Frozen Semen:
By Daniel J. Martin, D.V.M.
What you have always wanted to know about frozen
canine semen, but were afraid to ask.
Glendale, Arizona. He owns and operates Dr. Martin
is a private practitioner in Northwest
Valley Veterinary Hospital and Canine Semen Bank. His major clinical
interests include canine reproduction and ultrasonography. His research
interests include canine embryo transfers and embryo and ovum
cryopreservation. Dr. Martin is a native of Wisconsin. He earned his BS
from the University of Arizona in 1982. He earned his D.V.M. from the University
of Wisconsin-Madison in 1988. Dr. Martin is a member of the newly
formed High
Desert Labrador Retriever Club, the
Labrador Retriever Club of Southern California, and the Papago
Labrador Retriever Club in Arizona. Dr. Martin and his wife Jeanette
established their kennel, Calderwood
Labrador Retrievers, and are active in breeding and showing.
Calderwood Labs recently produced a litter of eight using 11 year old
frozen semen from Ch. Wingmaster’s Raisin Kane. Dr. Martin may be
contacted at (623) 979-8000 or at DJMartinDVM@mobile-vetservice.com
for information relating to the topics covered in the article.
Questions
& Answers
Are
there different ways to prepare frozen semen?
Currently two techniques are used to freeze canine
semen: the straw system and the pellet system. The earlier work done
with frozen canine semen in 1968-69 involved the use of canine semen
that was frozen much like that of cattle semen. These methods were
modified techniques using bovine glass ampules and bovine straws. The
successes with these methods were poor at best with no conceptions
occurring out of 28 inseminations.1,2,3 Successful inseminations with
frozen canine semen did not occur until 1969, using a system completely
different from the straw system. This system consisted of freezing semen
in pellets. Unlike the bovine straw and ampule techniques, the pellet
system allowed changes in temperature to occur extremely rapidly, thus
enhancing sperm viability. With modifications to the freezing technique
and extending media, rapid freezing and thawing procedures were
developed with conception rates as high as 92%. It
was later determined in an eight year comparative study (1971-1979),
supported by the American
Kennel Club (A.K.C.), that the pellet system provided significantly
greater protection for the sperm cells during freezing. Significantly
more sperm cells were recovered with greater motility after thawing.
Recovery rates were 20% to 60% higher when compared to those of the
bovine 0.5-ml and 0.25-ml straws, bovine glass ampule, and the bovine
"Magic Wand," a larger version of the bovine straw.
Current users of frozen semen using straws have reported
conception rates up to 73% with vaginal inseminations, and conception
rates up toward 85% when surgical inseminations are performed.
Unfortunately, many of these conceptions include litters with small
numbers. Many of the facilities using pelleted semen are reporting
80-90% conception rates with vaginal inseminations. These litters are
averaging normal or slightly above normal reported average litter sizes.
Why
is all this information important to me as a breeder?
The bottom line is that when you use frozen canine
semen that has been prepared with a pellet system, the semen will be of
better quality after it is thawed. Canine semen samples preserved in
pellets, have higher percentages of live, progressively motile sperm
cells. These sperm cells are healthier and are much more capable of
crossing through the cervix, moving into the uterus and into the
oviducts where fertilization occurs. Therefore, inseminations can be
performed vaginally instead of surgically and yet conception rates and
litter size remain high. No anesthesia, no surgery and no recovery time
are needed.
Doctor,
what are your breeding statistics in Labrador Retrievers?
Although I deal with all breeds of dogs at my
facility, the largest response, and use of their dog’s frozen semen,
is from the Labrador Retriever breeders. Our documented conception
rates, birth rates, and litter sizes for Labrador Retrievers using
pelleted frozen semen with vaginal inseminations are as follows:
Conception rate (litters conceived and carried to term) 91.6%.
Conceptions may occur and be documented by pregnancy ultrasounds at
20-28 days of gestation, but they may not be carried to term and should
not be included in this statistic. Live birth rate 89.5%. This is the
number of actual living, breathing puppies out of the total number born.
Average litter size, 6.1 live puppies.
What
are some of the benefits of frozen semen?
Clearly long term storage is the biggest benefit.
