Pouring Liquid Nitrogen to Fill a Freezing Box

Frozen semen preparation and use

– An overview of frozen semen – Part 1 of a two-part article which appeared in the “Canadian Morgan” magazine in the November 2000 issue.

By Jos Mottershead
Part 1; published in “Canadian Morgan” Magazine, Nov/Dec 2000 issue.
Part 2 is in this link
(“Click” on photos to see an enlargement)

The phrase conjures up a variety of emotions in the horse breeder, not the least of which is a negative response, often as a result of a lack of knowledge. As more Breeds permit the use of frozen semen, there can be no doubt that breeders will gradually lose their fears, and start to be able to appreciate the full value of frozen semen.

The use of frozen semen is a collection of dichotomies – the convenience of having the semen already “on hand” at breeding time, compared to the smaller window of opportunity for its successful use; the simplicity of the breeding process compared with the requirement for absolute attention to detail; the price per insemination dose for frozen semen being cheaper than for cooled semen, compared with the requirement for a larger initial outlay for the freezing; and so it continues…

This article will explore the generalities of the freezing and insemination processes, and look at some aspects in greater detail, although it should be borne in mind that there are a variety of different protocols for freezing, and it is therefore impossible to cover them all here.

Stallion variability

One vitally important point to note for both stallion and mare owners is that not all stallions have sperm that will survive the freezing and thawing processes. This is probably one of the biggest stumbling blocks seen with frozen equine semen. To compound this problem further, there is no predictable correlation between live cover, fresh or cooled semen fertility, and frozen semen viability. And one can add to that the fact that there is currently no single definitive test that has proven successful in testing thawed frozen semen for fertility other than pregnancy rates established with it’s use.

This is not to say that the failure rate is dramatic enough to prevent its use being feasible. Roughly 30% of stallions have sperm that will result in good quality thawed semen; a further 40% will have “acceptable” post-thaw results, and only 30% would be considered possessors of subfertile frozen semen. In other words, 70% of stallions have semen that is capable of achieving pregnancies once frozen and thawed [more recent statistics reflect improved technique, extender and knowledge levels and are considered to be 75%]. The process must however be carried out, and pregnancies achieved, in order to know what percentage group any individual stallion falls into. Research is being continually carried out to attempt to overcome these problems, and as the use of frozen semen becomes more popular, so the faster results will be achieved. There can be no doubt that the addition of the world’s largest breed the AQHA to the ranks of breeds that permit registration of foals produced by frozen semen use will dramatically increase the speed with which advances are made.

The freezing process


A brief overview of the freezing process reveals that sperm are separated from the seminal plasma; a freezing extender is added at a certain volume; the re-extended semen is packed into “straws” at a known sperm concentration; the straws are frozen using liquid nitrogen, and are then submerged and kept stored in the liquid nitrogen until they are needed for use. Semen is reputed to be viable for 10,000 years once frozen, but realistically pregnancies have been achieved with sperm that have been stored for 10 to 12 years. [Since this article was written, at Equine-Reproduction.com, LLC we have established pregnancies with 30 year-old semen.]

Straw size

CASA Screen

CASA should ideally be used for semen evaluation

Semen is collected from the stallion in the same manner as is normal for fresh or cooled use, usually using an AV, although other methods may also be used. Once collected, a basic semen analysis is performed identifying such features as sperm concentration per millilitre; total gel-free volume; total numbers of sperm present; and progressive motility. These figures are then used to establish the numbers of straws that may be frozen from that ejaculate. Different protocols call for different numbers of sperm per straw, depending upon both the straw size and the particular protocol being used. “Straws” are short tubes commonly made of PVC. The two sizes of straw most common in North America are the half-millilitre (which is also seen in the cattle industry) and the “macro” straw which contains either four or five-millilitres. There are varied opinions as to which size straw yields the best results.


