis of Eosin Y stained sperm on fixed, coverslipped slides and manually counting incidences of morphological changes either to the head or tail of the sperm cells.

Automated analysis can be performed by one of two systems available at the present time: The Hamilton Thorne Computer Assisted Sperm Analyzer from Hamilton Thorne Research or the CellSoft Computer Assisted Sperm Analyzer from CRYO Resources.

The Biology of Male Reproduction

Sperm cells are produced in the seminiferous tubules of the testes encased by large Sertoli cells which provide support for the meiotically dividing germ cells. The primary germ cell is the Spermatogonium which passes through a series of cell divisions to produce first primary spermatocytes, then secondary spermatocytes and finally spermatids. Each spermatid eventually matures into one spermatozoa (or sperm cell) during which process it sheds its cytoplasm which forms a droplet that travels down the length of the forming flagellum until it disappears in the mature sperm cell. Sperm cells in the testes are not yet capable of movement, they gain this ability as they pass through the epididymis in their maturation process. After passing through the cauda epididymis the maturing sperm cells enter the vas deferens which is a muscular duct which leads eventually to the urethra. The sperm in the vas deferens is mature and capable of motility.

Sperm cells consist of a head which is made of a nucleus and an enzyme filled vesicle called the acrosome, a midpiece which contains mitochondria to power the flagellum, and a thin tailpiece. In immature sperm cells the cytoplasmic droplet may be seen at the junction of the midpiece and the tailpiece. This droplet is sometimes visible though not pronounced in rats, more pronounced in mice, and quite pronounced in hamsters.

Sperm Susceptibility

Sperm cells are very susceptible to changes in temperature and pH and can live and move only in a very narrow range of either. Outside of the acceptable range the sperm very quickly lose motility and die. Further, since they have no cytoplasm they cannot provide energy for movement from stored energy sources and need an external energy source which they can absorb. In a live animal this is provided by the secretions produced by the seminal vesicles which add considerable volume to the semen, but on a slide this source is absent. In a phosphate buffered saline (PBS) solution the phosphates can offer some source of energy, but not for long. Another option is to let the sperm incubate in a cell growth medium such as M-199. In the sample preparation for the Hamilton Thorne Computer Aided Sperm Analyzer the sperm from the cauda epididymis is allowed to swim out into a dish of M-199 medium warmed to 36 ± 1 degree C and balanced to a pH of about7.2 by nicking the epididymal tubules at the point nearest the entrance to the vas deferens (where the most mature cells are found). If called for by protocol the sperm sample can also be obtained by placing the vas deferens into the dish of prewarmed and pH balanced medium. If the pH or temperature is out of range the sperm will quickly curl up and twitch slightly, then eventually relax and die.

In properly balanced and warmed media the healthy sperm exuded by the contractions of the nicked tubules or vas deferens will quickly swim out and disperse into a diffuse cloud. Non-motile sperm, even in properly warmed and balanced media, will not disperse but will remain in a thick thread or blob on the bottom of the dish. If the sperm does not very quickly disperse it is important to insure that the temperature and pH have not shifted out of range for some reason. If it appears that the temperature or pH has shifted out of range this must be noted in the raw data as this may have adversely affected the motility of the sample.

Once the sample has been obtained and incubated it is important that the slide it is placed on is clean, and that the coverslip is clean, in order to obtain the best possible image and to prevent the sperm cells from sticking to the surface of the slide or coverslip. Also it is important that the coverslip be placed on the slide in a manner that will prevent bubbles of air from being trapped beneath it causing areas which will not produce a good image. The quality of the image you obtain will directly relate to the quality of the analysis you produce.

The Hamilton Thorne Computer Assisted Sperm Analyzer (CASA)

The Hamilton Thorne CASA consists of three separate systems combined to perform a single complex function. For a user to be able to fully understand the Hamilton Thorne CASA it is necessary to understand each of the individual systems of which it is comprised.

The first system is the optical system consisting of a phase contrast microscope and video camera. This system is what provides the raw analog video image of sperm that the Hamilton Thorne digitizes for analysis. The optical system is accessed through the sliding panel on the right hand side of the Hamilton Thorne case. The microscope inside is provided with a warmed slide stage, two separate light sources, and a turret with the capability of holding up to four lenses (the standard installation holds three lenses). Behind the microscope is a video camera of the charge coupled device (CCD) variety. Between the microscope and the camera is an ultraviolet (UV) filter block which can be slid in place or out of place, depending on which light source is to be used. Beneath the condenser on the microscope are the light sources: a standard light source and a xenon strobe light source used for the UV florescence scans of IDENT stained sperm samples.

