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.

TWO DIFFERENT SIZES OF SEMEN STRAWS
The upper straw is the larger 5 ml straw (with the upper scale on the ruler in inches), the lower the .5 ml straw (lower ruler scale in centimeters)

In North America, the most commonly used packaging systems for frozen stallion semen are the five milliliter "macro" straw and the one-half milliliter straw. 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 inseminators point of view. The one-half ml straw on the other hand, 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. For the sake of this article, we will merely discuss thawing protocols and the differences between the two systems and not attempt to pass judgment. This article will also be 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, as they will have tested various protocols, and again, there is a degree of "stallion variability" in which time and temperature works best.

5 ML STRAW USED FOR EQUINE AI

Frozen semen is stored in liquid nitrogen (LN2), in specially designed vacuum containers that maintain a constant minus 196oCelsius throughout the main part of the body cavity of the container. In the upper part of the container, below the neck and still in the body of the container, the temperature rises to between minus 180o to minus 150o degrees Celsius, and this is still an acceptable temperature for the storage of semen. If even only 10 to 12 centimeters of LN2 remain in the tank, because the tank is designed to continually circulate the gas, it will still maintain the low temperatures. Damage will start to occur to the frozen semen if it is exposed to temperatures greater than minus 80o Celsius for longer than 4 seconds. These temperatures occur within the neck tube of the LN2 container, where in fact, the temperature will rise above 0o 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 must be plunged back into the LN2 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 not known to have resulted in pregnancies.

All of the above information will apply in exactly the same manner to the "LN2 dry shipper", in which your semen may have been delivered to you, as it does to a regular LN2 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 with a sterilizing cleaner, such as "Betadine", 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 LN2 in either end, and place the straw into the water bath. The 5 ml staws can be transferred directly. The whole transfer should not be allowed to take longer than 4 seconds. As there is a danger of a straw exploding during the transfer, it is recommended that protective eyeware 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 (37oCelsius), 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 bowl can be used for the half ml straw, and a tall glass 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 submerged at once, 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 possibly becoming more common than the macro straw, has a conundrum associated with thawing protocols by itself. Experimentation has shown that the protocol resulting in the best results is to submerse the straw in 70o C. water for 7 seconds, and then transfer it rapidly to another water bath at 35o C. for at least 5 further seconds. It must be stressed that temperatures and timing of this method in particular are extremely critical, because of the high temperature of the initial water bath. 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 and it's 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 38o C. for 30 seconds. Obviously, as this is almost the temperature that is the final aim, exposure for a marginally longer period will not be detrimental, but exposure for periods greater than 5 minutes is not recommended.

As sperm in the half ml straw is typically frozen at the concentration of 400 million per ml, each straw will hold approximately 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. Each straw should be thawed rapidly, and emptied into a sterile, non-spermicidal container such as a 15 ml centrifuge tube, that has been pre-warmed to 37o Celsius. 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, it is desirable to briefly check a small drop of each straw's thawed semen for motility before adding it to the centrifuge tube. Any straws showing a gross negative abnormality from the average post-thaw motility should be discarded. Once all required straws are thawed and added to the container, the semen should be draw 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 allow for 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, 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 I would feel more comfortable checking each straw for post-thaw motility with unproven semen. It should be borne in mind that freeze/thaw results can vary even within a single "batch" of semen, and very definitely from separate freezes, and with the time of the year.

The 5 ml "macro" straw requires a higher temperature than the "field use" half ml 38os, as it is a thicker straw. This straw must be submersed in a 52o C. water bath for 52 seconds. During that time, it should be removed long enough to invert, when the air bubble that is in the center upon freezing, is seen to float to the top of the straw. Occasionally, two inversions are required. At 52 seconds, the straw should be removed and placed in another water bath at 35o C. for at least 5 seconds. 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 may in fact hold enough sperm for an insemination dose. As with the half ml straws however, it is still easier to empty the contents into a centrifuge tube, and draw the semen into the syringe from there. Again, checking each straw's contents prior to placing it in the tube is advisable.

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.

© 2000 Jos Mottershead and Equine-Reproduction.com.
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