Frozen Semen--Easy As 1, 2, 3
by Carol Austin
Have you heard that breeding mares with frozen semen is difficult and expensive? Or that conception rates are much lower than with live cover or fresh cooled semen? Or that veterinary costs are higher? Don’t let misconceptions dissuade you from trying the most convenient, cost-effective method of breeding. Frozen semen conception rates are now comparable to those of live cover and fresh cooled, and more stallions than ever are being frozen, making it possible for you to breed to the world’s elite performance horse stallions. As with any breeding method, however, there are techniques for the use of frozen semen that result in optimal success. By understanding three basic requirements, you will have the best chance for achieving breeding success with frozen semen.
1) A good mare candidate. Breeding success depends most upon the mare. All a stallion can do is produce the sperm that will fertilize the egg, and no more. After that it’s up to the mare to sustain a pregnancy. A relatively young mare is the best bet. A youngish mare who has a foal on her side, is having her first true heat (as opposed to a foal heat, which has only a 35% or less conception rate) and who had no difficulties foaling, is the best choice. Already a proven producer, she is likely to have a normal cycle, and, because of her youth, usually has good uterine tone and normal hormonal functions. A young maiden mare is also a good choice, providing she has not recently come out of hard training. It often takes such mares a year or more to recover from the stress of hard use and become efficient in their cycling. An older mare who has been a reliable producer and has a foal at foot will also often conceive with frozen semen. But, the older a mare gets the less reproductively reliable she becomes. She may have scaring from infections, loss of uterine tone, declining hormone levels and other difficulties. These mares must be evaluated on a case-by-case basis. An older maiden mare (such as a 17 or 18-year-old performance mare at the end of her career) is a dubious choice. This type of mare can be difficult and expensive to get in foal, although some of them do end up conceiving. But it’s a gamble--don’t risk more than you can afford to lose. The worst mare candidate for breeding is any mare 17-years-old or older, who was barren the previous year. Such mares have a greatly reduced chance of ever getting in foal again, even with live cover breeding. Realize also that, although it’s rare, some mares who are reproductively sound in all respects, and have no apparent problem, will not conceive with frozen semen. Their systems simply "don’t like it." No matter which type of mare you choose to breed, have her in good health and do a pre-breeding reproductive soundness exam. It will be money well spent.
2) Quality semen. After the selection of a good mare candidate for breeding comes the purchase of quality semen. There are many factors that affect the quality of a stallion’s semen, so let’s assume that semen from a stallion who freezes well (which not all do) was properly collected, processed and frozen. When you inquire about a stallion’s semen you should ask:
a) how many straws are in a dose,
b) how many sperm are in a dose (600-800 million sperm per dose is the industry standard, but new insemination techniques may allow success with fewer sperm per dose),
c) what is the post-thaw motility of the sperm (35% is an industry minimum, 65% to 85% or better is expected in a quality product),
d) has the semen been tested disease-free, and,
e) what is the stallion’s conception rate with frozen semen. This is important, as there are some stallions who meet all the above criteria, but who fail to impregnate mares. With young stallions who haven’t had the opportunity to prove themselves as "frozen sires," you may just have to take a chance with semen that meets the other criteria. If you do, it’s wise to have a backup dose of an "old reliable" frozen sire to use in case the mare doesn‘t settle. It‘s probably best, when using an unproven sire, to use a mare who has settled with frozen semen, so you‘re not working with two unknown factors. Dealing with reputable producers and brokers of frozen semen is another way of insuring success. They know how to collect, process, handle, transport and store semen to insure quality. They can also provide you with advice and information that will help you achieve a conception. Semen brokers are in a position to hear from many people about their successes and failures with different stallions, and most will pass that information on to their clients.
