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.1996 by Hoflin Publishing Ltd. ©
Dr. Martin is a private practitioner in Glendale, Arizona. He owns and operates 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 and Answers-
Doctor, what are your breeding statistics in Labrador Retrievers?
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Is there anything special that I need to do when I have my dog collected for freezing?
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What records does A.K.C. require a breeder to keep regarding frozen semen?
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Why can't you just look at vaginal smears and determine when to breed my bitch?
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Everybody is recommending and doing progesterone testing. Are all tests the same?
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Guidelines for selecting a canine semen freezing and storage facility:
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 breeding's.
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 breeding's 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 breeding's 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 breeding's 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 breeding's 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 breeding's. 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 breeding's. 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!
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 breeding's 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 and 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 and 24. Bitches #2 , #3 and #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
breeding's 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.
