It is recommended that a “test cool” be performed on semen from a stallion for which no cooled semen data is available. A test cool compares semen extenders and evaluates sperm motility following cooled storage of semen at a standardized sperm dilution (25 to 50 million motile spermatozoa per ml) in the storage-shipping container to be used during the breeding season. The goal is to determine if semen from the stallion cools well and to identify the semen extender that optimizes motility during cooled storage. It should be noted that evaluation of the motility of cooled semen is not always a good indicator of true fertility. The only measure of true fertility is the pregnancy rate of mares bred. There are stallions that have good fertility with fresh semen and good motility characteristics after cooling, but have poor pregnancy rates with cooled semen. In general, it is estimated that 70 to 80% of stallions will have semen that cools adequately. It is also anticipated that sperm motility will decrease approximately 20 to 30% after 24 hours of cooled storage for most stallions.

A test cool consists of the following procedures:

  • Semen is collected from the stallion to be evaluated
  • A semen evaluation (gel-free volume, concentration, motility) is performed
  • Gel-free semen is diluted to a concentration of 25 million progressively motile sperm per mL in each semen extender to be tested (it is recommended that 3 to 4 different extenders be evaluated)
  • 40 mLs of each type of extended semen are placed into the same kind of storage container that is to be used during the breeding season (i.e. Whirl-Pak® bag, baby bottle liner, 50 mL syringe, centrifuge tube)
  • The storage containers are placed into the same type of passive cooling device that will used during the breeding season
  • Extended semen is cooled for 24 hours
  • The passive cooling device is opened, the storage container is gently mixed and a small aliquot of extended semen removed for evaluation
  • The storage containers is immediately placed back into the passive cooling system, if the temperature can be maintained at 5 to 8° C (41 to 46.4° F), or place into an insulated container within a refrigerator at 5 to 8° C (41 to 46.4° F)
  • Each aliquot of semen is warmed up in a 37° C (98.6° F) incubator or water bath for 10 minutes
  • Motility characteristics (i.e. total and progressive motility) are evaluated after warming and recorded
  • The evaluation procedure is repeated at 48 hours after collection
  • Motility characteristics are compared between the semen extenders tested

Ultimately it is determined if semen from the stallion “qualifies” for use in a cooled-transported program and which extender is most suitable for preserving motility. In addition, it is determined if additional tests or procedures, such as evaluation of different extenders, passive cooling systems, or removal of seminal plasma (centrifugation) are necessary.

Stallions that are determined to be “poor coolers” may benefit from removal of a majority of seminal plasma in the ejaculate by centrifugation. However, removal of all seminal plasma after centrifugation results in poor motility after cooled storage. Therefore, it is recommended that 5 to 20% of the seminal plasma should remain with the sperm pellet after centrifugation. The remainder of the seminal plasma supernatant is removed (i.e. using an aspiration system) and discarded. An appropriate semen extender is then added and the spermatozoa are resuspended.

In addition, during the breeding season it may be advantageous for a stallion owner or manager to periodically hold back a dose of semen, store it in a passive cooling system, and evaluate the semen 24 hours later. In this way the stallion owner or manager can keep track of motility characteristics of the shipped semen in case a potential problem arises. This may help differentiate between problems with the stallion, problems encountered during shipment, and problems on the mare breeding farm.