Thawing and preparing frozen semen for artificial insemination.

By Jos Mottershead

In order for maximum reproductive efficiency to be gained from the use of frozen equine semen, it is essential that the product be frozen, stored and thawed correctly. It is important to note that even if all these facets are carefully covered, reliability of frozen equine semen is still immensely variable from stallion to stallion.

Semen StrawsIn North America, the most commonly used packaging systems for frozen stallion semen are the one-half milliliter straw (shown at top in image) and the five milliliter "macro" straw (shown at bottom in image). There is tremendous dissension among freezing centers as to which size straw yields the best result. The macro straw is possibly easier to handle during the freezing process, which can be carried out completely manually and an entire insemination dose can be frozen in a single straw or two, which is convenient from the inseminator's point of view. That a single dose of semen is contained within the one straw also makes it more difficult to "split doses" between multiple mares (as the mares would have to be synchronized almost perfectly relative to ovulation), which is positive from the perspective of the stallion owner (reducing the risk of multiple mares being bred from a single breeding contract) but negative for the mare owner who has purchased semen "by the dose" with no usage restrictions. The one-half ml straw can be used with mechanical processors, which is useful in cases of mass production, and yields a more equal freeze throughout the straw as the diameter is considerably less than that of the macro straw. This article is limited to the general parameters, and if a different thawing protocol is recommended by the freezing technicians for some semen that you are in possession of, please follow their recommended protocol. They will have tested various thawing protocols, and there is a possible degree of "stallion variability" in which suitable duration and temperature works best.

Semen TankFrozen semen is stored in liquid nitrogen (LN2) in specially designed vacuum containers that maintain a constant minus 196° Celsius (-320°Fahrenheit) throughout the main part of the storage cavity of the container. In the upper part of the container immediately below the neck but still in the body of the container, the temperature rises to between minus 180° to minus 150° Celsius (-292 to -238 °F), and this is still an acceptable temperature for the storage of semen. If even only one inch of liquid nitrogen remains in the tank, because the tank is designed to continually circulate the cold gas, it will still maintain an adequately low temperatures (note however that it is not recommended to allow the tank to get to this low a level! Tanks should ideally be filled when liquid nitrogen levels reach about half). Damage will start to occur to the frozen semen if it is exposed to temperatures greater than minus 140°C (-220°F) for longer than 4 seconds. These temperatures occur within the neck tube of the nitrogen container, where in fact, the temperature will rise above 0° Celsius at the top. This means that great caution must be taken whenever there is a need to raise a canister containing semen in the tank, such as to carry out inventory checks, or to establish which straws one wishes to remove. As a "rule of thumb", the semen straws must not be raised above the point where frost appears in the neck of the container, for longer than 4 seconds, and once 4 seconds of exposure is reached, the canister should be plunged back into the nitrogen to the bottom of the tank for at least 30 seconds. This practice will result in minimum damage to sperm. Inseminations of sperm that have been thawed and re-frozen are known to have reduced or no pregnancy rates.

Vapour ShipperAll of the above information will apply in exactly the same manner to the vapour ("dry") shipper in which your semen was delivered to you as it does to a regular storage tank.

The mare to be inseminated must have undergone any required palpations or ultrasounds, and be prepared for insemination, having had a tail-wrap applied, and her perineum washed prior to the semen being thawed. Ideally she will be restrained in palpation stocks, and it is essential that once her perineum is washed, her tail is not allowed to come back into contact with it.

Raise the canister containing the semen straws to be used up to the frost line of the tank, and remove the straw that is to be thawed. If using the half ml straw, give a brief, decisive shake (such as you would a mercury thermometer to lower the mercury) to remove any residual liquid nitrogen in either end, and place the straw into the water bath. The 5 ml straws can be transferred directly. The whole transfer should not be allowed to take longer than 4 seconds. As there is a risk of a straw exploding during the transfer, it is recommended that protective eyewear be worn at any time frozen semen straws are handled. This is especially important with straws that are sealed with glass or steel balls, rather than PVC powder.

Raise the canister containing the semen straws to be used up to the frost line of the tank, and remove the straw that is to be thawed. If using the half ml straw, give a brief, decisive shake (such as you would a mercury thermometer to lower the mercury) to remove any residual liquid nitrogen in either end, and place the straw into the water bath. The 5 ml straws can be transferred directly. The whole transfer should not be allowed to take longer than 4 seconds. As there is a risk of a straw exploding during the transfer, it is recommended that protective eyewear be worn at any time frozen semen straws are handled. This is especially important with straws that are sealed with glass or steel balls, rather than PVC powder.

There has been much experimentation to establish the "best" thawing protocols for frozen stallion semen, and it has been unanimously agreed that a fast-thaw procedure in hot water is best. Other methods tried included air-thaw, cold water, and ice bath. The aim is to reconstitute the sperm to body temperature or close to it for insemination. Obviously with the difference in size of the straws, there will be a difference in times and temperatures required to achieve this.