IEC Centra CL2 Centrifuge, showing swinging bucket interior

IEC Centra CL2 Centrifuge, showing swinging bucket interior

Once the concentrations are established, a centrifugation extender is added to the rest of the semen to afford it a degree of protection and it is centrifuged to remove the majority of the seminal plasma. It’s one of nature’s little jokes that seminal plasma is detrimental to sperm’s well being over an extended period of time. Some horses’ seminal plasma seems to be more of a problem than others, and it is hypothesized that this is one part of the reason for variability of success rates seen from stallion to stallion. A larger volume centrifuge is used, usually holding several 50-ml vials, and the semen is spun at a moderately high speed, usually producing G-forces in the region of 300 to 800 G. [Since this article was published, centrifugation cushions have been developed, which allow spinning at a standard 1,000 G for 20 minutes with about a 95% recovery] There is also variability from stallion to stallion as to how hard or long the semen needs to be spun. Obviously one wants the maximum harvest of sperm from the semen possible, but if spun too hard or too long, damage will occur to the sperm, and they will not be viable after freezing and thawing – or possibly even before! A soft pellet of sperm is desired, and once that has been produced, the seminal plasma is removed. Some protocols call for the removal of all the seminal plasma, and some for all but up to 10%.

Packaging concentrations

The math carried out to establish the total numbers of sperm in the ejaculate would also have provided the means to establish the correct number of straws to be frozen from the ejaculate. The half-ml straws are typically frozen at the rate of 200-400 million sperm per ml, and the “macro” straws are commonly packed to hold a total of 600-800 million sperm per straw. This would mean for example that an ejaculate containing a total number of 12 billion sperm would be capable of producing up to 120 half-ml straws or 20 five-ml straws [note that the half-ml straw typically requires 8 straws for a full insemination dose while a single 5-ml straw contains one dose]. Note that the figure used in most protocols is not progressively motile sperm, but total sperm per ejaculate. The only function that the progressive motility figure serves at the pre-freeze stage is to determine whether the ejaculate is worth processing. Ejaculates with a progressive motility pre-freezing of 50% or lower would not usually be considered worthwhile freezing, except under exceptional circumstances. The number of progressively motile sperm post-thaw will be evaluated, and it is this figure that will determine the numbers of straws needed for an individual insemination. More of that later though.

The actual number of straws capable of being frozen from a single ejaculate is entirely stallion-dependent, as the individual stallion will produce different numbers of sperm from ejaculate to ejaculate, and at different times of the year. The average generally given as a guide, is that there will be sperm numbers sufficient to produce enough straws for between 5 and 15 insemination doses, but again, I must stress that this is very variable, and factors such as stallion age, frequency of use and general fertility will all come into play.

Freezing extenders

Once the number of straws to be frozen is established, it is easy to determine the amount of freezing extender that is required by subtracting the volume of the sperm pellets remaining after centrifugation from the total volume of re-extended semen required for the straws to be frozen. Say that there were 6-ml of sperm pellet, then with the above numbers of straws to be frozen, one would need to add 54 ml of extender to produce 120 half-ml straws, or 94-ml of extender to produce 20 straws of the 5-ml size.

Semen extenders are variable in actual ingredients, but all contain ingredients that serve a common purpose. There will be a nutrient, a buffer, a cryoprotectant agent and antibiotic. A typical nutrient seen is a sugar, such as glucose or sucrose, which serves to provide energy source for the sperm. Buffers are added to balance pH and osmolarity of the solution, and the sugars also serve this purpose. The cryoprotectant most commonly seen is glycerol, although others such as DMSO [or a combination of the various Amides (Methylformamide, Dimethylformamide etc.)] have also been used with varying success. The cryoprotectant assists in stabilizing the cell during the freezing and thawing process. If one imagines the effect that is seen when a glass jar full of water is frozen (the water expands as it freezes, and the jar may crack), then one understands one of the most important stresses that sperm is subjected to during the freeze/thaw process. The cryoprotectant actually causes the removal of (and in the case of glycerol, replaces a little of) the water found within the cell, so that there is not the same expansion factor upon freezing, and the cell membrane does not rupture. Egg yolk, which is a common ingredient in a wide variety of frozen semen extenders, serves several purposes. It has nutrient value for the sperm during the freezing and thawing process; acts to some extent as a buffer; and also most importantly “coats” the sperm cell, and thereby prevents changes to and losses of some of the lipoproteins present in the cell membrane that can occur during the freezing and thawing processes. Various antibiotics are added to control bacterial content that may be present in the raw semen. It should be noted that the antibiotics will only protect against bacteria and not viruses, for which being frozen in liquid nitrogen may be an ideal storage medium. For this reason it is desirable that stallions to be frozen be tested EVA (equine viral arteritis) negative prior to collection.

