A Progesterone Test For Dogs? Exploring CANINE Ovulation Testing

What is a Progesterone Test For Dogs? 

Timing is everything when breeding, but what can you expect from a progesterone test for dogs when you’d like your dog to be expecting? Progesterone is an important hormone in the canine ovulation cycle and when measured via scheduled blood tests and interpreted correctly, it can be used to help determine the optimum breeding time. 

Progesterone Test For Dogs

Vaginal Cytology vs. Progesterone Testing For Dogs

The main methods of monitoring estrous cycles in dogs are through the collection of vaginal swabs and the interpretation of vaginal cytology or the collection of blood samples and the measurement of progesterone hormone concentrations in the blood. 

Vaginal Cytology 

A veterinarian performs a vaginal swab through a speculum, places the swab onto a glass slide, and then uses a microscope to examine what types of cells are present in the vaginal lining. The vet then interprets the cell composition and morphology to determine what stage of the estrus cycle is represented. 

Several vaginal swabs are needed to accurately determine the progression of the estrous cycle and the optimal breeding window period. Successive smears can help differentiate between proestrus and diestrus. 

Vaginal cytology is fairly straightforward, cost-effective, and usually well tolerated by most bitches. Most veterinarians are equipped to provide vaginal cytology and that’s why it is commonly used in practice. 

The four different stages of the canine estrous according to vaginal cytology are:

Proestrus 

Most owners notice this as the first sign of the breeding cycle and it is often referred to as the dog being “in heat”. It lasts for about nine days on average but there can be significant variation. This is also when male dogs begin to show interest in the bitch but she is nonreceptive to their advances. 

The cytology seen is mostly mixed cell types with red blood cells.

Estrus 

This is the time when a female becomes receptive to the male and can last an average of nine to 11 days depending on behavioral signs. The fertility window occurs during this period and this is the stage when the female is receptive to the male. The bloody discharge decreases and the vulva becomes soft and responds to stimulation. 

Vaginal cytology shows predominantly cornified (flattened) epithelial cells. 

Diestrus 

The female becomes non-responsive to the male and behavioral signs diminish. This stage lasts for about two months.

Cytology shows a shift back to basal cells, with fewer red blood cells than in proestrus.  

Anestrus 

This is the period between estrous cycles and can last for about four months depending on breed type and size. There should be no vaginal discharge or enlarged vulva, but if a discharge is noted it is important to visit a veterinarian as there could be underlying uterine pathology.

The vaginal cytology shows just basal cells.

Progesterone Levels

Progesterone levels rise slowly and stay high throughout the reproductive cycle, so in order to know when to breed, one must measure the level of progesterone sequentially to determine the best time to breed.

Proestrus 

Initially, the progesterone levels of the bitch during this period are less than 2ng/ml and this is considered the baseline. The first progesterone level test should be run on days five and six of this cycle.

Estrus

As the cycle progresses the bitch will enter the canine estrous cycle and her progesterone levels will begin to rise above 2ng/ml. This is considered to be the initial rise and its only significance is that it indicates that progesterone levels need to now be closely monitored to determine when the bitch will ovulate. 

Serial serum samples should be drawn every one to three days and when the progesterone levels reach over 5ng/ml, ovulation is thought to occur.

Diestrus 

After ovulation, progesterone levels will continue to rise to about 40 to 50 ng/ml (the average range can be between 10 to 90 ng/ml). Progesterone levels will rise regardless of the fact that she has been bred or may or may not be pregnant.

Progesterone levels remain elevated for at least 57 days unless there is an ovarian or uterine pathology.

Anestrus 

Progesterone levels begin to fall to the basal level of below 2ng/ml until the next cycle begins, which is referred to as anestrus.

How to Interpret Canine Progesterone Levels

There are two ways to measure progesterone levels in the blood:

  • ELISA tests based on color changes
  • Laboratory tests

ELISA Tests

The straightforward ELISA test provides results in-house in about 30 minutes and is based on a color change that reflects the concentration of progesterone in the blood. This progesterone test can also be done at home if you have the correct set of skills and equipment. 

visual representation of an ELISA test

The color chart starts at bright blue which reflects progesterone levels below 1ng/ml. This means that levels are not high enough to warrant daily testing so every second a sample is drawn and only once the color changes to light blue will daily tests be recommended. 

Once the light blue color begins owners can start to plan insemination between 3-6 days. A faint blue color indicates that progesterone levels are over 2.5 ng/ml but below 8 ng/ml so ovulation has not yet occurred. Prepare to breed or inseminate in one to three days depending on the color.

Once the test becomes white it indicates that ovulation has taken place as progesterone levels are over 8ng/ml and mating or insemination must occur immediately. 

COLOURPROGESTERONE ng/mlRECOMMENDATION
Bright Blue0 – 1Retest every 2 days until a LIGHT BLUE color is obtained 
Light Blue 1 – 2.5Retest every day until a FAINT BLUE is obtained
Plan to inseminate in 3-6 days
Faint Blue2.5 – 8Repeat test daily 
Breed or inseminate in approximately 1-3 days
White>8Mate or inseminate immediately 

Laboratory Tests

Immunoassay or chemiluminescence assays are used in labs and provide more accurate values compared to the semiquantitative values of an ELISA test.  As exact progesterone levels are attained, breeding decisions can be made more confidently when planning the ideal time to mate or inseminate.