The "water bath" that is used need not be fancy. In fact, a mug or small Styrofoam box can be used for the half ml straw, and a tall glass or plastic container such as a pasta storage jar can be used for the 5 ml macro straw. It is important that the entire straw can be fully submerged, and that there be sufficient water surrounding the straw to avoid a great loss of temperature upon the water's exposure to the frozen straw.

The half ml straw, which is more common than the macro straw, has a slight conundrum associated with thawing protocols. Experimentation has shown that the protocol resulting in the best result is to submerse the straw in 70°C (158°F) water for 7 seconds and then transfer it rapidly to another water bath at 35°C for 5 further seconds. It must be stressed that temperatures and timing of this method in particular are extremely critical. Consequently, although this method will usually result in a higher post-thaw viable sperm yield, it is not really a practical method for anyone "in the field", where access to a laboratory with hotter water and accurate measuring equipment is often not available.

The easier, and more favoured method for thawing the half ml straw therefore, is to submerge it in a water bath at 37 or 38°C for a minimum of 30 seconds. Obviously, as this is almost the temperature that is the final aim, exposure for a marginally longer period will not be detrimental. Straws may be transferred from the storage tank or shipper into the same thaw container with little delay in between (remember the water will be cooling as each straw makes contact), however the entire dose of straws should not be transferred "em mass" by simply emptying the goblet into the water, as if straws come into contact with the water while also in contact with each other, they may freeze together temporarily, negatively impacting the regularity of the thawing process.

As sperm in the half ml straw are usually frozen at a concentration of 200-400 million per ml, each straw will hold approximately 100-200 million sperm. Depending upon post-thaw motility rates of the sperm, this will mean that thawing between four and eight straws will commonly be necessary to achieve the desired insemination dose (it is usual to target around 800 million total sperm per dose, but post-thaw motility can affect this number). The straws should be thawed as described, then emptied into a sterile non-spermicidal container such as a 15 ml centrifuge tube, that has been pre-warmed to 37°C. The container must be kept at body temperature during the thawing process of the multiple straws, in order to maintain the temperature of the already-thawed semen contained within. Although an incubator is preferable for this, a warm hand or pocket may prove more practical in the field. If a microscope is available, and if multiple technicians are available to prevent delay, in particular if the semen has unknown (or unreported) post-thaw values, it may be desirable to briefly check a small drop of each straw's thawed semen for motility before adding it to the centrifuge tube. Any straw showing a gross negative abnormality from the average or reported post-thaw motility may be discarded and replaced with another if available. It should however be remembered that any sperm not inseminated is guaranteed to not cause a pregnancy, so discarding without replacement, or deciding not to inseminate based solely upon poor post-thaw results is typically not recommended! Once all required straws are thawed and added to the container, the semen should be drawn reasonably slowly up into a warmed all-plastic insemination syringe, and inseminated as soon as possible into the mare.

"Minitube" produces a "Universal" insemination pipette which will allows the removal of an emptied straw from within the holder of the pipette without removing the pipette from the mare's cervix. This means that multiple straws can be loaded into the pipette one after the other, and the semen deposited into the mare without the need for pre-mixing the semen in a centrifuge tube or similar container. This is a convenient method for use with semen that has proven fertility, but can make checking semen for post-thaw motility more difficult.

The 5 ml "macro" straw requires a higher temperature than the "field use" half ml straw, as it is a thicker straw. The larger straw must be submersed in a 45-52°C water bath for 45-52 seconds depending upon the recommended protocol, which should accompany the semen delivery. Better results have been seen with semen from straws that are thawed vertically rather than horizontally. During thawing using the vertical method, when the air bubble that is placed in the center of the straw during freezing is seen to float to the top of the straw, the straw should be removed from the water quickly and inverted then replaced, causing the bubble to be back at the bottom. Multiple inversions are often required, and it is done to keep the semen moving within the straw, which reduces the possibility of heat shock of sperm immediately adjacent to the outer surface. At the end of the thaw period the straw should be removed and placed in another water bath at 35°C for 5 seconds, or any surface water immediately wiped off with a piece of paper towel or a cloth. This will stabilize the outer temperature of the straw, and prevent those cells closest to the outside from overheating.

Sperm is typically packed into the macro straw at a rate of between 100 to 200 million per ml. This means that a single straw will be likely to hold enough sperm for an insemination dose. After thawing, empty the contents into a centrifuge tube and draw the semen into the insemination syringe. Again, evaluating each straw's contents prior to placing it in the tube is desirable.

The thawing and use of frozen semen is not difficult, but as with any reproductive procedure, the more care and attention that is paid to the process the more successful the outcome is likely to be.


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