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International handling of frozen Semen
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The first pregnancy in a mare inseminated with frozen stallion semen was reported in 1957 and, since then, improved cryopreservation and insemination protocols for stallion semen, as well as breed registry acceptance, have led to a wider utilization of frozen semen in the international equine breeding industry.
One of the main advantages of frozen semen is that when properly maintained at liquid nitrogen temperature (-197°C), spermatozoa can remain viable for an indefinite time, provided the spermatozoa respond favorably to the freezing process.
In our laboratories, equine semen stored frozen for more than 22 years was used to inseminate mares with good results and achieved pregnancies.
In order to maintain maximum fertilizing potential, spermatozoa must not be exposed to fluctuations in temperature and must be maintained below the critical temperature (approximately -130°C) at which thermally driven chemical reactions proceed. Exposure of cells to temperatures above -80°C can initiate some degree of thawing and then recrystallization of ice, as well as reorganization of membrane proteins and lipids. This can lead to cell damage and impaired fertilizing potential. To maintain cells at these ultra-low temperatures, packaged frozen semen is typically immersed in liquid nitrogen. To allow for such storage, cryogenic insulated containers are commonly used in the equine frozen semen artificial insemination (AI) industry. Most equine frozen semen is currently being packaged in 0.5 ml straws for freezing.
It is essential that all personnel working with liquid nitrogen are aware of two critical facts concerning this substance:
- Liquid nitrogen is extremely cold. At atmospheric pressure, liquid nitrogen boils at -196°C.
- Liquid nitrogen produces a large amount of nitrogen gas. One liter of liquid nitrogen vaporizes into almost 1 m3) of nitrogen gas.
Either of these two properties can produce personal injury or property damage and therefore caution must be used when handling liquid nitrogen to prevent injury or damage.
Equine frozen semen is typically transported in a nitrogen vapor-phase container (dry shipper). These cryogenic containers maintain near liquid nitrogen temperatures (typically -180° to -195°C) for days or weeks without the use of hazardous liquid nitrogen. Vapor-phase containers work by absorbing liquid nitrogen into a thick layer of hydrophobic absorbent material that surrounds the inner cavity of the container where the semen is stored. A long holding time is achieved through the superior insulation afforded by the double-walled aluminum shell, which is filled with insulating foil and vacuum sealed.
Vapor-phase containers must be properly ‘charged’ by filling with liquid nitrogen to the point of saturation of the absorbent material and then pouring off the excess liquid nitrogen. Such vapor-phase shippers can be transported with a ‘non-hazardous’ classification throughout the world, which significantly reduces shipping costs. A word of caution is offered, however, concerning the use of vapor shippers; because the package is being shipped with a nonhazardous classification, it is critical that all of the liquid nitrogen is poured out of the container prior to transport. Transport of a hazardous material without the proper declarations and appropriate labeling can lead to significant fines as this is a violation of IATA (International Air Transport Association) regulations and may be in breach of certain state laws.
Vapor shippers are fairly durable; however, it is highly recommended that they be placed inside a solid outer shipping carton that will protect the container against physical damage or loss of vacuum due to rough handling during shipping.
Occasionally, semen is shipped in liquid shipping containers. These containers maintain semen for a longer period of time provided an adequate liquid nitrogen level is maintained. There is no absorbent material in these containers therefore if they are tipped and the liquid spills out, the container will quickly warm to room temperature and the semen will thaw. When shipping in a non-vapor, liquid container, the package must comply with IATA regulations with regard to the type of packaging, the declarations on the shipping documents, and appropriate labeling of the package. Furthermore, the individual who packs the container and signs the hazardous materials declarations must have undergone specific training in the use of the hazardous material in question. When packaging frozen semen for shipment, extreme care should be taken to avoid exposing straws to elevated temperatures.
One of the main benefits of using frozen semen is the fact that it can be transported to the veterinarian or mare owner in advance thus eliminating the anxiety and logistical headaches associated with cooled semen breeding. Ideally, the semen can be ordered prior to the mare’s estrus and transferred upon arrival to a storage container at the farm or clinic.
When transferring frozen semen after arrival, the technician should take care not to expose the straws to room temperature by lifting them into the warm neck of the tank or transferring across any significant distance between the shipper and permanent storage.
The following paperwork should be included with each international frozen semen shipment:
- Instructions for thawing and handling the semen.
- A transaction report or frozen semen shipment which includes the following information:
- stallion’s name and registration number;
- number of doses included;
- collection date, lot number, or straw ID code and number of straws per insemination dose
- number of total and progressively motile sperm per insemination dose, plus total sperm per dose;
- expected post-thaw semen motility;
- Mare management recommendations:
- Instructions for return of container;
- Applicable breed registry insemination or frozen semen distribution certificates;
- Export Health Certificate issued and signed by an Official State Veterinarian of the country of origin.
An export health certificate must always accompany frozen semen shipped from one country to another.
This certificate states that the semen exported/imported was produced in an approved laboratory, under the sanitary rules of the receiving country. For example, the European Union (EU) member states have established requirements for approved equine AI and semen production facilities as well as donor stallion health testing requirements for frozen semen produced for use in the commercial market and eventually exported within the EU. Producing frozen semen out of a stallion tested for health requirements (i.e. Equine Infectious Anemia, Equine Viral Arteritis, Equine Herpevirus, Glanders, Dourine, Contagious Equine Metritis, etc.) avoids the risk of infectious diseases international spreading.
Unfortunately, when importing frozen semen, different countries ask for different semen donor stallion health requirements. This could refer to both the infectious diseases against which the stallions must be tested or to the time of testing (i.e. prior, during or after the semen is frozen for export). For this reason it is therefore very important that, before freezing equine semen for export, the import rules and regulations of the receiving country are verified. These rules and regulations are usually and easily found on the State
Health Department website of the receiving country.
These regulations are primarily concerned with preventing disease transmission through transported frozen or cooled semen but do not make any recommendations concerning semen quality standards. As a result, the quality of semen that a mare owner or veterinarian receives for insemination is highly variable and often well below minimum standards to expect a reasonable outcome of success. This is true for both cooled and frozen semen. However, the frozen semen technology itself is often blamed for negative results from poor quality semen that should never have been used in fulfillment of a commercial contract.
Nevertheless, the World Breeding Federation for Sport Horses (WBFSH), a non-profit federation of sport horse studbooks from around the world has proposed minimum quality standards for frozen and fresh semen being exchanged between participating WBFSH studbook members. For frozen semen the minimum standards recommended by a WBFSH veterinary committee are that each insemination dose of frozen semen should contain a minimum of 35% progressive motility and a minimum of 250 million progressively motile spermatozoa after thawing. This is certainly a step in the right direction toward restoring faith in the quality of semen provided by member facilities and providing some reassurance to breeders who purchase semen.
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