The second system is the computer hardware system consisting of a digitizing board which provides the digitized version of the video signal which the Hamilton Thorne system actually analyzes, an IBM PC style central processing unit (CPU), an external magneto-optical disk drive, and a printer. The digitizing board converts the video image into a digital format that the CPU can manipulate and feeds that digitized image into the random access memory (RAM) of the system for analysis. This image file and the data derived from it can be stored under user control in a number of formats to a number of locations. The digitized image, which is the primary raw data, is saved to the magneto-optical disk drive (drive D) which contains removable media in the form of a removable optical disk cartridge. The derived files can be saved as clinical reports which are saved to the magneto-optical disk drive (drive D) or ASCII export files which are saved to the internal hard drive (drive C) and can later be copied to a floppy disk (in drive A) to be transferred to the Data Management department for tabulation and statistical analysis. Derived reports can also be printed directly to the attached printer.

The third system is the software system which includes DOS, a Windows operating system, and the proprietary Hamilton Thorne IVOS software. The user does not have to have any great familiarity with much of this software but must have a full understanding of the Hamilton Thorne IVOS software and the Windows Program Manager in order to properly manipulate the system.

In addition to the Hamilton Thorne CASA one additional system is needed to undertake sperm analysis, and that is the biological system consisting of the trained user and the properly prepared sperm sample. Without a good sample it is not possible to undertake a good analysis and even with a good sample it is not possible to undertake a good analysis without a properly trained human operator. Because of this, it is important for the operator to have a basic understanding of the biology of the male reproductive system and the affect of sample preparation and media on sperm motility.

Acquiring the Images with the Hamilton Thorne IVOS

A good analysis requires attention to several factors that affect the quality of the image produced. First, the image must have sufficient clarity. This is why it is necessary to calibrate the phase annulus adjustment prior to each study. If the phase annulus is not properly adjusted a clear, bright image cannot be obtained. Also since the concentration calculation depends on the use of an accurate value for the screen area it is important to calibrate the magnification scale by use of a standard 100 micron grid, and to maintain consistency in the depth of chamber by using the same chamber for each concentration analysis. The UV filter block must also be in the proper position for the type of light source used and the proper objective lens must be in place.

In order to insure a good motility sample it is important to insure that the slide stage temperature does not fluctuate out of range and that the sample on the slide is fresh. For this reason only five fields at a time can be acquired for motility analysis and for each set of fields the slide must be loaded with a fresh aliquot of sample preparation (A new software update from Hamilton Thorne Research has corrected the slide stage temperature update problem- check your version of the software to see if it updates the slide stage temperature during analysis scans). Care should be taken to choose the fields to acquire as quickly as possible so the sample will still be fresh during the scan. Fields must not be chosen based on activity of the sperm on the slide, but only based on factors which could make it difficult for the IVOS to analyze the sample. Examples of reasons a field could be passed over during selection would be too high of a concentration of sperm, to low a concentration of sperm, excessive debris in the field, excessive "clumping" of sperm (heads stuck together), or bubbles. Non-motile sperm are NOT a valid reason to skip a field for analysis, though they are a reason to check for other factors that should be noted, such as out of range temperature or pH.

The Info Screen

The data entered in the "info" screen is very important and should be checked before running any sample. This is where the animal number and group number are entered. It is also where the analysis setup being used can be found and changed. It is very important to be sure that analysis is run under the proper and validated setup parameters, otherwise the data will not be valid and a serious SOP deviation will result. Also this is the area where the tissue weight for the cauda epididymis is entered for concentration analysis. Without the proper weight the result will be incorrect and the sample will need to be reanalyzed from the disk image. Finally, remember that there is a comment area on the bottom of this screen. Any factors which may have adversely affected the analysis should be entered in this area of the info screen before the file for the animal is printed or filed. In addition, any files which are analyzed from the image on the optical disk should be labeled as such in this field, along with the date of necropsy if the reanalysis is on a later date (A new software update from Hamilton Thorne Research has allowed the image date and time to be printed on reports making the notation of date of necropsy unnecessary- check your own version to see whether it prints the image date and time).