From carbon decay studies, it appears that dog semen may be stored in
liquid nitrogen for virtually thousands of years without loss of
viability after thawing. The ability to breed to
a stud when he is not available. If your stud is heavily booked for
breeding, or out on the show or trial circuit, breeding conflicts may
arise. Frozen semen will ensure his breeding potential for future
generations, even in the event of a catastrophic disease, unexpected
sterility, or death. Frozen semen allows you an alternate breeding
option, making your stud accessible at all times. Preserving
a certain gene pool can obviously be of great benefit when you are
dealing with an outstanding dog. Preserving the gene pool of a dog with
hereditary problems can be of great value as well. A known carrier of
Progressive Retinal Atrophy (PRA) could have his semen collected and
frozen for future use in DNA studies or for test breedings.
You may be able to increase the marketability of your dog in
areas of the country where he will never travel by shipping frozen semen
there instead. The ability to breed several
bitches on the same day in different places. Frozen
semen breedings eliminate the need to transport bitches to the stud dog.
Thus diminishing transportation risks to the bitch, boarding costs, and
stress on the bitch which could alter her ability to conceive.
Frozen semen breedings ensure that your stud will not
contract an infection from the bitch. Brucella canis, herpes virus,
mycoplasma, and other bacterial infections could be devastating to a
breeding operation. The ability to perform long
distance or international breeding is another added benefit of frozen
semen. Prolonged shipping times, custom clearances, and quarantine
requirements often make it impossible to use chilled semen , or to ship
the animals. Frozen semen remains frozen and viable for several weeks
when packed and shipped properly. Frozen semen will make these breedings
a reality. Whenever considering an international breeding, plan ahead!
Each country has its own regulations regarding the importation, and
registration of litters born through artificial insemination of imported
semen. Many times we will receive calls for these types of services at
the time the bitch begins her estrus cycle. This is too late to make the
preparations necessary for this type of breeding. These breedings need
to be planned several months ahead of time to ensure adequate time to
meet each country’s importation/exportation requirements.
When
is the best time to have my stud dog collected?
Obviously the ideal time to freeze your dog’s
semen is when he is in top form and health. Usually between two and six
years of age is ideal for most dogs. Many times a stud’s reproductive,
show, or performance value is not determined for a long period of time.
By the time his value as a stud has been determined he may be older,
ill, or starting to fail reproductively. Any dog, regardless of age, can
be evaluated for freezing. Dogs up to 15 years of age may be capable of
producing viable sperm cells. Having their semen successfully frozen is
possible. If the sperm evaluation parameters are poor, you should
realize that only a small number of viable sperm will be able to be
stored. This small number of sperm may not be sufficient to create a
pregnancy. Several collections and freezes may be required to obtain an
adequate number of viable sperm in order to breed even one bitch.
Other than
age, what can affect my dog’s sperm?
Something many clients do not understand is that
there are many factors that affect a dog’s ability to produce semen.
In a normal dog there may be differences in the quality and quantity of
the semen from one ejaculate to another even though everything else
remains unchanged. Young, mature dogs will usually have higher sperm
counts and better quality semen than older or immature dogs.
An ill or stressed dog will suffer reproductively with
decreased sperm counts and decreased sperm quality. It may take a
minimum of six months for the ejaculate to return to acceptable levels
after the dog has recovered. Many drugs and chemicals have effects on
spermatogenesis. Environmental stresses such as heat, shipping,
overcrowding, competition, and being dominated by another dog or person
may cause decreases in sperm counts. The larger
breeds tend to produce more sperm per ejaculate compared to the smaller
breed dogs. The smaller breed dogs may need to be collected several
times to obtain adequate sperm numbers to breed one bitch. The
dogs libido (sex drive) is the last key to collecting a good semen
sample. When a dog’s libido is high, we obtain high quality semen with
maximized sperm concentration and motility. By simulating a natural
breeding as closely as possible we obtain the most sperm to work with.