3) An experienced veterinarian. When it comes to artificial insemination with frozen semen, a good veterinarian is one who has had experience using it, who stays abreast of the latest developments and technologies and who is willing to go the extra mile to check your near-to-ovulating mare at regular intervals around-the-clock, to be sure she is inseminated within the proper "window of opportunity." Veterinarians all have their areas of expertise and, if you can, you should use one experienced with artificial insemination using frozen semen. When selecting a veterinarian to do the insemination, ask him or her how many mares they have inseminated with frozen semen, and what their first-cycle conception rate is, as well as their overall rate. The conception rate for frozen semen ranges from 55 to 75%, the same as live cover. If the conception rate of the person you are interviewing falls well below that, reconsider your choice. If they don’t have as much experience as you’d like, but you feel comfortable using them anyway, go for it. It’s a skill that can be quickly mastered by an interested veterinarian willing to bring him or herself up-to-speed on the latest information and techniques. As long as they are willing to check the mare frequently and inseminate at the proper time, they have a good chance of success.
And that’s all there is to it. Now that you understand the three basic requirements for achieving breeding success with frozen semen, here’s hoping you have the fourth and final ingredient--good luck!
All Text and Articles Copyright of Carol Austin
Useful Information
At stud farms all over
Europe each year, semen is collected and frozen for exportation. It is the
culmination of a years-long process that begins when breeders bring young
stallion prospects to be inspected by representatives of their breed
registries. Foals are inspected and then re-evaluated at two years of age for
correctness of conformation, movement and free-jumping ability. At age three
they are performance tested throughout eleven-month, 100-day or 30-day stallion
tests, to assess soundness, disposition, trainability, and to receive scores on
dressage and jumping ability. Only the top stallions are purchased for breeding
purposes. Upon reaching eight years of age, stallions are evaluated again, this
time for their ability to sire performance horses. This successful breeding
program of over 200 years' duration continues to produce international
performance stars of the highest quality, as well as esteemed riding horses of
suitable character for amateurs. American breeders now have access to these
superior equine genetics via frozen semen from Superior Equine Sires, Inc.
Purchase enough semen. We recommend buying three doses per
mare, for three heat cycles. (Average conception=2.5 cycles.) Frozen
semen is sold by the dose. Each dose contains 600 to 800 million sperm. This is
packaged in eight, 0.5cc straws, or three to four 5.0 straws, the
recommended amount for an insemination. Frozen semen is stored in liquid
nitrogen at minus-350 degrees F. At this temperature all biological processes
are stopped until the semen is thawed and the sperm cells come back to life.
Frozen semen will last indefinitely if properly stored. However, if the semen
warms at all before thawing, it can be ruined.
Use stallions who freeze
well. Some
stallions freeze better than others. Establish that the stallion whose semen
you purchase has a good conception rate. If he is an unproven horse, or, a
horse known to have marginal conception rates, but whom you want to take a
chance on anyway, have a backup plan. We are happy to share our knowledge of
semen viability and conception rates on the semen we handle, and would
appreciate any information about your experiences with the stallions we offer.
Handle and thaw the
semen properly. Handling
and thawing of the semen can have a great influence on semen viability. Any
time semen is out of the tank for more than three seconds, cellular damage may
occur. Be sure that your veterinarian has the proper thawing instructions for
the size of the straw(s). Standard water bath thawing time is, 45 seconds at
122 degrees F.(50 degrees C.) for 5.0 ml straws, or, 30 seconds
at 98 degrees F.(37 degrees C.) for .25 or 0.5 ml straws. But, thawing
protocols vary according to how the semen was frozen. Best results occur
when thawing protocol is followed precisely.
Breed a reproductively
sound mare. A young
mare who had no trouble delivering the foal at her side, and who is not being
bred on her foal heat, is the best candidate for breeding success. Another
excellent prospect is a young, maiden mare who has not recently been in hard
training. Any mare who has been difficult to get in foal, or who has failed to
conceive via live cover breeding or artificial insemination using fresh-cooled
semen, is a bad risk. An aged maiden mare (fifteen or older) is also a poor
risk. If you are unsure of your mare's breeding soundness, have a veterinarian
do a breeding soundness exam on her.
Have a good teasing
program. This is
essential. Do not rely on guessing or hormonal manipulation.
Use an experienced veterinarian.
Ask how many mares
he or she has inseminated using frozen semen, and what their conception
rate is, and select from among those with the best results. Success with fresh
cooled semen does not necessarily translate into success with frozen. Some
veterinarians are better than others at the insemination process (timing).
