Frozen Semen Processing and Quality Control

Due primarily to the fact that breeders select stallions for breeding based on performance, confirmation and pedigree with little regard for reproductive efficiency, the general stallion population includes numerous valuable stallions with poor reproductive efficiency. A stallion may be subfertile in a frozen or cooled semen program and exhibit normal fertility in fresh AI or natural mating conditions. It is nowadays almost impossible to collect objective and reliable fertility data from the field, like first cycle, per cycle and even seasonal pregnancy rate. This happens particularly when mares are inseminated with cooled or frozen semen shipped internationally. Monitoring fertility of breeding stallions in AI programs with cryopreserved semen begins with the identification of the semen freezing technique that allows to produce the best possible semen quality from a given stallion. In fact, providing the equine breeding industry with high quality semen should lead to achieve the optimum level of fertility in the field. In order to guarantee the highest level of fertility from a stallion, semen parameters should be regularly checked by performing a strict post-thaw quality control on each batch of semen that is processed for freezing. This abstract will focus on how semen should be processed for freezing, so to obtain the optimum semen quality that will inevitably help the practitioner to obtain the highest level of fertility for a given stallion.

Processing Semen for Cryopreservation

The first critical aspect of a successful semen preservation technique is to obtain a high quality sample without damaging the fragile sperm during collection or processing. This may seem obvious and simple yet in our work with breeding farms and clinics we find that this is where many of the problems occur. The ideal sample for semen preservation (either cooling or freezing) is one that contains a high concentration of sperm and therefore a low ratio of seminal plasma to spermatozoa and exhibits a high percentage of progressively motile sperm with a low percentage of morphological abnormalities. This can be accomplished by managing the collection frequency for the stallion such that the number of days sexual rest prior to collection is optimum to obtain an ejaculate with the desired characteristics and limiting the amount of sexual stimulation prior to collection. Ideally, the stallion should be stimulated just enough to elicit a good erection and result in ejaculation obtained on a single mount. Another management tool that we find very useful is to “void” as much of the pre-ejaculatory fluid as possible prior to placing the stallion’s penis into the AV for collection. This fluid contains no sperm and only serves to dilute out the sperm in the collected semen with fluid that is far from an ideal media for incubating sperm.

Sperm can suffer sub-lethal damage from many sources and this damage may not be obvious during initial evaluation but only be revealed after the sperm are stored for a period of time as is the case when transporting cooled semen. Factors that can adversely affect sperm motility include:

1. Toxic residues on collection equipment such as soap residues and minerals in tap water. To avoid this it is recommended that whenever possible disposable AV liners and semen containers be used and if latex reusable liners are used they are cleaned and rinsed thoroughly with deionized water to remove residues.
2. Contamination of semen with high osmolarity, water soluble lubricants. This can be avoided by use of water soluble lubricants osmotically balanced with semen, use of minimum quantities of lubricant in the AV and changing the semen collection container after each unsuccessful mount when multiple mounts are required to obtain an ejaculation.
3. Temperature shock caused by exposing the semen to either too high or too low temperatures. Ejaculated spermatozoa are very sensitive to cold and heat shock and every precaution should be taken to avoid temperature deviations from the optimal range of 34-37C prior to diluting the semen in extender. Once extended, sperm should not be incubated at 37C as prolonged incubation at this temperature will reduce sperm motility. 4. Excessive bacterial contamination. It is essential that good hygiene be practiced when obtaining samples to avoid contamination with bacteria from inadequately cleaned collection equipment including the AV, phantom, wash bucket, etc. [...]