BREEDING DOGS 101 - TIMING & PROGESERONE
by Jessica Strauss

The information provided in this article, is for breeders who are serious about the success of their breeding program. Arming yourself with basic knowledge of the canine reproductive system and of progesterone testing, are keys to a stress free and successful breeding experience.

Estrus, The Heat cycle: The stages of the canine heat cycle are anestrus, proestrus, estrus, and diestrus. Anestrus is the period of time when there is no activity, and the female dogs reproductive system is in "hibernation". This stage lasts from 90 to 180 days. The next stage is proestrus. This is where we see the first signs of her cycle beginning, such as blood spotting, vaginal swelling. This stage lasts 9 days average (range 3 to 17 days). She is not receptive during this time, but male dogs WILL be interested. The next stage is estrus, which lasts 9 days average, (range 3 to 21 days). During this stage, the LH surge will occur, and ovulation will follow. The bitch will be receptive and breeding will occur. The final stage is pregnancy or diestrus, which lasts for 60 days whether the dog is pregnant or not. Progesterone levels will continue to rise to anywhere from 10ng/ml to 80ng/ml, and then drop off around the 60th day. The bitch goes back into anestrus, and progesterone levels stay at under 1ng/ml for the next 3 to 6 months.

My Experience: In my experience, the time from the beginning of a heat cycle, to the time of the beginning of the next cycle, is between 20 and 22 weeks (if the bitch is not bred). So when tracking the girls' heat cycles here, and planning our upcoming breedings, I will count 20 weeks from the beginning of her last cycle and mark that date on my calendar, as a starting point to start watching for her next cycle to start. There are, of course, exceptions, like our Shelby, who was what we call an "8 monther".

Pre-Breeding Vet check: There are a number of complications that can occur when trying to breed. It is important that the bitch have a full examination with her Veterinarian, to ensure she is healthy, and that she is able to breed. One issue that can occur, is vaginal stricture which will prevent any natural breeding's. Here is a link to several complications that can occur: [link].

Progesterone tests - when to start: Female dogs ovulate when the estrogen level is decreasing and the progesterone level is increasing, which is the complete opposite of most other animals and people. It is best to start progesterone tests within the first 5 days of the very beginning of proestrus, and then every 2 days after, especially for bitches that you have no previous progesterone history on. Progesterone levels during proestrus are typically under 1.0 ng/ml, until the day before the Luteinizing Hormone( LH) surge, which is what triggers ovulation two days later. There is a test to determine the LH surge, but it needs to be done every day, making it impractical. The LH surge only occurs for a period of under 24 hours, so it can be difficult to catch. The LH surge can be estimated, when doing progesterone tests though. On the day of the LH surge, progesterone levels will be between 2-3 ng/ml. The day after the LH surge, progesterone levels are 3-4 ng/ml. So, if you did a progesterone test on Monday and it was 1.0ng/ml, and another on Wednesday which came out at 3.8 ng/ml, you will know that the LH surge probably occurred sometime on Tuesday. Ovulation occurs 2 days following the LH surge, when progesterone levels have reached 5.0 ng/ml.

Once the bitch has ovulated, it takes up to 48 hours for the eggs to mature. So, in our example testing above, we estimated the LH surge occurred on Tuesday, which means ovulation is estimated to occur on Thursday. The eggs won't mature for another 2 days, and then die if not fertilized, 3-4 days later, but we can start our first attempts at breeding Thursday. Sperm in fresh semen, survive 5-7 days after insemination, following the 7 hour period ("capacitation time") following ejaculation required for them to mature. Ideally, natural breedings should take place in our example breeding, on Thursday, Friday, Saturday and Sunday, if possible, but a Thursday and Saturday breeding would probably be equally effective. If only one natural breeding was possible, I would have that occur on Saturday, which is an estimate of when the eggs are mature, and they still have 3-4 days survival. See below for timing adjustments that would need to be made for the different type of Artificial Insemination options.

