Breeding Programs with Cooled and Frozen – Thawed Stallion Semen

Starting a breeding program with either cooled or frozen-thawed equine semen requires knowledge and expertise in collecting and evaluating semen from stallions. With mares breeding with cooled or frozen semen also requires intensively planned breeding management. Cooled or transported semen is not frozen semen, and frozen semen is not cooled or transported semen, although they are often confused or discussed indistinctively.

Cooled or transported semen is semen that has been packaged in a special container for cooling to refrigerator temperature of 39 to 43 degrees Fahrenheit (4 to 6 degrees Celsius) during transport for insemination of mares at distant farms. Cooled semen will only last for 24 to 48 hours, depending on the type of container used for shipment. If the semen is of good quality and is handled adequately, fertility rates with cooled semen are similar to those obtained by performing artificial insemination with fresh semen. Any breeding manager with expertise in handling stallion semen can start a cooled or transported semen program, after minimal training at a referral facility or by a veterinarian. Some universities offer short courses for veterinarians and horsemen interested in cooling stallion semen for transport.

Frozen equine semen is that usually packaged in 0.5-, 2.5- or 5-ml (cc) straws and stored in a liquid nitrogen tank at –320 degrees Fahrenheit (-196 degrees Celsius). In theory, frozen semen can last forever (like a diamond). However, freezing semen for storage is a much more complex procedure than just cooling semen for immediate transport. Freezing semen requires specialized equipment and specialized training. Fertility with frozen-thawed semen is very poor even with good quality semen and very intensive breeding management techniques are used. For this reason, stallions are often taken to specialized centers or referral facilities for this process.

Not all breed associations allow the use of transported or frozen-thawed semen in order to register the offspring. When in doubt, you should always check with your breed registry prior to deciding whether to breed your mare with stored semen.

Considerations

Every stallion owner or manager can start a transported semen program if shipment of semen is required to breed mares at distant farms. However, minimum requirements should include a breeding shed to collect semen and a laboratory equipped for on-farm semen evaluation. Minimal equipment includes a microscope to evaluate semen, some sort of machine designed to count sperm concentration, an incubator to keep all equipment at body temperature of 98.6 degrees F (37 degrees C), and miscellaneous lab materials to handle and evaluate semen, such as pipettes, microscope slides, plastic cups and graduated cylinders. Additionally, some training in the basics of semen handling and packaging is required.

The semen quality of the stallion is also an important consideration. Any stallion entering a transported breeding program should receive a complete breeding soundness evaluation by an experienced veterinarian specializing in reproduction. A cooling test of the semen should be done on-farm prior to performing any shipments.

Advantages of a Transported Semen Program

  • Avoids the stress of shipping a mare and foal for breeding
  • Increases the gene pool for the particular breed
  • Reduces the use of genetically inferior stallions
  • Eliminates the cost of boarding a mare and foal at a breeding farm
  • Increases the book of mares for the stallion in a given breeding season
  • Reduces the risk of transmission of venereal disease or contagious diseases resulting from boarding the mare and foal at a different farm

    Disadvantages of a Transported Semen Program

  • Requires some training in semen collection and handling techniques, more so than with artificial insemination programs with fresh semen
            
  • Requires an initial investment in semen collection and handling equipment, unless an artificial insemination program is already in place
  • Requires purchasing semen cooling equipment         
  • Carries possibility that poor quality semen may not survive the cooling/shipment process.        
  • Requires mare in estrus or ovariectomized mare to stimulate stallion for semen collection        
  • Requires more intensive mare breeding management
  • Carries risk of fraud, as when the semen is used for breeding a mare different than that stipulated in the stallion service contract

    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.

  • 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.
  • 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.

    The Equitainer

    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
  • Coolant cans
  • Ballast bags

    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.

    Extenders

    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.

    Advantages
            

  • Many of the advantages of using frozen-thawed equine semen are similar to those stated for cooled-transported semen.
            
  • An added advantage of frozen-thawed equine semen is that you can have it at the farm in a liquid nitrogen tank 'ready to go' and therefore can use it as soon as the mare is ready to ovulate without having to coordinate the time of shipment in relation to mare readiness.
            
  • Semen from the stallion can be used while the stallion is at shows or even after his death (please note that some breed associations will not allow registering a foal bred with frozen semen after the death of the stallion).