Using an estrus bitch or a bitch induced into estrus also helps to
provide the maximum potential for sperm collection. We
have collected ejaculates that have provided as many as twelve
inseminating doses and as little as one half inseminating dose. On the
average three to six inseminating doses are obtained from any one
collection. Most Labrador Retrievers will produce enough sperm in one
ejaculate to have four to eight inseminating doses prepared. We
recommend using two inseminating dose for each bitch bred.
Is
there anything special that I need to do when I have my dog collected
for freezing?
When you present your dog to have his semen
collected for freezing you will need to provide some information and
documentation. Each facility has different ways of doing this, so you
will need to check with your particular facility. At
our semen bank facility, we request that you bring your original A.K.C.
registration for your dog. We will make a photocopy of it for our record
and another will be sent with our notification to A.K.C.
of your authorization for us to collect, freeze, and store your dog’s
semen. If your dog is tattooed or microchipped, these must be verified
and entered into our records. We will take three (front, and both sides)
Polaroid photographs of your dog. We highly
recommend written verification of a negative Brucella canis test no more
than thirty days prior to collection and freezing. I have recently
started recommending a negative mycoplasma test of the ejaculate as
well. A complete preventative screen would also include a negative
canine herpes test and a clean semen bacterial culture as well.
Remember that your dog will not become infected by having his
semen frozen, however the prepared semen product will be inseminated
into the bitch. This could potentially infect the bitch were the male to
have an infectious disease. One safeguard here is that there is an
antibiotic in our frozen semen extender that is effective against the
most common bacterial agents that we deal with in the reproductive tract
as well as mycoplasma.
What
records does A.K.C.
require a breeder to keep regarding frozen semen?
The following information is listed in A.K.C.
Form ALAIFZ which can be requested from A.K.C.
Records to be kept by owners of donor-dogs:
In addition to the records required to be kept by
owners and breeders, as provided in A.K.C.’s
pamphlet "Regulations for Record Keeping and Identification of
Dogs," owners of dogs from which semen has been collected, frozen
and stored, shall include the following: A.K.C.
registered name and number of donor-dog Date dog
shipped to collector Name and address of
collector Number of breeding units collected,
frozen and in storage Location of semen storage
Transfer of ownership of semen
Transfer of ownership of semen requires:
Records required to be kept by owners of dogs from
which semen has been collected and stored must also note transfers of
ownership of semen. Such records to include: Authorization
of transfer Number of breeding units transferred
Date of transfer Name and address of
new owner The A.K.C.
shall be immediately notified of transfer of ownership of frozen semen.
Records to be kept by owners of semen of donor-dogs when breeding
has been arranged:
Identification of semen (Breed, A.K.C.
registered name and number of donor-dog, date semen collected)
Number of breeding units authorized for shipment and
insemination Date of Shipment To
whom semen shipped A.K.C.
registered name and number of bitch to be inseminated Name
and address of owner of bitch
Records to be kept by breeders:
In addition to the records required to be kept by
owners and breeders, as provided in A.K.C.’s
pamphlet "Regulations for Record Keeping and Identification of
Dogs," owners (or lessees) of bitches inseminated shall include the
following: Name and address of veterinarian who
handled insemination A.K.C.
registered name and number of bitch inseminated Date(s)
of insemination Identification of semen (Breed, A.K.C.
registered name and number of donor-dog, date semen collected)
Your veterinarian performing the frozen semen insemination
should provide you with A.K.C.
form ALAIFZ. This is the form that is required for registering a litter
whelped as the result of artificial insemination using frozen semen. On
the back of this form are the specific record keeping requirements.
What happens
when I have my dog’s semen collected?
