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Reproductive Medicine

• What is progesterone testing?

  • Progesterone is perhaps the best indication of ovulation other than luteinizing hormone (LH) assay. Unfortunately the LH surge, which is the actual stimulus for ovulation is a short event. The pituitary gland releases the hormone, and in 24 hrs the surge of LH may be gone, and you’ve missed it. This therefore requires daily testing, and for that reason can be quite expensive. Some vets will do progesterone levels, and then LH assay when they see a rise to confirm ovulation. This is a good confirmatory test, but often not necessary. The old method of looking at vaginal smears for cornification was an effort to determine how long the bitch has been under the influence of elevated estrogen levels which start to rise after follicle stimulating hormone (FSH) is released from the pituitary gland to tell the ovaries to mature follicles for egg release at ovulation. Unfortunately the time between FSH release, maturity of the ova, and actual ovulation can be variable, and is not accurate enough for some procedures done today such as fresh chilled inseminations, or frozen semen inseminations. Therefore vaginal smears are simply not accurate enough for these methods. The reason is simple. Semen from a natural “tie” or side by side AI may live on average up to 7 days, while fresh chilled semen may only live for 48 hrs, and frozen semen for only 12-24 hrs. One must be very accurate when timing the procedure to allow for egg and sperm to meet while still alive.
    
    Progesterone levels rise about 24-48 hrs before ovulation, and continue to rise after well into the luteal phase( post ovulation). Therefore it is a good hormone to test for and quantitative (actual number evaluation) of progesterone levels are accurate to assess when ovulation occurred. Progesterone test levels are usually somewhere between 3-6 nanograms/ml at ovulation. Many vets like to use the figure of 5 nanograms, and this is a good “estimate”, but not completely precise. There is a bit of art involved with monitoring the trend of rise, and it requires a level of experience to confirm ovulation. Therefore a veterinarian experienced in progesterone timing is a real asset. The in house “blue dot” kits, such as Status Pro by Synbiotics (formerly ICG), and Target by Biometallics should be avoided for important timings such as fresh chilled and surgical inseminations. They are often misleading and not always accurate. You also do not get a quantitative value, but a color change only. Save these tests for natural breedings where the dog and bitch will have plenty of exposure to each other for several ties during the timing process.
    
    One of the most beneficial side advantages to quantitative (number readings) progesterone testing is the ability to predict whelp date. Whelping occurs typically 63 days post ovulation +/- 24 hrs. This is a hard concept for most breeders to grasp. We all think of gestational length and whelp date in terms of breeding dates and therefore conception, but this is not accurate. This is why most literature will give you a range of day 58 -72 post breeding for whelp date. With quantitative progesterone testing we are almost always accurate to within 24 hrs. or so. For English Bull Dog breeders, and other bitches requiring c-section, this is an invaluable tool. In our clinic we will tentatively schedule a caesarian section for any bitch we’ve progesterone timed on the day before she is due and the actual due date even if the owner plans a natural whelping. This is to allow for a dedicated time slot in case of need for an emergency section. It is often very disappointing when your trusted veterinarian is too booked to perform a section if you’re having a dystocia ( difficult whelping) and has to refer you elsewhere. Those breeders needing to schedule time off from work also find this benefit helpful.

   

• Why is progesterone testing a valuable tool in breeding?

  • (see previous topic)

   

• Does it make sense only for artificial insemination?

  • There are reasons some like to do only artificial inseminations:
    
    One is disease spread between dog and bitch. If proper testing and evaluation are done on both ( brucellosis, for both, vaginal vault examination for inflammation and disease for the bitch etc) there should not be a reason for doing only artificial insemination. Natural breedings are the way we all have procreated for millions of years. If due diligence has been done to prevent disease, and it is convenient to both parties, this is not a reason to do only AI.
    
    Another reason for AI is to be able to evaluate the dog’s sperm quality before the breeding. This can be done by an experienced veterinarian, and then an AI done if the sperm is viable. However, it can also be done prior to breeding with a collection, and evaluation when the bitch first goes into heat, and the veterinarian can give his/her blessing to proceed with natural breedings. This evaluation should always be done soon before the proposed breeding . A dog that has produced a litter 3 or more months ago, may not be viable right now. Spermatogenesis (sperm production) is a dynamic, constantly occurring event ( the cycle of initial spermatogenesis to maturity is 2 months) and can change over several months.
    