    Disadvantages

  • Freezing semen successfully requires not only very good quality semen to start with, but also expensive specialized equipment and extensive training.
  • The frequent checks required for optimal breeding timing in the mare can result in a very expensive veterinary bill.
  • Results with frozen-thawed semen are suboptimal and quite variable among stallions and mares. Fertility rates vary from 0 to 80 percent when using frozen-thawed semen, with the most important variables being the particular stallion and breeding management.

    The Basics of Freezing and Thawing

    It is important that you seek professional advice if you are intending to start a freezing program. Freezing stallion semen requires specialized and expensive equipment, and there are many different techniques and extenders used to freeze semen.

    As a general guideline, most extenders to freeze stallion semen contain a cryoprotectant to buffer sperm during the freezing process. These are usually egg yolk or glycerol or, most commonly, a combination of both.

    Prior to adding the freezing extender, semen to be frozen is centrifuged and the seminal fluid is separated from the sperm cells. Then the sperm cells are rediluted into the freezing medium and packaged into plastic straws. These are either placed into an automated freezer machine or over liquid nitrogen vapor prior to plunging into the liquid nitrogen for storage in a special tank. Frozen straws may last for indefinite periods of time as long as the temperature conditions in the storage tank are kept constant.

    Straws used for insemination of mares should not be thawed until the mare is prepared for insemination. This is usually done by plunging them into a water bath at a given temperature, which may range from 98.6 to 122 degrees F (37 to 50 C), for variable periods of time. The thawing method depends largely on the freezing method, and therefore straws for insemination are always shipped with a sheet explaining appropriate thawing instructions. These should be strictly followed for optimal results.

    Insemination Dose

  • The minimum recommended dose to achieve reasonable success for insemination with fresh and cooled equine semen is 500 x 106 (million) progressively motile spermatozoa. But remember: even though it only takes one, the more sperm used to inseminate a mare the better the chances for impregnation.
  • The adequate breeding dose for frozen-thawed semen has not been worked out. Most specialists in the field, however, recommend a minimum of also 500 x 106 progressively motile spermatozoa based on post-thaw motility. This is a controversial topic, and pregnancy rates with frozen-thawed semen are not predictable even when using optimal numbers of progressively motile sperm.

    Insemination Volume

    Some veterinarians and breeders claim that large insemination volumes will cause uterine infections in mares, and may insist on shipping no more than 50-ml of extended semen. Unfortunately, this may compromise the number of viable sperm available to inseminate the mare and thus pregnancy rates may be reduced. There is no research that supports this and, in the case of cooled semen, you should always send as much available extended semen as you can possibly fit in the shipping container.

    For example, a research study in pony mares compared pregnancy rates using 30 and 120-ml of extended semen, when both volumes contained an sufficient number of progressively motile sperm. Pregnancy rates were not statistically different, and where slightly higher in the group inseminated with the 120-ml treatment. One mare developed a significant post-insemination uterine infection, and that was after using 30 ml of extended semen!

    Mares that are susceptible to postbreeding infections may get infected regardless of insemination volume. A typical example is when breeding with frozen-thawed semen. Even though insemination volumes are as small as 0.5 to 5 ml, some mares may still develop significant post-breeding infections due to an inflammatory reaction against the sperm itself.

    Insemination Timing

    Good breeding management of the mare to be bred with cooled or frozen-thawed semen is as important as good management of semen at the stallion's end. The key to good breeding management is timing of insemination in relation to ovulation.

    Any mare in a breeding program should be closely monitored for signs of heat, by teasing, and then her reproductive tract closely followed by palpation and ultrasonography per rectum to ascertain the optimal timing for ordering semen (in the case of cooled semen) and/or insemination.

    The use of reproductive hormones such as Prostaglandin F2a, to "short cycle" or bring the mare into heat, and human chorionic gonadotropin or deslorelin (Ovuplant) to induce ovulation, will facilitate management of mares for breeding with transported or frozen-thawed semen.

    In general terms, cooled semen should be inseminated within 48 hours of ovulation. The closer to ovulation the better, but 24 to 48 hr will do.

    In the case of frozen-thawed semen, optimal pregnancy results are obtained when breeding within 6 hours of ovulation. If the semen quality is excellent, one can stretch it up to 12 hours, but I do not recommend it. Insemination with frozen-thawed equine semen beyond 12 hours before or after ovulation will yield extremely poor pregnancy rates.

    Research has also shown that it is always better to breed before rather than after ovulation.

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