Innovative Cryopreservation for Transported Horse Semen

Cryo-Progress:
A Unique Freezing Technology (UFT)

Although great improvements have been made to conventional liquid nitrogen vapor techniques, there have been relatively few true technological advancements in the field of semen cryopreservation. The Unique Freezing Technology (UFT) operates on a principle radically different from conventional liquid nitrogen vapor cryopreservation methods.

With UFT, samples are frozen by submerging them into a liquid vat. The proprietary fluid used in UFT is a modified organic solution that is FDA approved. Unique characteristics of this fluid are that it can be maintained at temperatures below 0oC without solidifying, it has a heat index similar to water, and a very high rate of heat dissipation.

UFT frozen doses are packaged in cryovials. This offers a number of benefits. First, current UFT protocols dictate thawing the vials at room temperature for 45 seconds before placing them in a 37oC water bath. Vials frozen in this system have been exposed to ambient temperatures for up to two minutes without detectable damage. UFT preservation in cryovials affords the opportunity to confidently work with and identify samples before they are thawed, as well as making the combination of volume from multiple straws unnecessary.

Extensive testing at a major university indicates that UFT offers substantial benefits for the cryoprservation of equine semen. These include the effectiveness of this system with a larger population of stallions, and superior protection against the damage caused by cryopreservation.

It is estimated that less than 24% of the entire stallion population produce an ejaculate suitable for conventional liquid nitrogen cryopreservation. University conducted research trials indicate that 75% of the stallions evaluated performed at a satisfactory level with UFT. This is because UFT minimizes the amount of damage that occurs during the cryopreservation process.

The thermal transfer involved with UFT is characterized by the dissipation of heat from the sample. This cools the sample very homogenously. The dissipation of heat applies much less mechanical stress to the cells compared to the thermal insult of blast freezing associated with conventional liquid nitrogen vapor freezing.

During the cooling and freezing process typical damage includes degradation of cellular membranes, altered metabolism, and cryolysis or perforation of the sperm cell by ice crystal formation. This damage is manifested as abnormal swimming patterns, loss of motility, or loss of fertilizing capability.

As the membrane integrity and metabolic processes of the sperm cell are disrupted, the sperm will accumulate further damage as a process called lipid peroxidation reaches toxic levels in the cell. The cascade of damage will ultimately lead to the premature capacitation and acrosome reaction of the sperm cell.

The acrosome is a sack of enzymes located at the top of the sperm head. These enzymes assist the sperm cell in the penetration of the egg. An intact acrosome post-thaw is crucial for fertilization. Research has verified that samples frozen in UFT express considerably less acrosomal damage than conventionally frozen cells.

Finally, the most visible evidence of the benefit of UFT over conventional liquid nitrogen cryopreservation is the percentage of increased post-thaw motility. In two university conducted trials the average percentage of increased post-thaw motility were 146.57%. For example, a stallion with a post thaw motility of approximately 18% in liquid nitrogen could reach a post thaw motility of approximately 45% by freezing in a UFT.

The samples for these trials were collected, cooled, and shipped to an offsite laboratory for cryopreservation. This resulted in lower than optimal post-thaw motilities for both liquid nitrogen controls and UFT cryopreserved samples. However, the increased post thaw motility achieved through the UFT system is evident. These results illustrate that UFT does indeed offer distinct advantages for the cryopreservation of stallion semen.