Straw packing

Once the correct amount of extended semen is packed into the straws, an air bubble is introduced, and they are sealed. The air bubble is of paramount importance, and is present to prevent the same “freezing glass jar” effect discussed above, although in this case, the bubble permits the expansion of the semen during freezing and prevents the rupture of the straw, or the “blowing” of the sealant at either end.


Half ml Semen Straw Freezing Rack

Half ml Semen Straw Freezing Rack

5 ml "Macro" Straw Straw Sealing Beads

5 ml “Macro” Straw Straw Sealing Beads

The half-ml straws usually contain a “wick and powder” sealant at one end, which upon coming into contact with moisture (the semen) becomes solidified, and seals the top end (the “open” end of the straw is immersed into the semen and suction is applied, sucking the semen into the straw until the sealant at the top end prevents further suction by becoming solid). The bottom end of the straw is then sealed by using either polyvinyl alcohol powder (which may then be dipped into water, and solidifies), or by inserting a small steel ball. The macro straws use steel or glass balls, and must have one end sealed prior to being filled. The semen is introduced into the macro straw by a pipettor that dispenses a pre-established amount. The open end is then sealed with another ball – often a glass one which may be of a variety of colours which later aids in identification.

Straw labelling

It is important that all the straws carry a variety of information written on the outside. The stallion’s name, registration number and breed; the date the freeze was performed; and the type of the extender used should be considered a minimum. Other information that may be considered suitable for inclusion would be the freezing location and company [required for export] and the antibiotic used in the extender. This information is essential for identification of the straws prior to insemination, and should never be omitted.

Cooling and freezing

Some protocols call for a slow cooling period for the packed straws, from room temperature to approximately 5° Celsius over the course of a couple of hours prior to the straws being exposed to the liquid nitrogen for the final freeze. If that is required, it may be carried out in an especially programmed slow cooling machine.

For the actual freezing process, two methods are commonly seen. A programmable freezer, and freezing in liquid nitrogen vapour.

The programmable freezing unit may also be capable of performing the slow cooling to 5° Celsius, and carries out the freezing process automatically once the straws are packed.


Pouring Liquid Nitrogen to Fill a Freezing Box

Pouring Liquid Nitrogen to Fill a Freezing Box

5 ml "Macro" Straw Floating Rack

5 ml “Macro” Straw Floating Rack

With the nitrogen vapour freeze technique, the straws are placed onto a freezing rack and lowered into a Styrofoam box (such as a picnic cooler) that contains at least 3 inches of liquid nitrogen. The freezing rack is designed to support the straws one inch above the liquid nitrogen, at which point the temperature is approximately minus 160° Celsius. After being held in that position for 10 – 15 minutes, the straws are plunged into the liquid nitrogen (the temperature of which is minus 196° Celsius – minus 320° F). There are a variety of freezing racks available, but I personally believe the best to be one that floats on the liquid nitrogen at a known level, as this will result in an even freeze. Fixed racks are dependent upon the surface supporting the freezing box being level. If that surface is uneven as one end of the straw will be closer to the nitrogen than the other, it will result in both the air bubble within the straw floating to one end, and an uneven overall freeze, both occurrences of which are highly undesirable.

Transfer and storage

5 ml "Macro" Straws Raised in the Neck of a Tank for Inspection

5 ml “Macro” Straws Raised in the Neck of a Tank for Inspection

Half ml Semen Straws in Goblet on Cane

Half ml Semen Straws in Goblet on Cane

Once the straws are frozen and have been submerged in the liquid nitrogen for 10 minutes, they are transferred to a liquid nitrogen storage tank, which is essentially an oversize thermos container that holds liquid nitrogen. The half-ml straws must be first placed in a “goblet”, (which is pre-cooled in liquid nitrogen), and they in turn are placed on a “cane”, which is stored in a “canister” within the tank. Failure to place half-ml straws in such a storage apparatus within the tank may cause them to be exceedingly difficult to retrieve for use, as they can float out of the canisters when a tank is filled and then be loose in the tank. The macro straws can be placed directly into the canisters for storage. There are only approximately four seconds available for transfer before thaw damage to the sperm occurs as a result of exposure to higher temperatures than the liquid nitrogen. From this it can be seen that care and respect in handling the frozen product is essential.

Go To Part 2

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