    If a dog has been determined to have a low sperm count, or high numbers or abnormal sperm structurally (sperm morphology) , AI such as trans-cervical, or surgical implantation may be suggested by your reproductive veterinarian to ensure a proper number of viable sperm make it to the eggs. Remember, in a natural breeding many sperm get lost in the vaginal canal, and cervix…These AI methods help “deliver” more sperm to the uterine horns( fallopian tubes) when there are fewer numbers, or fewer normal sperm, and one cannot afford to lose the few good sperm to the vaginal vault or cervix.
    
    As mentioned in the first 2 questions, if fresh chilled semen is to be used, or frozen semen, AI is obviously the only way to perform the insemination. For fresh chilled regular vaginal AI’s done with proper progesterone timing are quite accurate if done properly ( proper deep deposition of the sperm with an appropriate insemination tube, feathering and 10 minutes of elevation after proper warming and evaluation for motility is done. For this reason the author feels a properly trained veterinarian with proper timing experience, equipment, microscope for evaluation, and experience handling fresh chilled semen is the most capable individual of ensuring a conception by these methods. The AKC has recently loosened its requirements on who may perform fresh chilled inseminations, allowing for breeders to ship, and inseminate themselves. While there is nothing wrong with this, the author feels that the percentage of success with fresh chilled semen will undoubtedly decrease without proper training, equipment, and microscopy skills unless the person inseminating has been properly trained in these methods.
    
    If frozen semen is to be used only 2 methods of AI seem to have a successful track record in the according to most reproductive veterinarians in the U.S. These are trans-cervical and surgical insemination. The trans-cervical method uses rigid endoscopy equipment, and has the advantage of requiring no anesthesia, and usually no sedation. The method is to advance the rigid endoscope into the vaginal vault to the level of the cervix and visualize the placement of a flexible pipette into the os or opening of the cervix. Insemination is done directly through the cervix into the uterus. The down side to this method is that there is a steep learning curve associated with it for the veterinarian, and there are some bitches cervixes that are very difficult to “thread”.
    
    The surgical insemination method requires anesthesia, and a very small incision to expose the uterus, and directly inseminate the “horns” or fallopian tubes. The disadvantage is the need for anesthesia. The advantages however are:
    
    1) quick simple learning curve
    
    2) the ability to handle and observe the uterus for health or disease such as cystic endometrial hyperplasia ( CEH) and other conditions, and
    
    3) the ability to monitor the filling of the horns with semen. Also many surgeons will hold pressure at the level of the cervix for 1 minute to “hold” the semen in the horns and prevent back flow to the vaginal vault. This allows for the mucous lining the fallopian tubes to “capture” the sperm.

   

• Please describe the timing of the test numbers with the fertilization and implantation of the egg cell on the uterine wall.

  • As mentioned earlier, ovulation occurs around 3-6 nanograms, let’s say 5 nanograms for purposes of this discussion. It is important to realize the canine egg is unique in that it requires another meiotic division, or polar body shed after ovulation before it is ready for conception. This means that the canine egg is not ready for conception until 24-48 hrs after ovulation. It continues to be viable for about 3 more days. (5 days in all after ovulation) . Values of progesterone are often 12- 18 nanograms when the inseminations should occur, as progesterone levels continue to rise after ovulation. This varies however with each individual bitch. Some have very sharp rises, while others may not rise as fast. Therefore a hard and fast number on which day to inseminate is not available to us. Again it is the trend and curve of the rise that is important, and therefore a veterinarian very experienced with this is a real asset.
    
    Actual implantation into the uterine wall takes place only after 15 -18 days post conception…The fertilized ova actually move freely about the horns during this time, and likely spread out to implant in both horns.

   

• Describe the process of how chilled semen is preserved and transported.

  • Fresh chilled semen is collected either in 3 fractions…seminal fluid, the sperm rich middle fraction, and then prostatic fluid. Only the sperm rich fraction is used and added to a special extender which has nutrients and buffers to allow the sperm to liver for extended periods. Some veterinarians will collect the first 2 fractions together and centrifuge them (this should be done with a variable speed centrifuge at very low speed (700 g’s or around 700-1000 rpm) , removing the fluid portion and replacing it with the extender. Semen can live 48 hrs, and sometimes up to 96 hrs. or longer, but most reproductive vets feel it should be inseminated anyway within 48 hrs. of collection. Interestingly, by centrifuging, and replacing the extender every couple of days semen has been maintained for 7 + days in the laboratory. This should not be regular practice in a normal clinic however.
    
