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New information, new instrumentation, and the availability of new skills needed to be incorporated in the article formerly found here, asking “Why AI” and “Why Not AI”. At the present time, trans-cervical insemination is rapidly becoming more widely available. More information is available about the reliability of in-the-office ELISA tests, and more practitioners interested in small animal reproduction are establishing breeder programs.
Information about veterinarians offering reproductive services can be obtained from the membership list of the Society For Theriogenology (animal reproduction). Their web site is at https://www.therio.org/ and you may contact them for member veterinarians in your area.
So, once again, let’s examine the subject of artificial insemination. There are many variations of AI to consider. There is dog-to-dog artificial insemination, fresh-extended (chilled) semen insemination, and frozen semen insemination. Techniques of AI include vaginal deposition, surgical implant, and trans-cervical insemination. Studies have been published with convincing evidence that every semen preparation – fresh, fresh-extended or frozen – will produce larger litters if semen is deposited directly into the uterus, especially with trans-cervical technique. That being said, fresh and fresh-extended semen give decent results with vaginal deposition.
Trans-cervical inseminations are done by endoscope or the “Norwegian catheter”. The rigid endoscope is more widely used than the Norwegian catheter, and has the considerable advantage of providing the operator and the bitch’s owner with a view of the actual deposition of semen in the uterus by way of a TV monitor. Those skilled in the use of the Norwegian catheter can get excellent results as well. Trans-cervical semen depositions do not require any sedation at all in nearly all cases.
The important difference between a trans-cervical insemination and a surgical implant is, obviously, that the trans-cervical does not entail the risks of general anaesthesia and surgery. Unless there is a pathological condition of the uterus, there is no benefit to doing a surgical. If the tissues of the uterus or the ovary are abnormal, an exploratory surgical procedure can be done on the bitch well before she is in heat, if hormonal studies, ultrasound or breeding history indicate a pathology that cannot otherwise be identified. Many abnormalities will make themselves known through hormonal studies, and may be treated medically rather than surgically.
Of the different types of semen preparation, it is again obvious that fresh dog-to-dog collection and insemination will give the best semen preparation. Fresh-extended or “chilled” semen has performed very well for many years, but not as well, statistically, as fresh semen collected at the time and on the same premises as the breeding. The worst statistics are for frozen semen, no matter what process is used for the freezing. The reason for this is that once frozen semen is thawed, it lives for a period of two to twelve hours. With fresh or fresh extended semen we’re looking at days, not hours, of longevity. Because of this difference, frozen semen should only be used with direct intra-uterine deposition, and trans-cervical is to be preferred to the surgical implant. It should be clear that the timing of the frozen semen deposition is extremely critical, while there is more latitude in the deposition of fresh or fresh-extended sperm. If at all possible, if the stud dog is still alive, fresh or fresh-extended semen should be used.
Now back to the earlier article:
I thought I’d call this column, Why AI, and give all the good reasons for artificial insemination as a part of a canine breeding program. Then the thought occurred to me that I’d also have to do one called Why Not AI, detailing some of the inappropriate circumstances used to justify the decision to breed this way.
There are several circumstances in which artificial insemination may be the best way of delivering semen to the bitch. Where all relevant factors are known and normal, in small and medium sized breeds of dogs, a conventional artificial insemination is, in my opinion, likely to be 100% successful. However, the caveat here – that all other conditions are known and normal – is a substantial one. This presupposes that both the dog and the bitch are clean of any infection, that they are under 5 years of age, that their hormonal function and fertility are normal, and the dogs are sound. If any of these factors is other than stated, the likelihood of success of any breeding activity, whether artificial or not, is reduced.
The valid reasons for using artificial insemination include the following:
- Use of frozen or fresh-extended semen.
- Protection and continuous evaluation of the stud dog’s semen
- An inexperienced stud dog
- A stud dog with a physical disability.
- Size incompatibility of the dog and the bitch (i.e. height).
- An overly aggressive or dominant bitch.
- Opportunity to evaluate the semen before insemination.
- Splitting an ejaculate between 2 bitches.
- Vaginal stricture or persistent hymen.
