Physiology of Cooled Semen

For equine semen to be transported over long distances or for longer than a few hours, it must be cooled to retain its viability. Cooling semen below 19° C (66° F) causes damage to the sperm cell membrane and reduction in fertility. The sperm cell membrane consists of intimately associated lipids and proteins that intermingle in fluid form. At and below 19° C, the membrane begins to gel as lipids within the structure begin to solidify and proteins clump. Membrane lipids become gel at various temperatures down to 8° C (46.4° F). Cell membrane function is altered by the distribution of proteins and lipids into separate clumps. At that temperature, the whole membrane becomes “gelified.” Irreversible partial damage to the cell membrane occurs if semen is cooled too rapidly (cold shock), and this results in loss of sperm motility and, in particular, circular swimming patterns. However, even slow cooling may induce some membrane damage, which may not be apparent when the cells are warmed but results in a shorter life span in the sperm. If the clumping of proteins and lipids does not allow the normal liquid-to-protein association to be reestablished upon warming, the damage is irreversible. Low-temperature storage causes premature capacitation and osmotic and DNA damage. Below 4° C (39.2° F), fragmentation of DNA results.

Even the best cooling techniques cause some damage, so the longevity of sperm is reduced after the semen is warmed. Like many aspects of dealing with stallion semen, sperm longevity is likely to be dependent not only on cooling technique but also on the stallion and the specific biochemistry of its sperm. The practical result is that one sample of cooled (or frozen) sperm has relatively good viability when warmed and stored at room temperature, whereas another may deteriorate quickly. Consequently, the resulting fertility in vivo is affected.

Although some semen samples can result in pregnancy when the interval from artificial insemination (AI) to ovulation is longer than 24 hours, other samples must be deposited in the mare much closer to ovulation. Frozen or thawed sperm have reduced ability to bind to oviductal cells, which substantially limits longevity within the genital tract.

Additional sperm damage occurs by exposure to reactive oxygen species (ROS). These molecules are produced during oxidative metabolism and are generated at an increased level by dead and abnormal or damaged sperm. Damage done by ROS production during freezing is stallion dependent. Removal of ROS before storage or preservation and even after storage increases the viability of sperm. Because ROS are important in sperm capacitation, the premature capacitation of cooled and frozen sperm by ROS production reduces the longevity of the sperm. Methods of gradient filtration have been developed to reduce the number of ROS-generating dead and nonmotile sperm in an attempt to increase viability. Ironically, the removal of seminal plasma necessary for storage removes antioxidants with it.

The Stallion

It is important to realize that not all stallions produce semen suitable for preservation. Although different preparation, cooling, freezing, and transport techniques may improve the quality of some semen, semen from certain stallions may always have reduced quality or very poor fertility after it has been cooled, frozen, and transported.

Bacteria

Bacterial cultures should be made from the external genitalia of any stallion used for semen collection to eliminate the possibility of transfer of venereal pathogens in the semen. Semen extenders contain antimicrobials primarily to limit or prevent bacterial growth while the semen is being stored above 4° to 6° C, rather than to eliminate pathogens. Antimicrobials may also limit the establishment of endometritis in susceptible mares. Various combinations of antimicrobials are used, and the stallion farm processing the semen should experiment with different combinations to determine which is best suited to each particular stallion. Some antimicrobials (notably polymyxin B) may be deleterious to certain semen samples.

For most semen to retain viability for longer than a few hours, it must not only be extended but also cooled. It is thus conventional, when overnight delivery is used, to cool semen to 4° to 6° C. This means that sperm cells will be damaged to some extent by the cooling process, in particular by the rate of cooling and the type of extender used. It would be preferable to transport semen that has been cooled to room temperature but not below 19° to 20° C (66.2 to 68° F) to avoid this damage. Although one study found storage at 15° C (59° F) in a commercial extender was superior to storage at 10° C (50° F) or 4° C (Leboeuf et al, 2003), another found storage at 20° C for 48 hours to be inferior to storage at 4° C (Moran et al., 1992). Unfortunately, little other research has been done to assess the viability (essentially the longevity once inseminated) of sperm kept at 20° C for extended periods. In the author’s laboratory, semen has been successfully stored (i.e., yielded pregnancy) for up to 18 hours at room temperature. By avoiding the need to cool the semen below 20° C, the longevity within the mare is retained to some extent. When semen can be delivered within a few hours or even on the same day, consideration should be given to storing it at more than 19° C rather than cooling it to 4° to 6° C.