The semen is collected from the dog by manual
stimulation. Experienced and confident dogs are our best candidates for
quality collections. Some dogs may be difficult to collect semen from,
and repeated attempts may be required. Once the
semen is collected it is completely evaluated. This evaluation includes
a total sperm count, progressive motility evaluation, sperm morphology
(what the individual sperm cells look like), pH, and an abnormal cell
count (prostatic cells, white blood cells, red cells). If
the sperm is found to be adequate for freezing, the semen is combined
with a specially prepared buffered freezing extender. This extender
feeds and protects the sperm while it is frozen and thawed. After the
freezing procedure has been completed, a single pellet is thawed for
sperm evaluation. The post thaw progressive motility is evaluated and
should result in a figure greater than 70%. Most of our frozen
collections have 75-90% progressive motile sperm after thawing. These
figures far exceed the post thaw motility (40-60%) of sperm thawed from
straws. I have recently added a test for
acrosomal integrity of the sperm heads after they have been thawed. This
will further help to calculate the actual number of sperm that have good
progressive motility and are actually capable of being effective at
creating a pregnancy. The acrosome is a cap on the head of the sperm and
it is filled with enzymes that break down the protective outer
encasement around the egg. Some of the sperm may have sustained damage
to their acrosomes. If the sperm are moving but do not have the ability
to break down this boundary around the egg they will not be able to
create a pregnancy. The frozen pellets are
packaged into insemination doses based on the total sperm count, sperm
morphology, post thaw motility, and acrosomal integrity. The cryogenic
storage tubes are individually identified and set up for permanent
storage. The frozen semen is maintained in
liquid nitrogen tanks at a temperature of -320 degrees F. This
temperature will allow the sperm to remain viable indefinitely. The
storage tanks do not require electricity, so power failures do not pose
a threat to the preservation of the sperm. The American Breeders Service
(A.B.S.) services the tanks on a monthly basis even though the tanks
could remain unserviced for up to a full year. The
entire freezing process take 2.5-3 hours. You and your dog are only
present for about 20-30 minutes. The initial visit takes a little longer
in order to complete the necessary paper work.
What is
involved with using my dog’s frozen semen?
The key to having success in any breeding program is
planning ahead. This is even more important when you are dealing with
frozen semen. I will discuss many things that I feel should be
considered in a breeding of this type. Not all of these items will be of
concern to you and your particular breeding, but will be included as
food for thought. Now that we have collected and
prepared a good quality pelleted semen product, we turn our thoughts to
the brood bitch. In situations where we have a
very limited quantity of semen, the dog is no longer living, or we do
not have a very large number of inseminating doses, it is very important
to plan a breeding with a proven brood bitch. A bitch that has been
reproductively normal in the past is best. What I mean by normal is that
she has normal estrus cycles, conceives with out difficulty, and carries
to term without complications. All bitches,
maiden or not, should be tested for Brucella canis and mycoplasma. These
infections are transmitted other than during mating and casual contact
could transmit these diseases from one animal to another without being
bred. I perform vaginal bacterial cultures and
sensitivities on all bitches prior to having chilled or frozen semen
inseminations. Appropriate antibiotics are administered throughout the
estrus cycle for all bacterial organisms. Some might argue this point,
however my thoughts are shared by many others who are having success
with their frozen semen breedings. The argument arises because there are
what we considered normal vaginal bacteria present in many bitches.
I feel that there are too many variables in dealing with
frozen semen breedings. By eliminating the bacteria as a potential
reason for a missed conception we can look for other problems if the
bitch does not conceive. Remember, we are using an aliquot of sperm
cells that have had anything but a normal existence. Frozen sperm cells
have had their natural abilities diminished, yes even in a pelleted
product. These sperm will only survive for 12-24 hours at best once
inside the bitch. This is a markedly shorter period of time when
compared to fresh semen which has been thought to survive for four to
six days inside the bitch. By providing a healthy, uncontaminated
reproductive tract we can do nothing but help these sperm to do their
duty. Arrange for proper fertilization timing.
Accurately interpreted progesterone hormone testing coupled with
accurate vaginal smear counts is mandatory when preparing for a frozen
semen breeding. As discussed above, thawed frozen semen survives for a
shorter period of time than fresh or extended chilled semen so
fertilization timing is critical. It is imperative that the progesterone
levels be interpreted correctly. In discussions with colleagues, I am
amazed at the number of ways these test assays are interpreted. The end
result many times is that the maximum point of fertility is not
identified, and the breeding is missed or a smaller than expected litter
is born. If you will be receiving frozen semen, allow an ample amount of
time to ensure that a dry shipper is available. We need to prime and
package the dry shipper and make shipping arrangements when the sperm is
needed. There is also the necessary paper work that goes along with the
transfer of the semen from our facility. The shipper will maintain the
semen for almost two weeks if it is left sealed until the sperm is
needed. You will need to arrange for your
veterinarian to be prepared to handle the frozen semen. The pelleted
frozen semen is easy to thaw and prepare for insemination. The only
special requirement is having a hot water bath for thawing the semen.