The most frequent circumstance in which an artificial insemination is requested, is when the stud dog doesn’t wish to breed the bitch on a given day. The fact that it is day 10, or 12 or 18 of the bitch’s season is often the basis for the breeder deciding that this is the correct day for the breeding. It may be thought to be the time that “all” bitches of this particular breed are most receptive; it may be the day, counting back from a prior litter, that the most effective breeding was done. It may be that for reasons of time constraints of the people or the dogs involved, show commitments, travel opportunities, and so on, it is desirable to try to cover a period of three or four days when it will be impossible for the two to mate, even though it is likely to be early. Sometimes, less frequently, it is done toward the end of true estrous, when the dog’s interest, once again, has dropped to a less than willing level, and due to the generally held belief that the later breedings are most likely to yield a litter, or a large litter, so one last attempt is desired.
There are many ways of estimating the proper day to deposit semen. An experienced and reliable stud dog’s nose is frequently the finest instrument for this. A ‘smear’, which is more properly termed a vaginal cytology, can be read with good success by an experienced reproductive veterinarian, and is best done every day, or every other day, from day 8 on. The next step up from this, and a tool that can be used by a relatively inexperienced veterinarian with good success, is the progesterone RIA test; this is the test that must be done by a commercial laboratory. Generally, it is possible to get the results the day after the blood is taken. In-the-office ELISA progesterone tests are only about 50% reliable, according to published studies. An example of this type of test is the frequently used ‘Target’ test. Likewise, the LH ELISA test is not very useful due to the tiny window of opportunity for catching the LH peak. The RIA progesterone test will give a precise number. With this number it becomes possible to extrapolate with good accuracy, once the level has reached a particular threshold value. Using an RIA test it is possible to time FES and frozen breedings with good success. It is absolutely critical that this kind of progesterone test be used for these kinds of breedings.
One technique I find very reliable, is the vaginoscopic examination of the vaginal mucosa. I find this gives me the very best real-time information on a bitch that hasn’t been available for repeated exams all through her cycle, or where the correct date isn’t known for the beginning of her season. Even this technique, however, works better when repeated exams are done. This gives a sense of the progression of hormonal events in a particular bitch. Progression through the estrous cycle generally follows a pattern that is similar in most bitches, once that threshold point is reached. The most common reason for failure of artificial insemination is breeding at an inappropriate (usually early) time, and failure to follow up with inseminations later at the correct time.
The bitch that never appears to stand for breeding, or to swell and loosen up enough to admit the male comfortably, is often simply not cycling properly. Before doing repeated artificial breedings with such a bitch, with poor likelihood of success, it is often more useful to check on the bitch’s hormone levels through one complete cycle, trying to identify a correctable problem. Often such problems respond to thyroid supplementation, and frequently, with younger bitches, once one cycle has been corrected in this way, and a pregnancy achieved, it is not necessary to continue the thyroid supplementation in later cycles. Sometimes, the bitch just needs a jump start. A frequent cause of failure to conceive by artificial insemination is the lack of a fully developed season in the bitch, and the failure to ovulate. Later in the pregnancy, there is the possibility of insufficient progesterone to maintain the pregnancy. This too can be successfully treated. Finally, your veterinarian may wish to give an injection to assure the release of all mature eggs at the appropriate time. Bitches that have extremely narrow vaginas may have hormone imbalances that need to be addressed, or may be too immature to cycle normally.
A stud dog that hasn’t been bred for a period of months or years is an unknown quantity as far as fertility is concerned. Even one that is actively bred may have developed an infection, especially if he is bred naturally. Ideally, an ejaculate would be evaluated before the actual day of breeding. If this hasn’t been done previously, an artificial insemination offers the opportunity to do a semen examination before being committed to a breeding with a sub-fertile male. Every year a fair number of bitch owners are saved by the discovery that the intended stud dog is sterile or sub-fertile. Just so, many stud dog owners are shocked to discover that their dog is in fact ejaculating either nothing, or occasionally, pure pus, instead of good quality semen. They then have the chance to respond and treat the stud before his infertility is irreversible. This is another example of the advantage of dog-to-dog AI’s, where semen is always examined before deposition. We now have therapeutic techniques that may return to fertility a dog that is ejaculating no sperm or no fluid at all. Such a dog should always be evaluated for retrograde ejaculation or testicular tumors.
For many stud dog owners, the decision to do AI’s only is made to protect the dog from infection from a bitch and from injury by a bitch. During the tie, the dog may be injured if the bitch throws herself about. Aggressive bitches may damage a dog’s libido. I know one dog that checks to see that the bitch has a muzzle on before he’ll mount. Breeders that do AI’s exclusively find that the continual evaluation of their stud’s semen is invaluable in picking up problems before they become serious.
