by SwineReproNet Staff
Before any further technical evaluation is performed, sub-sampling of the ejaculate should be made with a well-mixed sample. Gentle swirling is required since sperm cells settle to the bottom with time. It should be mentioned that shaking or swirling too vigorously could cause damage to the sensitive attachment region for the sperm head and tail.
Estimating Sperm Concentration Hemacytometer
Evaluation of a semen sample with the hemacytometer (cell counting chamber) provides a method for accurate determination of sperm cell concentration. An accurate estimation of concentration will allow extension rates that optimize boar utilization and fertility of extended semen. The materials needed for this procedure should include a good quality microscope (~$1500), a hemacytometer (~$100/ea). The sample dilution system can include a red or white blood cell dilution system (Unopette, ~1.00/ea) or a hand-pipet system. The Unopette white blood cell dilution system is desirable since no additional equipment is needed, sperm cells are killed, and pipetting and dilution errors can be minimized. In this system, a sub-sample from the ejaculate is taken by mixing the ejaculate well and placing 2-3 drops on a microscope slide. The Unopette system allows a 10-20 ml sample to be obtained with a Unopette capillary pipet. The semen sample in the capillary tube is then added to the Unopette dilution chamber and mixed (Figure 1 and 2). The diluted semen sample is then applied to both chambers of hemacytometer. After 5 minutes, the sperm cells settle onto the glass and counting of sperm at 200-400 magnification can begin. Since sperm are almost transparent under light microscopy, a phase-contrast scope can facilitate sperm visualization. When using a standard light microscope, the iris diaphragm should be closed to improve sperm visualization. Sperm are counted in 5 diagonal squares. Sperm heads are not counted if they touch the right or bottom triple lines of the squares (Figure 3). These sperm are not included since they contribute to over-estimation of the concentration when multiplication is performed. Both chambers of the hemacytometer are counted. Sperm numbers from each side should be within 10% of each other. If not, clean the hemacytometer and repeat sample addition (it is not necessary to repeat the dilution step). When sperm counts are within 10%, average the two numbers. If a 1:100, or a 1:200 Unopette dilution was used, simply add seven zeros to get sperm concentration/cc. This is a simplified way to determine concentration. The actual formula is ((sperm in 5 squares x 5; (or total sperm in 25 squares)) x dilution rate (100 or 200) x hemacytometer chamber volume (10,000 or 104) = sperm/cc) ). Example: (25 sperm average (in 5 squares) x 5) x 200 (dilution factor) x 104 = 250,000,000 sperm/cc. The total time required for this procedure is about 10 minutes per sample. Errors can occur with this procedure due to pipetting error and improper chamber filling (under or over-filling).
Sperm Morphology and Abnormalities
The fertility of a semen sample is related to the proportion of abnormal or percentage of normal sperm. As the proportion of abnormal cells increases, the fertility of the sample declines. Abnormalities occur in the head, midpiece and tail region. The type of abnormality, and the location can indicate when and what type of injury or insult occurred to the boar during sperm cell formation and maturation.
Defects in the head of the sperm can include an abnormal shape. The most common of which are either a head that is too small or too large. Other abnormalities include defects in the acrosome integrity. An intact acrosome allows aperm to binding and fertilize the egg.
Defects in this region may include an offset tail, (although for boar sperm this is normal) or the presence of a droplet, indicating an immature sperm cell.
Defects in this region are evident from insults during the maturation stage during the trip through the epididymis. Missing, bent, or coiled tails are a frequent defects that arises during improper processing and storage.
Sperm motility is the factor most related to fertility. Unfortunately, this is the most subjective assessment to make. It has been reported that differences in fertility are evident only when motility declines below 60%. Progressive forward motion (forward motion with large circles is also O.K.) is the assessment to make and should evaluate all non-motile and motile sperm collectively as a percent.