Also, the method of transferring frozen semen between shipping and storage
tanks, and subsequently handling and thawing, can have a great influence on
success. The information, resources and technologies for artificial
insemination using frozen semen improve daily. Soon, most equine veterinarians
will be successful with the process.
Timing is everything. Your mare's
follicle must be monitored every few hours when she's close to ovulation.
Though it was once common practice to inseminate before ovulation, or both
before and after, many veterinarians now prefer to inseminate post-ovulation.
It is believed that insemination within six hours after ovulation produces the
best results, but some have had success inseminating up to eight or ten hours,
and even up to twelve hours post-ovulation.
Handling, Transfer & Storage of Frozen Semen
by Carol Austin
Many breeders are opting to
handle and store their own frozen semen, both to avoid storage costs and for
the convenience of having their semen close at hand. Handling and storing
frozen semen is not difficult, but it must be done properly and safely. With
the right equipment and practice, anyone can handle and store their own frozen
semen.
Basics: Frozen semen is packaged in straws labeled with the stallion‘s name,
which are put into cup-like plastic cylinders called goblets, which are then
snapped onto aluminum canes for storage. The canes have a label on top, where
the stallion’s name is written in permanent marker (a fine point
"Sharpie" marker works well for this). Straws, canes, and goblets are
handled with straw tweezers. Protect fingers from nitrogen burns by wearing
thin gloves.
Storage Tank:
The first item one needs
for storage of frozen semen is, of course, a storage tank. There are many makes
and models of tanks, but all are essentially the same, being large cylinders of
stainless steel or aluminum, with an inner vacuum lining somewhat like that of
a thermos. There are from one to six storage canisters inside. A canisters sits
well down inside the tank, and is pulled up by a long handle which notches into
a groove in the neck of the tank. A used storage tank can be purchased from
representatives who service semen storage tanks for dairies and beef
operations. A. I. supply companies and frozen semen brokers also sell used tanks.
The main difference between an older, used tank and a new one is the hold-time,
or the frequency with which the tank needs to be supplied with liquid nitrogen.
A new tank can be topped-off every four months, while a used one will probably
need to be serviced every two months. As long as a tank is not frosting up on
its exterior after filling, it is a secure tank. All tanks should be checked
weekly for signs of leakage and measuring of the nitrogen level. This is done
by inserting a measuring device down the center of the tank. A tank should be
kept about half full, and its level never allowed to drop below three inches.
Equipment and Supplies:
Liquid nitrogen--available
from welding and medical supply shops or from "door-to-door"
suppliers
Liquid nitrogen
measure--available from suppliers or make your own--use wood, plastic or
metal--no cylindrical measures
Thin latex or cotton
gloves--to protect fingers from nitrogen burns
Long-sleeved shirt--to
protect skin against accidental splashing of nitrogen
Safety glasses--for eye
protection
Straw tweezers--plastic
(may be best for beginners) or stainless, for handling the individual straws,
goblets and canes
Assistant--working between
two tanks requires at least two people
Filling Containers:
Fill the vessels slowly to
minimize contraction and expansion stresses. Never seal the vessel tightly, as
it is designed so that no internal pressure builds up. The stopper must not be
too tight fitting, or the container may be damaged or burst. Never fill the
container more full than the bottom of the neckline. When measuring nitrogen
use metal, plastic or wood. Never use a tube, as the nitrogen may spurt up
through the tube and splash out. Dispose of nitrogen on the ground, never on
concrete or blacktop surfaces.
Tank maintenance:
Never clean with abrasive
cleaners, use mild detergent and water and always wipe dry. Remember
that the liquid nitrogen is evaporating constantly and must be replaced
periodically. The evaporation rate will depend upon the use, age and condition
of the vessel.
Handling the Container:
Do not drop or tip the
container. Do not slide, roll or walk the unit, because the vibration or shock
can result in partial or total vacuum loss. If it’s too heavy to carry, use a
roller base.
Caution:
One liter of liquid
nitrogen produces 25 cubic feet of gas, displacing oxygen in the surrounding
area. Always work in a well ventilated area. Call for medical help immediately
in the event someone is seriously splashed by nitrogen, or becomes dizzy or
loses consciousness after exposure to liquid nitrogen.
