Overview of Semen Handling and Analysis

Semen Handling

Sperm are motile and vigorous cells, but also fragile and susceptible to damage and killing by several environmental conditions. When collecting and handling semen it is critical to avoid exposing sperm to two types of insults:

• Exposure to toxic chemicals: Keeping collection equipment clean and disinfected is important, but soap and disinfecting chemicals are quite potent spermicides.
  
  Take great care to rinse the inner liners of the artificial vagina and collection tubes with deionized water to remove such agents. It is usually best to use new, sterile collection tubes. Finally, be certain to lubricate artificial vaginas with a lubricant known to be non-toxic to sperm.
• Thermal stress: Sperm are sensitive to both heat and cold. Rapid chilling of semen results in a phenomenon called "cold shock" that is often manifest by abnormal sperm motility and morphology. Short periods of exposure to temperatures just a few degrees above body temperature will usually kill large numbers of sperm.
  
  To avoid thermal stress, the collection tube end of the artificial vagina should be covered with an insulating cone. Additionally, microscope slides, coverslips, stains, extenders and pipets used to handle and examine sperm are best maintained on a warming plate prior to use, as shown below. If you are performing a large number of analyses, a heated microscope stage is also a valuable piece of equipment.

Semen from most species is not damaged by exposure to room temperature (20-22 C) for an hour or two. If longer periods of maintenance is required, it is best to dilute the raw ejaculate in a buffered nutrient solution - usually called an extender - and cool it slowly to refrigerator temperature (4-5 C). A large number of extenders have been developed, usually for use in freezing semen. They are similar in having an energy source (e.g. glucose), buffers to maintain pH (e.g. Tris or citrate) and a source of protein (e.g. 20% egg yolk).

A simple method for preparing extended, chilled semen is to dilute the raw semen with warm extender, suspend the tube into an empty beaker through an insulated lid (e.g. styrafoam; see image to right), then place the beaker into a refrigerator. The air in the beaker will cool slowly, thereby avoiding cold-shocking the sperm.

Extended, chilled semen is frequently transported for insemination, providing a useful alternative to either freezing or immediate use. To maintain temperature, special containers are made just for semen transport. The "Equitianer" pictured at right contains coolant cans and insulation to keep the semen sample cool for overnight shipping.

Semen Analysis

A large number of assays have been developed and advocated for assessing semen quality. Inevitably, at least three core parameters are evaluated:

• Sperm concentration and total sperm in the ejaculate. Total sperm is determined by multiplying concentration (sperm per ml) by ejaculate volume (ml).
• Sperm motility is usually assumed to be the percentage of sperm that are progressively motile. A progressively motile sperm swims briskly forward in a relatively straight line, as opposed to moving in circles. In some cases (e.g. with human sperm), total motility, or the percentage of sperm that are wiggling at all, is also recorded.
• Sperm morphology. The best samples of semen inevitably contain some sperm that have abnormal structure, such as bent tails or misshapen heads. To obtain a standard measure of sperm morphology, the percentage of sperm with normal shape and size is determined. It can also be useful to classify the abnormal sperm as to type of defect.

Methods used to determine concentration, motility and morphology are detailed on subsequent pages. Examples of other tests that are sometimes used are seminal pH, ability of sperm to bind or penetrate zonae pellucidae, and rate of dispersal in gel. Recognize that none of these tests actually measure fertility; rather they provide a useful index of potential fertility for that ejaculate. Further, there are many instances of pregancies arising following insemination of semen that has terrible quality, and conversely, some males with excellent semen quality are infertile for reasons that remain obscure.