It is important to continue with progesterone testing after the 2.0ng/ml mark, to have a good estimate of the bitch's ovulation date. It will be important to have an accurate due date at the end of the pregnancy. A bitch's due date is 63 days from ovulation, regardless of breeding days. If the bitch has any difficulty whelping, this date will be critical for the timing of a caesarian section.

Following a successful breeding and fertilization of eggs, it takes 17-18 days for the eggs to implant in the uterus. If implantation does not occur properly, or the placenta does not grow normally, the fetuses are reabsorbed. This can occur if her progesterone level drops significantly.

Split Heat Cycles: Another reason it is important to continue with progesterone tests after the 2.0 ng/ml mark, is it's possible for a bitch to hit the 2.0ng/ml level and then have her progesterone levels decreases again, and go back out of proestrus. This is called a "split heat". This has happened to us several times, and is fairly common in young females. If a split cycle occurs, she will usually go back into proestrus, in 6-8 weeks.

The Canine Pregnancy Test: The Relaxin blood test can be run at 28 days after ovulation. It is a good idea to run another progesterone test at this time as well, which I will explain later. I have found the relaxin test to be very reliable in determination of pregnancy, and in determination that the breeding did not result in pregnancy. We have done over 20 relaxin tests over the past 7 years, and all have been accurate. The possible results for relaxin tests are "POS" for a pregnant dog. "NEG" for no pregnancy, and the third option is a "WEAK POS", which we got when Breeze was pregnant with just one puppy. There can be inaccurate results if the Relaxin test is run too early in the pregnancy. The inaccurate result would be a false negative. The hormone, relaxin, is only found in the blood during pregnancy, so a positive relaxin test is an absolute for pregnancy. A dog would not have relaxin in her blood for any other reason.

I suggest running another Progesterone test at the same time the relaxin test is done. A bitch must have a minimum progesterone level of 5 ng/ml to maintain a pregnancy. The normal range is 20ng/ml. If the progesterone level drops below 2ng/ml, the puppies will be lost. If a bitch is running at a 5.0ng/ml or less, daily progesterone treatments (IV or oral) can be given, but this is when it is critical to know the date she ovulated, and a whelping date can be calculated accurately. Progesterone treatments must be stopped prior to that date, so the progesterone level can drop and the whelping can occur. Whelping starts between 24 and 48 hours of the progesterone level dropping below 2 ng/ml.

It is possible to predict an upcoming whelping date in a normal pregnancy, using progesterone tests. This is similar to estimating a delivery date through the temperature drop in your bitch. I will caution that we had one experience in which we did a progesterone test on a bitch who was due any day. The progesterone level was at 2.1 on a Tuesday evening, so we assumed we had a minimum of 24 hours, since "the book" says the progesterone will drop to under 2.0 from 24-48 hours before whelping. Well, the puppies started being born the next afternoon (in my car!) while we were eating Christmas Dinner at my parents house. Apparently, Venus didn't read the book!

Natural breedings and types of Artificial Insemination:

• Natural Breedings should start 2 days after the LH surge, or 2 days after your progesterone testing has reached levels of 2.0 to 3.0ng/ml. If only one breeding is possible, the ideal date to breed would be exactly 4 days following the estimated LH surge.
• Artificial Insemination using fresh chilled semen: Fresh chilled semen is only viable for approximately 48 hours. Insemination should occur 3-4 days after the estimated LH surge, or 3-4 days after the 2.0 to 3.0 ng/ml. It is best to have the semen placed directly into the cervix to increase chances of conception.
• Artificial Insemination using frozen semen: Frozen semen is only viable for under 24 hours, once thawed. Insemination should occur 5 days after the LH surge, and also calculate 72 hours following the 5.0ng/ml mark, to get the timing as close as possible. The only reliable way to use frozen semen, is by surgical AI, where the semen is placed directly into the uterus.

Doc, in the photo here, was produced by two Artificial Insemination's using fresh semen. Doc is named after his Veterinarian, to whom we owe the success! His mother, Shelby, was at 5.7ng/ml on the day of the first AI which was 3/31/10. We estimated the LH surge to be on the 28th or 29th. The second AI was done on 4/2/10, and her puppies were born on 6/1/10.