Transferring Semen:
The frozen semen will
arrive in a shipping container, often a short hold-time container that will
need to be promptly unloaded and returned to the semen broker. If you are
unskilled at transferring frozen semen, try to work with a veterinarian or
other skilled technician on your first attempt. Some shipping tanks will arrive
with the straws already in goblets and on canes. These are relatively easy to
transfer. Loose straws require more skill.
Important:
No matter how your semen
arrives, it is critical to remember that when frozen semen is exposed to
thawing temperatures for more than three seconds, cellular damage may occur.
Therefore, it is important to work quickly. If you have not completed the
transfer in three seconds, dip the canister back down into the tank to re-chill
it, and try again.
If your straws of semen
arrive loose in canister, you must first transfer them into goblets, and then
snap the goblets onto canes for storage. Pre-chill the goblets and canes before
use, and have them ready in a canister.
Steps to Transfer Semen:
· Arrange the tanks, and chairs for the handlers
to sit on, in a well-lighted and ventilated area.
· Keep the canisters you are working with two
inches below the neck of the tanks at all times, and dip them back down into
the nitrogen every few seconds to keep them cold.
· With the canisters pulled up to two inches
below the neck of the shipper, grasp a straw with your straw tweezers, identify
it, remove it and transfer it into a chilled goblet. Immediately re-submerge
the canisters.
· Next, have your assistant pull up a canister
containing a chilled cane marked with the stallion’s name, snap the goblet
onto it, and re-submerge. Some people combine these steps, by having
the goblets attached to the canes before transferring the straws.
· Remember to take it step-by-step. First,
the straws need to be identified. Next they are sorted into goblets. Finally,
the goblets are snapped onto canes. Re-submerge every three seconds and/or
between each step. Make sure all the equipment is pre-chilled. Soon you will
be handling and transferring frozen semen like a pro!
All Text Copyright of Carol Austin
Several Different Protocols
For Breeding
Important: When frozen semen is exposed to
thawing temperatures for more than three seconds, cellular damage will occur.
Equipment and Supplies for Transferring Semen--
--Thin
latex or cotton gloves--to protect fingers from nitrogen burns
--Long-sleeved
shirt--to protect skin against accidental splashing of nitrogen
--Safety
glasses--for eye protection
--Straw
tweezers--plastic or stainless, for handling the individual straws, goblets and
canes
--Assistant--working
between two tanks requires at least two people
Steps for Transfer Semen--
1--Arrange
tanks and chairs for handlers to sit on, in a well-lighted, well-ventilated
area.
2--Keep the canisters being worked with two inches below
the neck of the tanks at all times. Dip them back down into the nitrogen every
few seconds to keep them cold. The neck is several degrees warmer than the
lower portion of the tank.
3--With
the top of the straws two inches below the neck of the shipper, grasp a straw
with your straw tweezers, identify it, remove it and transfer it into a chilled
goblet from the other tank. Immediately re-submerge both canisters.
Preparing the Mare—Before insemination, prepare the mare’s
perineal area aseptically, using a sterilizing cleaner such as Betadine. If the
tail contacts the area before insemination takes place, clean it again. The
mare should be palpated at least every
12 hours when ovulation is close.
Thawing--Thawing instructions
are provided for each dose. Thawing protocols vary according to the method used
to freeze the semen, the reaction of a particular stallion’s sperm to the
freezing and thawing process and to the type of extender used. It is very
important to follow thawing instructions precisely, as they correspond to the
freezing method used and represent the best thawing method for a particular
stallion’s semen. If you do not receive thawing instructions, please call for
assistance. It is never appropriate to put frozen semen into boiling water or
to set it out on a counter to thaw at room temperature, as has reportedly been
done. Only when the mare is ready to be inseminated, is it time to thaw the
semen. Standard water bath thawing times are 45 seconds at 122 degrees F. for
5.0 ml straws, or, 30 seconds at 98 degrees F. for .25 or 0.5 ml straws. Dry
each straw carefully—water is spermicidal. If you did not receive thawing
instructions for the semen you received, please call us.
