CRYOPRESERVATION OF EQUINE
Successful cryopreservation of semen (frozen semen)
was first performed in the early 20th century with bull semen and is
extensively used in the cattle industry today. The first pregnancy resulting
from frozen stallion semen actually occurred in 1957 in the US, when
spermatozoa were taken from a stallion's epididymis, frozen, thawed and
inseminated into a mare. Interest has rapidly grown to transform equine semen
cryopreservation throughout the world into a successful industry.
Equine semen is less tolerant of the freezing and
thawing process than bull semen. Furthermore, not all stallion semen freezes
alike; no single technique or magic formula exists to freeze all equine semen.
Semen from some stallions fares better than others with certain freezing media.
Some stallion's semen does not freeze well and may require extensive testing
and laboratory procedures in attempt to preserve the spermatozoa. Furthermore,
at different times of the year, and even at different collections, tolerance of
the freezing and thawing process may show some variation in an individual
WHY FROZEN SEMEN?
The greatest advantage to the use of frozen semen
probably belongs to the stallion owner. However, both mare and stallion owners
have reasons to benefit from its availability. Several reasons
semen is insurance against injury or death of the stallion.
international shipment of semen, thereby enhancing the equine gene pool in all
from different hemispheres can be bred even during the stallions "off" season.
This may represent an additional source of income to the stallion
stallion's show schedule need not be interrupted for semen
of the stallion is minimized since most freezing is performed after the
breeding season. Furthermore, the use of frozen semen prevents low fertility
due to heat stress in the summer months since semen can be preserved at a
different time of year.
semen may allow a stallion with behavioral problems to be gelded.
precise timing of insemination becomes possible because semen may be shipped
many days prior to ovulation and thus, be available for insemination at the
optimal time. This eliminates emergency collections and ensuing anxiety that
semen may not arrive in time to cover ovulation.
in Customs for international shipment or due to airline difficulties do not
affect the viability of the frozen semen.
- There is
often a reduced cost for shipment of semen necessary for a single heat cycle.
With fresh cooled semen, collection and shipment fees can run as high as $250
or greater/shipment and often more than one shipment may be requested for a
Finally, frozen semen offers the same advantages as
fresh cooled transported semen in that it:
the ability to breed to any stallion regardless of distance.
- Eliminates the transportation and board fees to the mare owner at the
the stud farm.
the possibility of injury to both mare and stallion.
management of the "difficult breeder", i.e. a mare that does not show heat or
stand for a stallion.
the chance of intrauterine infection since gross contamination is removed at
the time of collection and antibiotics are added to the semen freezing
the chance of infection to the stallion.
There are some disadvantages to frozen semen use as
- There is
remarkable variability in the ability of stallion spermatozoa to withstand the
freezing and thawing process.
- There is
considerable initial expense involved for the stallion owner. However,
frozen semen is actually quite cost effective when compared over a breeding
season to other methods of breeding.
- In some
stallions, conception rates are lower with frozen semen compared to fresh
cooled semen. With education, familiarity, and subsequent improvement of
post-thaw insemination technique, this appears to be improving.
- Veterinary involvement may be more labor intensive, and thus, more
costly for the mare owner.
number of capable inseminators continues to grow; however, it can be a source
of frustration to both mare and stallion owners in finding an individual who is
both willing and trained to use frozen semen.
Semen collected into an artificial vagina is rapidly
processed for cryopreservation in liquid nitrogen vapor at -196oC. It may be
packaged in several different forms--straws (1/4 ml to 5ml), packets, ampules,
etc. The 5 ml straws are generally considered more "user-friendly"; therefore,
unless otherwise requested, semen is packaged and stored in these macrotubes. A
test straw from each frozen batch is thawed, cultured for potential pathogenic
bacteria, and examined for motility and velocity.
Straws that are processed at Honahlee, PC, available
for commercial use, are estimated to contain a minimum of 200 million
progressively motile spermatozoa and >25% motility unless otherwise stated.
Straws that do not meet these requirements are available to the stallion owner
for use in their own mares.
PREPARATION OF THE
Dead spermatozoa accumulate in the reproductive tract
of the stallion when he is not bred or collected every few days. Therefore,
these dead sperm must be "flushed out" during the week before freezing to
ensure optimal quality of the semen to be frozen. Also at this time, several
cultures of the reproductive tract are taken, and examined for potential
pathogenic growth in order to determine appropriate antibiotics for the
freezing media. Basic semen evaluations are performed during this preparatory
week in order to evaluate sperm number and motility. Lastly, this week allows
stallions to become familiar with semen collection methods, thereby reducing
the semen variability when collecting for a freeze.
