Breeding Programs with Cooled and Frozen - Thawed Stallion Semen
Dr. Sylvia Bedford-Guaus
Non-disposable containers. Non-disposable containers are cheaper and probably more suitable for cases in which only an occasional shipment of semen is required. For large breeding operations, where semen from a given stallion is regularly being shipped, probably non-disposable containers are more suitable and economical in the long term. These are sent to the mare's owner with the semen shipment, and then must be returned once the mare has been inseminated.
Transported/Cooled Semen Containers
Cooling containers can be purchased from several suppliers that sell general equipment semen for collection and breeding purposes. Cost is usually the most important consideration when choosing the type of container to be used. Containers may be disposable or non-disposable.
Disposable containers. These containers are usually made with a Styrofoam box. They do not always yield predictable cooling results and the semen is more influenced by environmental temperature changes than when packaged in a non-disposable container.
This is the non-disposable container of more widespread use. It cools semen slowly to a temperature of 39 to 43 degrees F (4 to 6 C), and it is durable and reliable. As long as it has not been opened it can hold semen up to 48 hours; however better results are obtained when semen is used within 24 hours of collection.
The Equitainer system includes:
The Equitainer or blue container itself
Isothermalizer, which is the container that is in contact with the coolant cans to provide an adequate cooling rate for the extended semen
Semen holding cup, which fits inside the isothermalizer
The range of volume that should be packed in an Equitainer is between 120 and 170 ml (cc), in order to achieve a reliable cooling rate and final semen temperature. If a minimum of 120 ml of extended semen is not available for shipment, then a ballast bag is placed in the cup next to the bag with semen to increase the total volume that is packaged in the Equitainer. Ballast bags (60 ml each, filled with purple fluid) can be purchased with the Equitainer system. Otherwise, water or extender can be placed in an adjacent bag and function as a ballast bag, as long as it is appropriately labeled so it won't be used for insemination.
The coolant cans should always be completely frozen before packaging semen in the Equitainer, and should have been kept in a freezer for at least 24 hours before use. The holding cup and isothermalizer should be at room temperature prior to use.
An extender is a diluent designed to extend the life of the sperm prior to insemination or during storage. Most extenders commercialized for stallion semen have a basic composition of dried-skim milk and some sort of sugar (most commonly glucose), based on the originally developed Kenney's extender. The extender can be easily made at the laboratory provided; the electronic scale and the ingredients are purchased. This may only be practical for large breeding operations. It also requires training in laboratory skills.
An antibiotic is added to the extender mix to prevent bacterial contamination during storage, which ultimately will affect sperm survival. The most common antibiotics presently used for semen extenders are ticarcillin disodium, amikacin sulfate, penicillin, or a combination of two of the above.
The brand of extender, and especially the antibiotic in the extender to be used, depends on the availability, cost and preferences. However, semen from some stallions may perform better in a certain type of extender and/or antibiotic and this should be tested prior to starting a transported semen program.
Preparing for Shipment
Once an ejaculate is collected, semen must be evaluated for volume and sperm concentration, in order to determine the total number of sperm in the sample. If several mares are to be bred, one must determined that enough sperm is given to each mare for an adequate breeding dose.
It is important to look at an extended sample to determine total and progressive sperm motility, or the percent of sperm that are moving or the percent of sperm moving in a forward trajectory, respectively. Estimating sperm motility requires training and experience. In general, it is considered that only those sperm moving forward are capable of fertilizing an egg, although this is a very simplistic view.
The final concentration of extended semen before packaging the sample for cooling must be always between 25 and 50 million sperm per ml (cc), to ensure maximal sperm survival without compromising fertility rates. Additionally, the final dilution ratio of extender: semen must be at least 2:1 (two parts of extender for every part of semen). This is a minimum dilution ratio, and higher dilution ratios are totally acceptable as long as the concentration of sperm is kept within range in the diluted sample.
Once the semen has been appropriately extended it is usually packed in plastic bags, most commonly in commercially available baby bottle liners or Whirl Pak bags, thoroughly closed and tightened with a rubber band. An alternative to rubber bands is to fasten the bags with a heat sealer, being careful not to heat the extended semen. In the case of Whirl Pak bags, cut the end of the bag with the wire to avoid puncture of the bag and loss of the sample during shipment. Prior to closing the bags it is very important to remove all air around the extended semen, which may induce oxidative damage of sperm during cooling.
Appropriately fastened bags are then placed in the sample cup and packaged in the cooling container for transport, always taking into account the appropriate final volume to be shipped (120 to 170 ml in the case of the Equitainer). The sample cup is then placed in the isothermalizer and the whole unit (isothermalizer with sample cup) on top and in contact with appropriately frozen coolant cans. It is advisable to include a sheet with the stallion's name and semen information with the shipment.
In some of the disposable cooling containers, extended semen is packaged into special syringes and placed in the container with a cooling pack. The appropriate instructions for shipment are included with each type of container.
Please keep in mind that the above rules are only guidelines and you should seek appropriate training before starting a cooling program in order to optimize your results.
The shipping container should not be opened until the mare is on the premises, scrubbed, and ready to be inseminated.