Young males often suffer from ‘performance anxiety’. Sometimes they can be helped with the bitch and a tie achieved. When this isn’t possible, rather than letting him lacerate, fatigue or overheat himself unduly without success, a skillfully managed collection can help the dog gain confidence and actually train him for a natural breeding career, rather than spoiling him for natural breeding. Some very dominant bitches can simply be too much for a more reserved or younger male.
When managing the virgin or young and underdeveloped male, care must be exercised not to force the dog beyond his comfort zone. Dogs of the same breed and age, may differ significantly in the size and apparent physical maturity of the reproductive apparatus. Underdeveloped physical development is often accompanied by an immature libido as well. An experienced reproductive veterinarian can collect semen from such a dog, and help to stimulate him and condition him for future natural breedings. However, if he just isn’t ready, it is better not to give him a bad experience which could later on result in an impotent adult. These individuals are easily identified by their lack of physical development and response to manipulation. It is very important to know when to stop with this dog, whether trying to induce him to breed naturally or artificially.
A dog with a missing limb or an injury to a rear leg or spine may not be able to stand on his rear to tie bitches. As long as the disability doesn’t reflect a genetic or structural unsoundness, artificial inseminations can keep this dog producing.
Given the absolute certainty of the ‘dormitory effect’ in kennels, it is possible to encounter the need to breed more than one bitch on a given day. In order to legitimately cover both bitches from one ejaculate, very good semen quality must be present. This practice requires an experienced evaluation of the semen if it is to be ethically attempted.
Some bitches presented for AI have vaginal strictures or bands of persistent hymen tissue that prevent the male from penetrating. Once again, it is best with this kind of bitch to evaluate her before she actually comes in heat, if the size of her vulva permits. Bands of hymen material can often be snipped before breeding. Evaluation of ring-like strictures will help determine whether or not a caesarian section may be necessary. Early evaluation by digital palpation and vaginoscopy can prevent a bitch from developing bad breeding behaviors due to pain when the male attempts to penetrate
Reasons NOT to AI include a bitch that is unattractive to the male by reason of incorrect timing or the odor of a severe vaginal infection. Obviously you don’t want to do a natural if the bitch has an obvious infection either. You may wish to consider whether or not to breed an extremely aggressive or vicious bitch. You need to differentiate whether it is unwillingness to be bred at a particular time, or a truly vicious animal. We see many individuals in some breeds where conformation (show conformation) of the bitch precludes natural breeding. In these breeds this conformation also affects the soundness of the animal in its normal activities as well. Artificial insemination because the bitch isn’t ready and one of the dogs is going to be unavailable at the appropriate time will only give the veterinarian, technique and stud dog a bad reputation.
Ideally, not only should you consider AI’s under a number of circumstances, but you should take advantage of this occasion to assure yourself that your dog or bitch has none of the problems that sometimes cause breeders to seek an AI. Finally, I have ‘scoped bitches with vaginal lacerations caused by inexpert attempts at artificial insemination. Many breeders feel they are very good at this technique. A qualified reproductive veterinarian is likely to give you a great deal of other insight as they perform the AI, and is not likely to injure your bitch!
FRESH EXTENDED SEMEN BREEDING
Fresh extended semen (FES) is semen which is collected at the time it is needed, for the purpose of being shipped. Frozen semen is semen which is collected for the purpose of being stored over a period of time, or for being shipped to distant places (for example Australia) where fresh semen would not arrive soon enough to be in good condition. Actually it used to be the case that even semen was quarantined upon arrival in Australia for 3 weeks. With the new canine quarantine laws at the present time, there may be a less restrictive policy on semen as well.
Fresh extended semen is processed in a similar way to frozen in that a ‘medium’ is used which protects and nourishes the sperm cells in transit. However, the freezing is avoided, thus assuring in most cases an over 40% better number of live and motile sperm cells. All extenders are not equal, and those who wish to freeze or ship semen should carefully research the subject, and not be swayed by advertising and other marketing techniques in making their choice. With the best of extenders, we expect fresh extended semen to be much more effective in fertilizing the bitch’s eggs than frozen semen from the same dog would be.
