Cryopreservation...Freezing Semen
By Karen Berk
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Equine
Cryopreservation is the process of collecting, processing, freezing and storing
a stallion's genetic material. It is perhaps the most misunderstood and
therefore backed away from technology in advanced equine reproduction. Research
is continuing at numerous universities and private enterprises to advance our
current knowledge, and smooth out what has become recurring problems.
At
this time, conception rates with frozen semen remain at about 50%. Even with
this, there are many critical factors for success. There must be proper
collection techniques, with a close to sterile environment. The stallion should
have a progressive motility, upwards of 50% (bare minimum). Middle to high
concentration is also preferred. Many aged stallions, beyond 13 years, while
developing varying degrees of testicular degeneration, produce larger volumes
of fluid with less sperm cells. It is from this group we receive the most
requests for freezing. Owners of young, virile stallions are not as concerned
at the moment for saving their stallions genetic material, thus insuring their
future. Often when we are called, it is too late. Many aged stallions can
continue to be successful with live cover, and even ship well with the aid of a
centrifuge, but lose potential to freeze enough straws per collection, to make
it economically feasible. It is therefore imperative to decide on banking semen
while the stallion is in his prime, if circumstances permit. Many stallions are
not suitable at any age. Young stallions may exhibit poor seminal quality due
to other factors such as: medications, physical condition, nutrition, over
breeding, testicular size, overwork, and heredity, to name a few.
The
freezing process begins similarly to a normal collection. We are using some
form of sperm counter to assess concentration, and preferably a phase contrast
microscope with a heating stage, centrifuge, incubator, liquid nitrogen tank,
and waterbath.
We start by washing the stallion thoroughly, making
certain he is dry. If using anything other than warm water for cleaning, it is
imperative to rinse thoroughly, making certain there is no residue. I recommend
doing this type of washing, the day before collection, and washing with warm
water only, prior to collection. A mild detergent such as Ivory works quite
well for removing smegma, as well as the sheath cleaning product, Excaliber.
Remember, be gentle. Do not force or pick off scales. Excaliber can be left on
for an hour, and when you return to finish the job, the scales are easily
washed away, leaving the penis non-irritated. And always, rinse, rinse, rinse,
and gently towel dry.
After collecting, the ejaculate is evaluated as
to concentration and motility, and extended in a centrifuge medium.
Centrifugation time varies, by the individual animal's ejaculate. A good base
to start at is 10 minutes at 400 G force. The centrifuge spins at extreme
speed, separating the liquid from the cells. The liquid is then aspirated off
leaving a sperm pellet on the bottom. It is important to leave a small amount
of seminal fluid, as this appears to be necessary to the cells survival. The
new concentration is then re calibrated by means of a hemocytometer, and re
extended in a freezing medium. This freezing extender is a base extender with
the addition of glycerol, egg yolk, and a food detergent, Equex.
Straws
are then filled depending on their size. If macro straws, 5 ml., are used, they
normally will accommodate 600 to 800 million progressively motile cells per
straw, or 1 dose. Half ml. straws are also common, but take 3 or as many as 10
straws, to support a dose. Straws are then placed on a freezing rack set just
above a level of liquid nitrogen. This allows the straws to initially freeze in
the vapors, producing less shock. After 20 minutes they are placed directly
into the liquid nitrogen which completes the freezing process. ½ ml
straws may take less time in the vapor. The straws can now be transferred into
a liquid nitrogen tank for storage. Properly cared for these will last forever.
Regular checks and maintenance of the tanks are a priority.
In
evaluating frozen semen, we can only test for post thaw motility.
Unfortunately, this does not mean fertility. Even very high motility is no
guarantee for conception. One of the problems being researched is that freezing
tends to destroy 1) the cells abilities to adhere to the egg, and, 2) the
enzymes needed to penetrate it, for capacitation and conception to occur. At
this time there is no test to confirm or deny wether any damage has occurred,
but research is currently being conducted to produce a test. Breeding a
reproductively sound mare, with a sampling of your straws is recommended. A
positive pregnancy test is your only warrantee. If purchasing straws, always
ask if they have had a successful test, and for numbers and references.