Stallions must be tested for Equine Viral Arteritis
(EVA) within a month of freezing or owners must furnish proof of vaccination
following a negative titer.
Vaccinations for other diseases (Influenza,
Rhinopneumonitis, Tetanus) are required before a stallion stays at Honahlee
unless semen shipment to foreign countries precludes the use of these
Frozen semen, after rapid thawing, should be
inseminated into a mare as close to ovulation as possible. Current research
suggests that a mare should be inseminated within 12 hours (ideally within 6
hours) of ovulation for best pregnancy rates. It is often recommended that
insemination takes place on either side of ovulation. Therefore, precise timing
of ovulation is critical to success and usually requires intensive efforts on
the veterinarian's part.
With some stallions, conception rates are lower with
frozen semen compared to fresh cooled semen. It is generally agreed that frozen
semen does not maintain viability after thawing as long as cooled semen but
there is a lack of objective data at this time to support this theory.
Furthermore, a great deal of variation probably exists between
In recent years, however, several studies have been
published that found no significant difference in foaling rates between mares
that had been inseminated with extended semen immediately following collection,
extended semen cooled for 24 hours, and frozen semen. With continuing
improvement in the freezing techniques and media, as well as the familiarity of
the inseminator with procedure, overall conception rates should continue to
The optimal number of progressively motile
spermatozoa necessary for pregnancy resulting from insemination with frozen
semen has yet to be determined. One straw is recommended per insemination.
However, the number of straws recommended/ heat cycle depends on the timing of
ovulation. In very general terms, 3 straws will cover 1 heat. Again in very
general terms, the average number of straws/pregnancy equals 6. Therefore, if
50 straws are available, a stallion owner might plan on covering 8-10
The number of acceptable straws produced by a
stallion is determined by individual stallion variation. Most 1000 lb -1500 lb
stallions produce between 10-20 straws per collection when collected on an
every other day basis.
There is no concrete evidence as to the length of
time that frozen semen can be stored in liquid nitrogen without losing
viability upon thawing. It is generally accepted that, over time, the quality
of semen will decrease by steady increments. However, foals conceived with
semen stored for 20 years, have been born in the past few years.
Again, there is tremendous variability in
pregnancy rates due not only to the stallion variability, mare fertility, and
the level of expertise of the inseminator, but to many factors that are far
beyond our control.
No guarantee is made concerning the fertilizing
capacity of the frozen semen.
It is the responsibility of the stallion and mare
owners to determine the policies regarding the transport and use of frozen
semen that have been set by individual breed registries.
Semen is stored in liquid nitrogen containers. This
service is available at Honahlee or the semen can be stored with the stallion
owner if a tank is available. These containers need to be "topped off" with
liquid nitrogen at regular intervals or they run the risk of "running dry" and
the sperm dying. There are several commercial outfits across the country that
store animal semen and ship it as well.
Frozen semen intended for international shipment must
be collected and stored at a USDA-approved facility. Honahlee meets these
Frozen semen intended for international shipment must
meet the requirements of the country to which it is being imported. Often, a
quarantine period and specific protocol are required. It is the responsibility
of the stallion owner to obtain a permit to import as well as ensure that all
criteria are met for export of the semen. Plenty of time prior to freezing
should be allotted for these procedures. The USDA/federal veterinarian can be
quite helpful in determining the protocols of various countries.
Honahlee is a USDA-certified facility, approved for
the collection and storage of frozen semen intended for international
In general, processed straws run between
$35-$70/straw (total cost). Again, there is tremendous variability based on
individual stallion variation. One can plan on spending between $2000-$3500 to
produce 50 straws, depending on the number of progressively motile sperm
produced by that stallion and the tolerance of the spermatozoa to freezing and
A breakdown of the cost follows:
|Cryopreservation (includes collection,processing, and
|Straw Charge (>25% Post-thaw motility)
||$20.00 per straw
||$10.00 per straw
||$5.00 per straw
|Semen Storage < 100 straws
|Handling fee for semen shipment: (includes canister
rental but not express mail or air freight charge)
|Health Cert.- International shipping
||$200.00 - $500.00 ea
|(a $800.00 refundable deposit for the vapor shipper)
Board, exercise, laboratory , training to the phantom, and or additional
handler costs are not incuded in the above price list.
Honahlee, PC is an equine facility located
approximately 20 miles from the center of Portland, Oregon. The practice of
veterinary medicine is limited exclusively to reproduction.
Dr. Lisa Metcalf owns and operates the facility. She
received her Master's degree in Reproductive Endocrinology from UC-Davis and
her DVM degree from The Virginia-Maryland Regional College of Veterinary
Medicine. Dr. Metcalf completed her internship at the latter and and has since,
divided her time between private practice, research in specific areas of
reproduction, and teaching.