There are various reasons for shipping semen instead of dogs. The possibility of death of the bitch due to shipping, for instance, as well as the stress which can precipitate herpes or bacterial infections and result in resorbtion of the fetuses. Sometimes the weather – hot or cold and snowy – is the reason to keep the bitch home. Sometimes holiday traffic. There stresses involved in shipping can be sufficient to prevent a litter being whelped, whether from Herpes or other factors.
As veterinarians in general become better at assisting breeders in the detection of the appropriate time to breed, the statistics for litters whelped from fresh extended semen will continuously improve. There are several ways of gaining information on timing breedings. The most important technique in timing estrous is a blood test called an “RIA progesterone”. The RIA or radio-immunoassay gives us a real number, not a range such as the in-the-office ELIZA technique tests produce. Once we obtain a number from the RIA test it is frequently possible to extrapolate that number to determine the best day. Sometimes 2 or 3 RIAs are required. It is not possible to handle the information from the ELIZA tests in the same way.
The next best timing technique, of course, is the response of the dog and the bitch to each other; obviously, this important indicator won’t be available to us in FES breedings. Vaginal cytology (“smears”) performed routinely every other day from day 8 on will be useful in the hands of a veterinarian experienced in reproduction. An instrument (‘Estron’) is available which measures the electrical conductivity of the cervical mucous, which works quite well if done daily throughout the estrous cycle. LH and Progesterone ELIZA kits are available to help those veterinarians without skill in reading smears, or other diagnostic instruments, but are much less useful than the RIA progesterone performed at a lab. Since progesterone is not a species specific hormone, these RIA tests can even be performed at your local human lab in a pinch. I believe the most widely used progesterone ELIZA in-the-office kit is one which started out as a sheep test.
The method I generally find most useful and reliable for most routine matings is the endoscopic examination of the cervix, which changes in appearance according to a very reliable schedule. The reference for this technique is : NORMAL CANINE VAGINOSCOPY by F. Lindsay and P. Concannon, published on page 112 in SMALL ANIMAL REPRODUCTION AND INFERTILITY, Edited by Thomas J. Burke, 1986, Lea and Febiger. However, when we’re shipping and timing of appointments, freight services, airline schedules and the availability of veterinarians and owners must all be taken into account, then we do the RIA test on day 8, scoping the bitch first to be sure we’re in the right vicinity time-wise. We are available to the vets on the other end to help them understand the reasons for using this test, and for help in interpreting the result from the lab and making the extrapolation.
Whatever method used, skill in estimating the progress of the estrous cycle is the greatest asset in producing puppies from chilled semen. Without this skill, the fresh extended semen breedings, as well as any other kind of artificial insemination, is likely to fail. This being the case, the bitch owner may need to research the availability of veterinarians with these skills in their general area. They may find themselves traveling a good way to obtain this service.
For best results
- It is necessary for the bitch owner and the stud dog owner to get their dogs a reproductive workup before attempting a FES breeding.
- Inform all involved – the veterinarians on each end as well as the stud dog owner – of your future plans a month or more before you expect your bitch to be in heat, and again on day one of the bitch’s season.
- Call your extender supplier to be certain that it will be shipped appropriately, firming up this date some days later on. Make sure of the availability of the extender.
- At the appropriate time, order 2 complete ‘kits’ by credit card and get them to the stud dog’s veterinarian for appropriate storage of the extender.
- Begin RIA progesterone testing on day 8 of the season -earlier if you aren’t certain you know when day 1 was.. Keep the stud dog’s owner and vet apprised of the RIA results as soon as they are available.
- When the bitch’s vet says you have 2 days to breeding, alert the stud dog owners and their vet that the collection needs to be done and shipped the next day.
- Ship by FedEx or regular air cargo based on which will ensure that the semen will arrive in time for you to get your bitch inseminated by your vet on the same day it arrives.
- Have a second collection and shipment made 2 days after the first, and inseminate again, using a full collection each time.
- Do not split the FES and do the insemination every day with half. The best place to store semen is either in the dog or in the bitch, not the refrigerator.
I hope I have persuaded you to plan ahead, and begin working on your planned FES breeding a month ahead of the expected insemination date. Failure of a FES breeding, or live breeding for that matter, can occur for any number of reasons having their base in lack of preparation. The expense of the kits and shipping, the collection, processing and insemination may be in the same ballpark as shipping the bitch, depending on the size dog being shipped. But the cost of the procedure is not the main expense. The expenditure of good will and availability of both veterinarians and both dog breeders is the main cost. The main difficulty, after timing the bitch’s season, is obtaining this willing participation. Don’t let the breeding fail through lack of preparation!