The sale and purchase of equine frozen semen, is also a new avenue for
most breeders. Frozen semen can be banked by the purchaser for any length of
time, including years. Live foal guarantees become difficult to promise, if not
impossible. The added expense of freezing and banking, is usually compensated
in the breeding fee, by being paid for up front, in full. The straws are then
released to the purchaser. Most stallions offering frozen semen also limit the
amount of straws, or cycles, of the breeding. If the mare has not settled,
additional straws can be obtained at a pre arranged cost. There are also
stallions, including some deceased, that charge another full stud fee, should
the mare not conceive on the allotted straws. There are pros and cons on both
sides. Considerable time and cost are engaged by the stallion owner, yet the
mare owner can pick and choose the right moment for insemination. The cost of
shipping, and the dry shippers themselves, specific nitrogen containers for
shipping, are costlier then shipping fresh chilled semen. But, you don't have
to worry about timing your mare's ovulation. The straws are available whenever
you choose, and your mare is ready.
Always get thaw instructions,
specific for the straws you have purchased, and a contact number for the
company who froze the semen. Only a few degrees, or 2 to 3 seconds, too long or
too short, will damage or destroy the cells. Competent companies, with positive
pregnancy results, are happy to give references. Always discuss the purchase of
frozen semen straws with your veterinarian. He or she may not be experienced
with working with frozen. It also is necessary to have a liquid nitrogen tank
available to either yourself or your vet to store the straws. A waterbath, and
straw forceps are also necessary to handle and thaw.
Insemination with
frozen has it's own specific needs. The mare must be carefully observed during
her estrus. Monitored by ultrasound throughout, and at least twice daily after
reaching a 35 ml. follicle. Ultrasound needs to be utilized every 6 to 8 hours,
nearing ovulation, in order to decide the closest point, prior or post
ovulation.. Most straws need to be administered within 6 hours of ovulation.
The ultrasound gives us a clear picture of the follicle beginning to lose
shape, just prior to ovulating. With most mares, ovulation occurs between 4 AM
and 10 AM. This can be an added burden on your veterinarian's time. Semen
previously frozen, does not have the life span of fresh chilled, within the
mare. Thus the need for insemination within minutes of thaw, and perfect timing
of the ovulation.
A new protocol was presented this year at the
International Reproduction Symposium at Solvang, California. It has been
researched and found effective, and simplifies the insemination timing. The
mare is observed at the start of estrus, and when ultrasounded at a 35
follicle, larger for certain breeds, HcG is administered. The mare is then
inseminated at 24 and 40 hours post HcG. One can then determine once a day,
utilizing ultrasound, the progression of her ovulation. This will definitely
cut cost to the mare owner.
It is actually the European, Iberian, and
South American markets, that have pursued the American breeders to become more
adept in the use and marketing of Equine Cryogenics. In Europe, the use and
shipment of straws, are as commonplace as fresh chilled is to us. For 20 plus
years it has been an accepted and successful practice. Recently they lobbied
the American Quarter Horse Association, so successfully, that at the AQHA
convention, they passed the usage of frozen semen, beginning in 2001. Because
of international customs regulations, fresh chilled was often unable to be
delivered within 48 hours, diminishing sperm quality severely, if not entirely.
To maintain a global genetic bank, readily available and accessible, their
reasoning won out.
This is also an excellent way to secure healthy
semen, for both exportation and importation, while also expanding your client
base, or, as a mare owner, expanding your genetic pool. Each country has their
own requirement for the importation of horse semen. A quarantine is generally
needed, along with a health certificate. The stallion while quarantined, may
use that time to be frozen. This ranges from 30 to 60 days. A quarantine may be
arranged at your own barn, a specific reproductive facility or University,
through your vet and the USDA. Semen frozen within the USA and shipped within
the country does not require a quarantine.
Heredity is a severe
problem we have created in the industry. In cattle, regardless of quality, show
career or breeding, a reproductively inferior animal is eliminated. As horse
breeders we continue to breed both stallions and mares with poor reproductive
histories. Using the advancements of veterinary science, we make up for what
nature has tried to cull. The result is a growing number of reproductively
defective, yet outstanding, show horses. Perhaps we need a wake up call to
breeders, owners, and horsemen, to recognize our responsibility to perpetuate
our breeds. Our standard must be as high for producing reproductive efficiency
as it is for creating an otherwise athletically and physically, superior
specimen.
Finally, the future of our industry to grow and be financially
healthy, on a global basis, is the utilization of the many advanced
reproductive techniques, such as freezing, embryo and oocyte transfer, gender
selection and frozen embryos. We as responsible horsemen, must continue to
educate ourselves, ask questions, and select our breeding horses responsibly.
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