Progesterone Testing & Vaginal Cytology

Sometimes the breeding process can be confusing. In our clinic, we require two different types of tests to determine if the female is ready for breeding. The information provided below is extremely important to understand if you are going to breed your dog.

Vaginal Cytology

A vaginal cytology is an evaluation commonly done. For many years, this was our only practical tool with which to time breedings.

The cells seen at the ideal time for breeding are a high percentage of cornified epithelial cells, with few red blood cells (RBC’s), white blood cells (WBC’s) or debris. The vaginal smear may also indicate the presence of other problems such as vaginitis. The test is simple, safe, inexpensive, well tolerated by the bitch approaching estrus, and very helpful in timing breeds.

Examination of a single smear can provide useful information, but can also be misleading. For example, it is often difficult to differentiate proesterus and disestrus from an isolated smear. It is most useful to evaluate multiple smears taken from the same bitch. These slides can be labeled with the date collected and stored for evaluation sequentially as she moves through her estrus cycle to monitor trends in cellular cornification.

Progesterone Testing

A quantitative progesterone blood test is the single most accurate method for timing breedings. The test is reported as a numeric result, usually ng/dl. Combined with observing the behavor of the dog and bitch, vaginal cytology, ovulation can be pinpointed with great accuracy in most bitches.

An exact progesterone level is also needed when the dog or bitch will be traveling a long distance for breeding, when fresh chilled or fresh semen is to be used, or when breeding dogs or bitches that have a history of being difficult to breed. In addition, an exact progesterone level for timing ovulation is necessary when a C-section is anticipated or when the bitch is near term and fetal survivablility needs to be assessed.

There are some veterinary clinics that have a high enough volume of progesterone samples that they have purchased equipment (Immulite, Tosoh, or Mini-Vidas) to run quantitative progesterone samples in their veterinary clinics. *We use MicroVet Cube which is comparable to Immulite.

Progesterone Interpretation

The canine’s reproductive system is unlike any other species. This is probably the most confusing part of canine reproduction. Many breeders and many veterinarians do not understand the cycle well until they see it graphed. Once you understand that progesterone rises slowly and stays high throughout the cycle.

At the start of the strous cycle, proestrus, the estrogen levels are rising. The hormone of interest, progesterone, of the bitch is at avery low level, less than 2ng/ml. This is often reffered to as “baseline”. During proestrus in a normal bitch that has started estrus spontaneously (without drug or hormone intervention), the first progesterone level should be run on day 5 to day 6 of her cycle.

As the bitch enters estrus, she approaches her fertile period and her progesterone level will rise above 2ng/ml. She will become increasingly attractive to the male and receptive to his advances. This slight rise is often called the initial rise. Its only significance is that you will want to monitor her progesterone levels more closely now as she is about to ovulate. Bitches are only fertile for a few short days during estrus, not the entire time they are in estrus.

As the progesterone rises above 2 ng/ml, continue to draw serial serum samples for testing, usually every 1 to 3 days.

Ovulation is though to occur when the progesterone level reaches 5 ng/ml. This may be the single most important concert regarding breeding in this book. The bitch’s ovary responds to the LH from the pituitary, which allows the release of one egg from each of many multiple mature follicles.

But stop: it is too early to breed if you are using fresh chilled or frozen semen. When the bitch ovulates, the eggs are not yet mature and ready to fertilize. Unlike in other species, the eggs mature over the next 48 hours before they are ready to fertilize. The timing of the breeding must occur when the eggs are mature and ready to fertilize and viable semen is in the oviduct, estimating how long the semen is anticipated to survive in the reproductive tract.

Breeding with fresh semen, by either natural breeding or vaginal AI with fresh semen, can be done on the day of ovulation, when the progesterone level reaches 5 ng/ml. Although the eggs are not mature yet, most fresh semen is viable enough to survive in the bitch’s reproductive tract until the eggs are ready.

Breeding with fresh chilled semen should be delayed until approximately 48 hours after ovulation. Fresh chilled semen usually will not survive as long in the reproductive tract as fresh semen. If the fresh semen is deposited vaginally or directly into the uterus 2 days post-ovulation, the eggs should be fertile when the semen appears in the oviduct.

Breeding with frozen semen should be delayed even longer, 60 to 80 hours after ovulation (Ovulation occurs when the progesterone is at a 5 ng/ml). It is thought most frozen semen will only live 12 to 24 hours (or less) in the reproductive tract. For this reason, the semen should arrive in the oviduct when the eggs are mature and ready to fertilize. If necessary to choose, it is better to breed slightly too late than slightly too early. most breedings with frozen semen are done by depositing the semen directly into the uterus, usually by surgical insemination but in some cases by transcervical insemination.

Once the progesterone level is 5 ng/ml we are still not done testing. Although it is believed that ovulation occurs around the time the bitch’s progesterone reaches 5 ng/ml, we still need to keep our eye on one more number; that number is a progesterone level of a 20 ng/ml. To assure that ovulation is complete and the progesterone level is high enough to maintain a pregnancy, delay either the final or the surgical insemination until the progesterone level has reached or exceeded 20 ng/ml.

After ovulation and breeding are completed, the progesterone level will continue to rise. The level typically rises to 40 to 50 ng/ml (the normal range can be 10 to 90) whether the bitch is bred, pregnant, or not. An elevated progesterone level only indicates that the corpus lutea in the ovaries can support a pregnancy, not that the bitch is pregnant. Bitches maintain this level unless that have ovarian or uterine pathology.

Greer, Marthina. “Chapter 3: Preparing to Breed.” Canine Reproduction and Nenatology. Jackson: Teton NewMedia, 2015. 46+. Print.