The usual rigid insemination pipette and all plastic syringes are the
only other requirements other than being familiar with proper vaginal
insemination techniques.
Doctor,
how do you determine when the bitch is to be bred?
We have to keep in mind that there are no tests
available to detect ovulation. The tests available at this time are
either retrospective (looking back to the point of ovulation) in the
case of vaginal cytology, or prospective (looking forward to the point
of ovulation) in the case of progesterone hormone and luteinizing
hormone (LH) blood assays. When a bitch comes into
season , we will perform vaginal cytology counts beginning in most cases
around the sixth to eighth day. When the superficial vaginal cells
become 80-90% cornified, then we begin testing the blood progesterone
hormone levels. These blood tests are performed every other day, or
daily as needed, until we reach 11.5 ng/ml or greater. Only when we
reach this level of progesterone do we begin breeding the bitch. We will
breed the bitch two times, usually one day after another.
Why
can’t you just look at vaginal smears and determine when to breed my
bitch?
Superficial vaginal epithelial cells become greater
than 90% cornified at a variable point two to four days before the rise
of LH. They will remain elevated through the entire estrus period up to
the first day of diestrus. (See figure 1.) If you looked at nothing but
vaginal smears to determine when to breed, you could breed up to five
days before ovulation and up to three days past the point of maximum
fertility. On the first day of diestrus there is an
abrupt change to normal vaginal epithelial cells. It is at this point
that a retrospective identification of the day of ovulation can be made.
By counting backward six days from the first day of diestrus, the day of
ovulation can be identified. (See figure 1.) This first day of diestrus
can also be used prospectively to count forward 57-58 days to the day of
parturition. Vaginal smears can not predict the LH surge or ovulation
before the fact, but by continuing the smears through estrus we can look
back and confirm that our inseminations were performed at the correct
time. Ask your veterinarian if he actually counts the
cells on the vaginal smears or if he estimates the numbers. Estimates
are only that!
What about LH testing?
LH tests should be helpful in predicting ovulation.
However, they have not been as helpful as they could be. These tests
need to be performed every day once the vaginal smear is greater than
90% cornified. The LH peak is very short in the bitch, sometimes less
than 12 hours. These peaks can be missed with testing done every 24
hours. Testing every 12 hours seems a little impractical though. You
would still need to perform the progesterone assays to confirm that
ovulation has occurred.
Everybody
is recommending and doing progesterone testing. Are all tests the same?
And do these tests really predict ovulation?
In many other species, progesterone hormone
production starts after ovulation, so a rise of progesterone in not seen
until after ovulation has occurred. The bitch is unique in that the
progesterone levels rise before ovulation and may actually coincide with
the LH rise. This is why so much emphasis has been placed on
progesterone hormone assays. Most of the progesterone
assays available to the veterinarian can only measure progesterone
levels of 2.0 ng/ml or up to 7.5 ng/ml and this is not adequate to
indicate where fertilization is to occur. The other problem with these
tests, although the veterinarian could interpret somewhat from them
anyway, is that the manufacturers instruct how to predict breeding days
based on the initial rise of progesterone of 2.0 ng/ml. The estimated
breeding days are determined to be four to six days from this initial
rise of progesterone. This may be true for most bitches. However, I have
documented many bitches that have been bred much later in their cycles
than the fourth to sixth day after the first rise of progesterone. If
they were bred on the fourth to sixth days following the initial rise of
progesterone they would have been bred too early in their cycles.
Figure 1. PROGESTERONE LEVELS VS. DAYS OF ESTRUS CYCLE
These six bitches had serial progesterone levels recorded
until their point of maximum fertility. All breedings resulted in
pregnancy using frozen semen. Bitch #4 most closely typifies the
"normal" estrus cycle and progesterone rise. Her progesterone
level began to rise at day ten and she was successfully bred on day 14
& 15. Bitch #6 represents a normal rise in progesterone but a very
early breeding. Bitch #1 represents a normal rise in progesterone but a
very long estrus cycle, being bred on days 23 & 24. Bitches #2 , #3
& #5 represent those bitches that have variations in the length of
time it takes for progesterone to reach that point we consider the point
of maximum fertility. These three bitches would have been bred too early
had we bred 4-6 days following the initial rise of progesterone.