File Management

The Hamilton Thorne IVOS produces three types of files which need to be managed. This is accomplished through the "Configure File" sub-screen of the "Print/File" screen. The type of file to be saved or printed can be selected in the main "Print/File" screen and automatic printing or filing options can be found in the "Preferences" sub-screen. The "Configure File" sub-screen is the area where the subdirectories to which files can be saved are able to be selected. The name of the new subdirectory can be entered for a type of file by using the pull down menu for that file type and selecting the appropriate drive (drive D for image or clinical files, drive C for ASCII files), and entering the name of the subdirectory in the appropriate space. Alternately the subdirectories can be created with the Windows File Manager program and then selected from the pull down menu on the "Configure File" screen.

For an average study the number of entries in a clinical or ASCII file do not require the creation of a subdirectory since these files are fairly small in size. By contrast the image files can be very large. Each image file consists of a list of coordinates for each object in each frame of the image with multiples of five frames in a file. In order to file an image the processor must go through each other file in the directory in order to assign the file number to the new image file. As more images are added to the directory this can take more and more time. By the time the directory holds over 300 fields there can be a significant increase in the filing time. Since the images for each field are filed before the next field is analyzed this can make the time a slide is on the slide stage too long to maintain good motility. Because of this each dayÕs analysis of either motility or concentration must be saved into its own subdirectory.

The subdirectories to which image files are saved are named in a way to make it as easy as possible to find and track any particular animal's image. The name of the subdirectory can be made of the six number code for the date of analysis (in the format mmddyy) followed by an "m" for motility, a "c" for epididymal concentration, or a "t" for testicular spermatid concentration.

In order to make it easy to retrieve the data for any particular animal it is important to keep a record of the file number to which the data for that animal is saved, especially since the Hamilton Thorne IVOS file naming convention does not necessarily use the animal number for filing but may use the study number. That is the purpose of a Data File Record sheet. These sheets should be filled out with the file numbers associated with the animal numbers for image files and the clinical report numbers associated with the animal numbers for clinical files. ASCII files do not need to be documented since all animals can be saved into one ASCII file by the study number and this file, which can contain the animal number, date, and time of analysis, is formatted to be loaded onto one spreadsheet so that the data is easily associated with the animal.

Troubleshooting

A. Sperm present, but not motile

1. Check the pH of the medium, it should be 7.2.
2. Check the temperature of the meduim, it should be ~36 degrees C.
3. Check the time passed since sample collection, it should be ~15 min. or less.
4. Check to insure that samples are collected as close to the instant of death as possible.
5. Check to see if there are any necropsy findings concerning the left epididymis, the left testis, or the seminal vesicles.

B. pH of medium is less than 7.1

1. For the sample in which the pH is discovered to be out of range note the deviation in the comments section of the info screen and insure that the correct pH is listed in the pH field on the info screen.
2. For subsequent samples place medium into the petri dish earlier, giving it more time to shift toward the alkaline side.
3. If placing the medium into the dish earlier does not allow the pH to shift back into range, then try adjusting the medium with 1N NaOH.

C. pH of the medium is greater than 7.3

1. For the sample in which the pH is discovered to be out of range note the deviation in the comments section of the info screen and insure that the correct pH is listed in the pH field on the info screen.
2. For subsequent samples adjust the medium with 1N HCl.

D. Temperature is out of range

1. Note the slide warmer temperature in the comments section of the info screen with the added comment that the medium temperature was out of range.
2. Adjust the slide warmer temperature and wait for it to come into range before preparing another sample.

E. Too much time has passed since the sample collection

1. Note that an excessive amount of time has passed in the comment section of the info screen.
2. Reduce the time for sample collection by insuring that the left epididymis is collected, trimmed, and weighed as quickly as possible and leaving all other steps of the necropsy until after the sample collection is complete.
3. Insure that no excess time passes between the time of death and the time of sample collection.

F. Necropsy shows findings concerning the left epididymis, the left testis, or the seminal vesicles.

1. Note the necropsy findings in the comments section of the info screen.

G. No sperm are present

1. Try another slide.
2. Check to see if there are any necropsy findings concerning the left epididymis, the left testis, or the seminal vesicles.
3. For IDENT stained sperm count samples check to see of the stain was properly mixed, i.e. was the crystal of stain stuck to the top of the stain kit vial? If the stain was not mixed invert the vial two or three times, tapping it each time, and then re-vortex and allow time to stain.