    It is important to note that the male should have a “chill test” done prior to a planned insemination unless he is regularly chilled and sent and proven viable for chilled semen. (essentially regular usage constitutes frequent chill tests!) In this author’s opinion, a full semen morphology (structural evaluation) should always be done on dog’s being chilled. 8-12 % abnormality is the norm for a healthy dog. An evaluation indicating 98-100 % normal should be considered suspect. Also it is inappropriate to send a shipment, and inseminate only ½ of the sample on the first day, hold the other half and inseminate it 48hrs later. This is sometimes done to save the money of a second shipment, and shipping kit, but is not beneficial. You have essentially cut the number of sperm in each insemination by ½, and the second sample has continued to age in the test tube. It is better to let it age in the bitch’s reproductive tract which is a much more conducive environment for its survival than a test tube, and ensure good numbers for each insemination, by sending a second sample which is fresher, and younger 48 hrs later. You have also increased the number of sperm introduced by sending 2 shipments.
    
    Transportation is usually by overnight carriers to allow insemination within 24 hrs. If a Monday insemination is required, then a Saturday collection for delivery Monday 48 hrs later is acceptable if the dog has been proven viable in a chill test for that long. Before the 9-11 tragedy many of us used same day counter to counter deliveries and were often able to inseminate a bitch on one coast the same day with semen from a dog on the other coast. After 9-11 only “known shippers” are allowed to do counter to counter delivery, and this is a hard designation to achieve. Many “known shippers” already had he designation before 9-11.

   

• What happens when it gets to the bitches vet? How do they unchill it?

  • When it arrives at the inseminating vet’s office, it is held unopened usually at room temperature until the insemination is to be performed. (preferably the same day it arrives, although it can wait another day ( 48 hrs after collection) if necessary and the dog is known to chill well). When ready for the AI, the vet will warm the semen in a water bath at 37 degrees centigrade and an activator fluid ( sometimes simple 9% saline) for up to 20 minutes if it is from the CLONE company. If it is from one of the other popular companies, Synbiotics ( formerly ICG), ICSB, or Camelot, the semen can be evaluated with a drop on a slide under the microscope after being placed on a slide warmer for a few minutes to evaluate motility. In the author’s experience ICSB, Camelot, and Synbiotics extended semen is often still very motile although slow before warming, while CLONE is usually completely non-motile until warmed for 5-20 minutes.

   

• How do you go about having your dogs sperm frozen for the future?

  • Contact a reproductive veterinarian convenient to you who has experience and a good track record of freezing and achieving viable pregnancies with the semen they have frozen. You should ask if full pre and post thaw morphology is done, and what % post thaw they recommend not saving the sperm. It is generally agreed that sperm post thaw which is less than 30-40% motile has a poor chance of achieving a pregnancy. It is also important to realize that 70% motile sperm with poor morphology, while it looks very viable and active may be completely unviable for achieving pregnancy.
    
    Progressive motility (or the ability of the sperm to swim forward in an appropriate manner) is also very important post thaw. This is usually measured on a scale of 1-5 with 5 being the best forward motility score.
    
    Costs may range from $300 to $500 dollars US. to have a freeze done, and this usually includes the first year of maintaining the sperm. Maintenance costs per year range from $90 to $120 after that. A common question is how long is frozen sperm viable? The answer is indefinitely…
    
    Some veterinarians also offer remote freezing in which the collection can be done by an experienced collecting vet, fresh chilled preferably in a non-glycerolized version of the freezing extender, and sent to the actual freezing center with which they work. This allows the collecting veterinarian to provide freezing capabilities without the expense or time requirements of freezing and storing the semen. It is important to note that most healthy dogs do well with this procedure, but some do better with direct freezing at the final freezing center.

   

• What is difference between Genotype and Phenotype?

  • Genotype is the genetic propensity for a trait. A dog may have the genes for a particular trait but not express them. Phenotype is the actual physical expression of that trait. For example a dog may have a familial history of hip dysplasia, and carry the genes for this condition, but environment, such as obesity in the first year of life, level and type of exercise, etc can influence whether the dog expresses the physical manifestation (or phenotype) of the disease. If said dog is kept thin the first year of life, not allowed to perform regular exercise, etc. he may not express the phenotype, and pass OFA films at 2 years of age… but he will still pass on the genes for dysplasia to his offspring because he has the genotype, or genes for the disease, despite passing OFA certification at 2 years of age.