Our facility uses the PreMate
progesterone assay. This has improved our ability to predict the
point of maximum fertility far better than any other in-house
progesterone assay that we have used. We have tried all of the test kits
marketed. This assay measures two levels of progesterone. The lower
level is 3.0 ng/ml and the upper level is 11.5 ng/ml. This upper level
value has been extremely valuable for identifying where fertilization
will occur. We will still look at the four to six day interval from the
initial rise of progesterone but, we will continue to monitor the
progesterone level until it rises to the 11.5 ng/ml level. This is when
we will inseminate the bitch for the first time. It is imperative that
this level be reached and slightly exceeded for our frozen semen
inseminations. It is very important to follow these rises of
progesterone after the initial rise to ensure that ovulation has
occurred and that the bitch has reached that point of maximum fertility
(See figure 1.). It has been assumed that if progesterone rises, that
ovulation occurs. However, luteinization may occur without ovulation
occurring, in which case the progesterone level will not rise as high as
when ovulation has occurred.
Guidelines
for selecting a canine semen freezing and storage facility:
Dog
breeders who plan to have their dog’s semen collected, frozen and
stored should realize that a facility that states it can freeze canine
semen does not necessarily have the ability to produce puppies after the
semen is frozen. To become an A.K.C.
approved center, a center must have the correct record keeping system
and semen storage container; the center is not required to be capable of
producing puppies with the frozen semen. Questions you
may want to ask a facility you are thinking of using are: 1.
How many puppies have you produced using your system?
2.
In what breeds have you produced using your system?
3.
What are your litter sizes?
4.
How many live puppies are produced using your system?
5.
Do you have any research of your own to support the number of breedings
you obtain from one ejaculate?
6.
Can you supply a list of professional and client references?
7.
What techniques do you use for artificial inseminations?
Non-surgical
with no tranquilization/anesthesia, non-surgical with
tranquilization/anesthesia, or surgical?
8.
What are your fees for freezing, storing, and shipping frozen semen?
9.
Where is the semen stored? The philosophy at our
facility for preparing and using frozen semen, is to provide you, the
breeder, with a product and service that you can rely on for your
breeding program. Should you decide to use your dog’s
semen at a later time, you know that it has been prepared in such a way
as to provide you with a product that will work . I can not tell you the
number of times I have heard from clients that have had their dog’s
semen frozen at another facility, have paid to store it for four or five
years or more, and now when they would like to use it find that the
semen is only 25% motile after being thawed! In almost all cases this
semen won’t work! If there were sufficiently high numbers of normal
sperm cells to begin with, there may be a slight chance that a product
like this may create a pregnancy following a surgical insemination. The
odds are stacked against you at this point though. Most of the time the
dog is no longer living or has become subfertile so there are no other
alternatives to turn to. At times I have had clients
elect to store semen that is of poor quality. These collections are more
for sentimental reasons, than functionality. However, the client is
informed when the semen is being prepared that it’s quality is such
that it will require a surgical insemination, and even then it may not
create a pregnancy. The client then decides whether or not to pay his
annual storage fee or to destroy the semen. We do not
feel we should be collecting and storing semen and collecting storage
fees from clients who have an inferior semen product, unless they have
decided that is how they want to deal with the situation. The
bottom line is that you need to discuss the possibility that the semen
may not be adequate to create a pregnancy. How does the collection and
storage facility deal with this? Will they collect and store the semen
regardless of the overall quality? Will they notify you as the sperm is
being processed if there is a problem? Ask to be notified of the status
of the sperm as it is being frozen. There are a few check points in the
preparation of the sperm that may indicate that the semen will not do
well in the freeze-thaw cycle. Sometimes we do not find out until after
the semen has been frozen. If the semen looks poor in some of the
initial evaluations you may not have to pay for the entire freezing
procedure.
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