Freezing Canine Ovarian Tissue. Samples for Cloning
Yes, we can freeze your bitch’s ovaries.
Freezing ovaries is less difficult than freezing canine semen.
Yes, eggs can be matured from frozen ovaries. Not all the time, and not by everybody. Not necessarily from the samples we have frozen.
Here’s the situation in a nutshell. The world at large thinks that there is a ‘canine over-population problem’. This being the case, very little funding ever goes into research which would allow the Dog Fancy and Ethical Dog Breeders to catch up to the world of sheep, cattle, horses, and homo saps. Imagine the response of PETA, were the AKC to fund a graduate student at the University of Georgia, where Dr. Richard Fayrer-Hoskins has what is essentially the only academic research program with emphasis on canines. So, when your local or breed club starts handing out money to the Baker Institute and other institutions, to pay for a graduate student’s room and board for a year, whether or not anything of real benefit to dogs is likely to result, you might think about mentioning the canine reproductive program at the University of Georgia. With techniques for collecting mature eggs, fertilizing them in vitro, implantation in a host dam, all worked out in great detail and done routinely for other species, the steps between the freezing of semen and ova, and the production of normal healthy offspring should fall in place very quickly once there is a program for this research funded. So, it’s up to us as breeders and exhibitors to think about advancing this knowledge base.
At the other end of the country, at the San Diego Zoo, Dr. Barbara Durrant has been developing an endangered canid program, which engages in freezing semen and ovaries of endangered canid species, among other things. Zoo researchers, working with the group at the U. of GA, have successfully matured ova from these frozen ovaries.
So, we can freeze ovaries at this time. We do this at the Canine Fertility Center in Ashford, CT. What we can’t do is guarantee that a given frozen sample will reliably result in ova which may be matured and fertilisable at some future date. However, the technique – while fussy – is still no more difficult than freezing canine sperm, which we can do very reliably. In the case of ovarian tissue, since the skills for maturing the eggs from the frozen ovarian tissue are only available in a couple of places, we can’t test these samples to see if they can produce suitable ova.
How do we get the ovaries? They are taken at the time of spaying, or as part of a euthanasia process. These are then transported as quickly as possible to the freezing center, in the appropriate solution and at the appropriate temperature.
The freezing is done according to Dr. Durrant’s protocol, and the ovaries are stored in liquid nitrogen tanks, just as the semen is.
The AKC does not have a program for overseeing the record keeping as regards this process, or for sanctioning breedings from banked ovarian tissue.
We at the Canine Fertility Center are a CLONE semen bank. We have undertaken to keep genetic material, ranging from blood clots through tissue samples and cheek mucosal brushes (the same way cells are obtained for DNA certification) for our clients for some years. Our record keeping and storage conditions are the same as we use for frozen semen. The objective is to have all our ducks in a row should the AKC get to the point of recognizing the efforts of early practitioners of this technique. We keep the records just as they specify for frozen semen. However, we cannot give you any certainty as to when the AKC will consider sanctioning such sample banking, and if, when they do, they will apply their sanction retroactively to banks which keep samples as we do. We can only say, that we are keeping records appropriately, and are processing and storing the tissues in what we understand to be the best manner available at this time.
Samples for Cloning
Samples including other tissues (somatic tissues) such as blood clots (the portion of the blood sample left after the serum has been removed), other body tissue specimens, and cheek swabs, will not be of any use for breeding puppies. Rather they are kept to provide material for further DNA analyses, or even for cloning at some future date, should these techniques become available. Those of us who have very small amounts of semen frozen on a great dog might think seriously about the best use of that semen; is it best to use it to try to impregante a bitch, or for genetic testing, or for cloning. Note: clones can’t be developed from an egg or a sperm cell. However, semen also contains some epithelial cells and some white blood cells from the donor and these have the complete genetic material.
Please understand that the techniques discussed above – maturing ova from frozen ovarian tissue, in-vitro fertilisation, and cloning companion animals – are not yet available for us to try. The point to taking these tissues and preserving them is very much one of nothing ventured, nothing gained.