Insemination—
*The
optimum time for insemination is from 12 hours pre-ovulation to 6 to 8 hours
post-ovulation.
--Prepare mare’s perineal area aseptically.
--Prepare a constant temperature water
bath for thawing semen.
--Remove straw from nitrogen container and
immediately place in water bath, exposing to thawing temperatures for no longer
than three seconds.
--Leave straws in water bath for at least
specified time.
--Dry off straw completely—water is
spermicidal.
--Hold the straw vertically so that the
air bubble is at top of straw, and cut of top of straw/sealing ball.
--Place open end of straw over sterile,
pre-warmed container (15 ml centrifuge tube) and cut off plug at other end, so
semen empties into sterile container.
--The extended semen is placed into an
incubator to maintain semen at body temperature, and then drawn up into a
pre-warmed sterile insemination pipette and inseminated just as would be done
with fresh semen.
Post-Ovulatory Protocol—
--35 mm follicle
--Continue to monitor follicle,
injection of hCG or GnRH (optional)
--Palpate at six hour intervals
--Inseminate within six hours
post-ovulation
Pre and Post-Ovulatory
Protocol—
--Presence of 35 mm or larger
follicle
--Give hCG or GnRH
--24hours post-injection,
begin twice daily palpations
--Inseminate with ½ dose 24
hours post-injection.
--Inseminate with ½ dose
within six hours post-ovulation
(Requires two doses per
cycle.)
--Examine the mare once daily
until a 35 mm pre-ovulatory follicle is present.
--Administer hCG or
GnRH.
--Inseminate with one dose
at 24 hours post-ovulating agent.
--Inseminate with one dose
at 40 hours post-ovulating agent.
Deep uterine/low-dose
insemination—This
relatively new method that uses fewer numbers of sperm deposited at the
uterotubal junction, and seems to have valuable applications with frozen semen
of sub-fertile stallions and when using sex-sorted semen. When used with good
quality semen containing high number of motile sperm, conception rates are
lowered. The deep uterine method may be used with all protocols. Extreme care
should be taken to maintain aseptic conditions throughout the entire
insemination process.
Post Breeding Endometritis—Post-breeding
endometritis occurs in 20% of mares. If uterine lavage is necessary because of
endometritis, allow at least four hours after insemination before lavage, to
give sperm time to reach the oviduct.
Did You Know?
FROM OVULATION TO CONCEPTION
·
At ovulation
the mare sheds an egg from her ovary, and the egg quickly travels into her
fallopian tube.
·
The mare is
inseminated post-ovulation.
·
Within minutes,
the sperm is drawn into the fallopian tube by uterine contractions, not swimming.
90% of mares conceive at this time.
·
After fertilization,
the embryo will remain in the tube for five or six days, before moving into
the uterus.
·
Following 10
days of moving around the uterus, the embryo becomes more stationary.
·
At about 37
days, the embryo attaches to the wall of the uterus by endometrial cups, which
are the beginnings of the placenta.
·
The placenta
produces progesterone, to help maintain the pregnancy. Before its development
and attachment, blood levels of progesterone support the pregnancy.
EARLY EMBRYONIC DEATH
The highest incidence of
early embryonic death occurs before day 11, when the embryo enters the uterus.
Often it occurs by day five or six. This happens in about 24% of normal mares.
The next highest incidence
of embryonic death occurs between days 14 to 40, in about 17% of normal mares.
Factors contributing to
early embryonic death:
·
When mares
are bred post-ovulation, the egg may not live as long as the sperm, and may
have aged and become defective.
·
Stress caused
by environment and management may cause lethal stress to the embryo.
·
Hormone deficiencies
and imbalances, the uterine environment, and the age of the mare can all be
contributing factors to early embryonic death.
DID YOU KNOW?
·
The national
average for first cycle conception using fresh cooled semen is 60%.
·
The national
average for first cycle conception using frozen semen is 50%.
·
The average
number of cycles per conception using frozen semen is 2.5 per mare.
·
The end-of-season
conception rate for both fresh cooled and frozen semen is about the same--70%.
(This rate is influenced by management and the number of cycles the mares
are bred.)