Cytology: Another option for estimation of ovulation is vaginal cytology. I find progesterone testing far more reliable, but prior to that option, cytology was used quite a bit in many mammal breeding applications. Cytology can also be used to back up any information you have received from your progesterone tests. Here is a great website to learn about vaginal cytology, and identification of cells involved in different stages of the heat cycle. [link]

Normal Semen: It is wise to have a semen evaluation done on any male dog who is intended to use for breeding, and especially when intended for artificial insemination. The 3 fractions of the semen (ejaculate: 1-6 ml, sperm: 100-500 million/ml, and prostatic fluid) are measured; the motility, or forward moving ability are measured (greater than 70% is good), and the veterinarian will also look at the structure of the individual sperm, which is called morphology. The head carries genetic material, the mid piece provides the energy for forward movement, and the tail works to propel, so all 3 parts are important, and 80% normal is considered a good evaluation.

Other Testing: It is also a wise idea to test your bitch, and the planned stud, for Brucellosis. This is a highly contagious disease, thought to infect as many as 8-10% of all breeding stock in the United States. Brucellosis could be the end of a breeding program, and even the possibility of having to euthanize infected dogs. One of the symptoms, is a bitch that appears to be infertile, or that can't stay pregnant, and aborts or absorbs her fetuses. Brucellosis can withstand freezing, so even frozen semen runs the risk of being infected. The Brucellosis test is relatively inexpensive, running in the $60 range. This test should be run prior to any breedings that occur between dogs outside your own kennel, or that have been bred outside your kennel.

My Experience with heat cycles and timing:

Now that we know how important timing is, to successful conception's, we are having much more success, especially when using artificial insemination. The biggest mistake when attempting to produce a litter, is to have chilled and surgical AI's done too early. Timing is less critical when using fresh semen, but you still need to be close. In the beginning, we made this mistake several times, and missed out on two breeding's, with Jessie and Nisga'a, and with Breeze and Stonecold, that could have been very beneficial to our breeding program!

Over the past 5 years, I have been documenting the start of heat cycles, progesterone levels, and breeding/AI dates, and have found most to be "textbook". The exception, is now I count 60 days from the first breeding, and mark that on my calendar as the earliest whelp date. And, place another mark on my calendar, for the 65th day following the estimated LH surge (or 63 days from the date of estimated ovulation). This is a really good estimated window of time for a whelp date, and may even work out to the same day. I put a 3rd mark on my calendar for 63 days following the last tie, so I know that if, by this date, we still have not had puppies, we need to head to the Vet! A simpler way to estimate your due date, is to add 2 months to the first breeding, and you will come within a day or two of your due date. Here is an example of this working out:

• Breeze bred 1/2, 1/3, 1/4. Puppies born 3/4
• Dallas bred 4/29 and 4/30. Puppies born 6/29
• Breeze bred 10/3. Puppies born 12/3
• Shelby bred 4/20, 4/21, 4/22. Puppies born 6/22
• Shelby AI'd 3/31 and 4/2. Puppies born 6/1

Do keep in mind that if running progesterone tests, to recheck a minimum of every other day. There have been several occasions where my girls progesterone levels have risen much faster than we anticipated. Dallas jumped from 3.0ng/ml to over 10.0ng/ml in less than 48 hours. Shelby was another who jumped from 1.8ng/ml to 9.6ng/ml quickly. Had we waited one more day to run another test, we could have missed our opportunity. Venus once had a progesterone level of 11.7ng/ml on day 7 of her heat cycle, so I emphasize starting your progesterone testing as early as day 5 of their cycles! In bigger cities, same day progesterone testing is available, but where we are, overnight is the fastest we get our results back, so that is another thing to consider when you are testing. You may not know until the next day, what your levels were the previous day!

Remember, the actual breeding of your dog is the least complicated of the entire breeding/whelping process. Let this article just be a small step in your research into the whole breeding experience, before deciding to jump in!