The main advantages of frozen semen are long-term preservation of genetic material, insurance against unanticipated loss of a stallion, access to frozen semen if fresh or cooled semen is not available, and ability to ship semen worldwide. Most major breed registries allow the registration of foals conceived with frozen semen, with the notable exception of the Jockey Club (Thoroughbred horses).
The general steps for cryopreservation of equine semen include mixing freshly collected semen with a basic skim milk extender, centrifugation to remove seminal plasma and concentrate the spermatozoa into a pellet, resuspension of the semen in a freezing extender containing a cryoprotectant, cooling to -120° C (-184° F) over liquid nitrogen vapor, and subsequent storage in liquid nitrogen at -196° C (-320° F). The post-thaw quality of spermatozoa is evaluated on at least one straw after every freeze. For frozen-thawed stallion semen, the percentage of progressively motile spermatozoa should be at least 30% at 10 minutes after thawing to be considered commercially acceptable.
Stallion semen frozen at the Equine Reproduction Laboratory is diluted in a freezing extender to a concentration of 200 million spermatozoa per ml. The diluted semen is subsequently loaded into 0.5 ml straws each containing a total of 100 million spermatozoa. A breeding dose consists of 8 straws and a total of 800 million spermatozoa.
A test freeze should be performed the first time semen from an individual stallion is frozen. It may be optimal to collect the stallion several times to deplete the extra-gonadal sperm reserves before performing the test freeze.
The test freeze would compare post-thaw motility characteristics between two or more freezing extenders, centrifugation techniques, cooling rates, sperm concentrations, and possibly different straw sizes and thawing rates. The goal would be to identify a customized cryopreservation protocol for the individual stallion to maximize sperm survival after freezing and thawing. Following the test freeze, one or more straws are thawed at the recommended rate and warmed to 37° C for 10 minutes to evaluate motility of the frozen-thawed spermatozoa.
Thawing Frozen Semen
It is recommended that frozen semen be thawed according to the guidelines provided by the facility or individual that initially froze the semen. In general, semen frozen in 0.5 ml straws may be thawed by placement into a 37° C water bath for 30 seconds.
Fertility of Frozen Semen
Pregnancy rates per cycle for mares inseminated with frozen-thawed semen are highly variable due to differences between stallions, freezing extenders, insemination dose, timing of insemination, number of inseminations, and reproductive status of the mare. Pregnancy rates can range from <10% per cycle for some stallions to >50% per cycle for other stallions. However, in general it is assumed that pregnancy rates with frozen semen will range between 30% and 45% per cycle.
Frozen Semen Storage
The Equine Reproduction Laboratory serves as a commercial frozen semen storage facility. Straws of frozen semen are held in labeled goblets within tanks or dewars of liquid nitrogen. Stallion semen properly cryopreserved can remain stored in liquid nitrogen indefinitely. Please contact us for information on options for short- and long-term storage of frozen stallion semen.
Shipping Frozen Semen
Frozen semen is transported from one facility to another using a “vapor shipper” or “dry shipper.” Vapor shippers are “charged” by adding liquid nitrogen (LN2) to the center and allowing the liquid nitrogen to be absorbed into the wall of the container. Once the container is fully charged, the excess liquid nitrogen is poured off and the liquid nitrogen vapor will keep the interior storage area of the shipper at a temperature of -196° C. The important principle of a vapor shipper is that it does not contain liquid LN2 (i.e. “dry”) and therefore it can be transported as standard non-hazardous cargo by overnight courier or commercial airline.
Freezing and refreezing for intracytoplasmic sperm injection
Our staff can also manage frozen semen for intracytoplasmic sperm injection (ICSI). Stallion semen can be frozen in ICSI doses by diluting the sperm into a very low concentration (i.e. 1 million sperm per straw), generating a large number of straws. Alternatively, an existing straw of frozen semen can be thawed and the semen diluted by adding more freezing extender and the diluted semen loaded into new straws and refrozen semen. The goal is to optimize the use and minimize the wastage of valuable frozen semen in an assisted reproduction technology program. Please contact us for additional information on refreezing stallion frozen semen.