Counting Spermatozoa - Part 1 By B. W. Pickett, Ph.D. Professor Emeritus, Colorado State University
A hemacytometer, originally designed to count blood cells, has been used for many years to count other types of cells. Toward the end of World War II, with the advent of artificial insemination in cattle, it became painfully obvious that other methods of counting spermatozoa had to be developed, because of the length of time it required for an accurate count to be obtained with the hemacytometer.
Since the late 1930's numerous scientific papers have been published describing how to use the hemacytometer to calibrate other instruments to estimate the number of spermatozoa more rapidly in a sample of semen than is possible with the hemacytometer. Consequently, today (2004), the number of spermatozoa are estimated in bull, boar, stallion, chicken, turkey and dog semen using electronic methods. However, the initial calibration of these instruments is done with a hemacytometer. In general, for an accurate calibration it requires 70 normal, clean semen samples ranging in count, per milliliter of semen, from 50 million to 1 billion, depending upon the species. Regardless of species, the important point is that the sperm concentration of the 70 ejaculates must be equally distributed over the range of expected counts regardless of frequency of collection of semen. Unfortunately, it requires 8 to 10 counts of each of the 70 samples to obtain an average count that is highly accurate. Of course a "quick and dirty" estimate can be made with fewer counts, but is not satisfactory for scientific purposes or any other purpose when accuracy is essential.
During the calibration procedure the same samples used for the hemacytometer counts are placed in the electronic instrument to be calibrated. Once the 70 samples have been through the process, a regression equation is "fitted", then the electronic instrument is used to estimate the number of spermatozoa per milliliter of semen. The electronic method provides a quick, simple, repeatable method, provided the semen sample is "clean". The presence of gel in the ejaculates of stallion or boar semen will cause the electronic instrument to estimate a larger number of spermatozoa than are actually present. The same is true of my sample containing extraneous material such as dirt, hair or cells other than spermatozoa. Consequently, it is not uncommon for an appropriately calibrated electronic instrument to overestimate the number of spermatozoa in a series of unselected semen samples, particularly if the electronic instrument in question was calibrated with a hemacytometer with too few samples selected over a narrow range, or if there were a majority of samples within a narrow range. To further complicate matters, the individual counting spermatozoa must have a good, appropriately adjusted phase-contrast microscope and be skilled in utilizing the counting procedure, as well as the instrument.
The method of counting spermatozoa with a hemacytometer is based upon the principle that the hemacytometer is loaded with a constant volume of semen between the cover slip and counting grid, or chamber. Accurate counts can be obtained with this method when used properly.
It is highly unlikely that a breeding farm would consider using a hemacytometer on a routine basis due to: a) the number of chambers that must be counted for reasonable accuracy, b) the time required to prepare the sample for counting with the hemacytometer, c) the time required to count the number of spermatozoa in the appropriate number of squares, d) procedure for counting the spermatozoa, e) preparing and counting a sample of semen requires considerable technical skill and equipment.
However, the hemacytometer is very useful in counting semen samples that have been placed in an extender and those that are contaminated with debris.