Progesterone levels are thought to double daily every two days once they start to rise and when they reach over 5ng/ml ovulation is predicted to occur.  

Interpretation of Tests

Once the blood level is over 5ng/ml it is still considered too soon to breed the bitch unless using fresh semen through natural mating or artificial insemination. Dog’s oocytes need 48 hours to mature before they can be fertilized so viable semen needs to survive in the reproductive tract to reach the eggs at the right time.

Tests still need to be done after the progesterone level has risen above 5ng/ml as we need to ensure that the level goes up to 20 ng/ml to ensure that ovulation is complete and that the progesterone level is high enough to support a pregnancy. 

The type of semen and the route of insemination depends on the concentration of progesterone levels as it forecasts the optimal time after ovulation that will yield the most fertile period.

20 Fascinating Facts About Canine Ovulation in Your Bitch

Here are 20 facts that might help you better understand the reasoning behind it.

Where to begin
Monitoring the estrous cycle closely is the first step in successful breeding. Keeping a breeding journal or making a conscious effort to record heat cycle events on the calendar is a great start. Schedule a veterinary consult around 6 days after the beginning of the heat cycle. Individual variation may mean that some bitches ovulate sooner than others so it’s important to start monitoring them as early as possible.

Get the numbers straight
Progesterone results are quantified in two different units, namely ng/mL and nmol/L. When communicating these numbers to your vet remember to include the units of measurement and if your vet is unfamiliar with the unit, you can use the following conversion factor: 1 ng/mL is equivalent to 3 nmol/L.

Once is not enough
A single test is not enough to determine when a bitch may ovulate. Due to individual variation, at least three to four tests over several days are usually necessary to establish ovulation.

Initially, daily vet visits aren’t necessary
Unlike hormones like LH that have their peak monitored, progesterone rises progressively throughout estrous and the key is to monitor when it rises above its basal value.

The basal progesterone value is low when its concentration in the blood is less than 1ng/ml. 

Should the initial tests yield a value of less than 1ng/ml then the next visit can be delayed by between 4-5 days.

Ovulation 

Illustrated below are the progesterone profiles of several bitches. The two main focus points are the “LH peak” which is when the luteinizing hormone is at its highest concentration and “Ovulation” when the eggs are released from mature follicles.

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Figure 1 – Canine ovulation timing chart 
  1. Timing it right

Ovulation occurs two to three days after the LH peak when the progesterone level reaches 2-3ng/ml.

  1. Ovulation detection is key 

Even if the LH peak has been passed, it is important to continue with progesterone monitoring to detect ovulation. There is always a risk that a bitch may not ovulate in what is referred to as an anovulatory cycle.

  1. Anovulatory cycles

When ovulation does not occur then no eggs are released and therefore there is no chance of fertilization. The exact cause of these non-productive cycles is still unknown, and even though progesterone rises as follicles containing oocytes have grown, it can suddenly drop and this means it cannot support the follicles and they simply resorb and no ovulation takes place. 

Bitches can have levels that rise up to just under 5 ng/ml and then suddenly drop so it’s important to maintain surveillance of progesterone levels to avoid fruitless mating/insemination. 

  1. Ovulation occurs when progesterone blood levels reach 5-6ng/ml.
  2. Size doesn’t matter 

Progesterone blood levels are not influenced by the type or size of a breed, so when looking at Figure 1, it is evident that all the different curves intersect at the same level of progesterone when ovulation is taking place.

  1. The estimated day of ovulation means more than the progesterone level

Although the level of progesterone is known at ovulation, the ideal breeding time cannot be gauged by a value of progesterone levels. This is due to the large amount of individual variation as well as several other factors and testing limitations. The breeding protocol should be determined based on the estimated day of ovulation. 

  1. What is the purpose of a progesterone test after ovulation? 

Ensuring that the progesterone levels rise high enough to support a pregnancy and to also rule out possible ovarian pathology like cysts is very important. 

  1. What is a progesterone plateau?

Progesterone will rise quickly after ovulation but if the progesterone stops rising and stays at a constant level that is known as a plateau. If the plateau persists for more than three days an ultrasound should be performed to rule out ovarian cysts. 

  1. Progesterone molecules are the same in animals as well as humans so we use human assays in canine medicine.
  2. Familiar interpreter 

Due to variability in certain labs and machines – it is important to rely on a machine operator or a lab technician that can provide proper insight into the result of the value obtained.

  1. In-house progesterone testing 

Some practices have invested in in-house machines that do not require couriering to an off-site lab which makes the test result much faster and more convenient to access.

  1. The time it takes to run a progesterone test is between 10-120 minutes
  2. Not all ovulations are created equally
    Just because a previous progesterone level test revealed ovulation to occur after a certain number of days, does not mean the next cycle will be the same. Cycles can vary by up to 40% so to ensure a predictable ovulation timing, levels should be done at every cycle.

How Progesterone in Dogs Contribute to Time of Breeding

Progesterone levels determine when a dog ovulates and therefore allow a mating schedule to be determined. This is especially important to breeders using sires from other breeders or for artificial insemination as it lays out when a breeder needs to start preparations.

The idea is to establish when progesterone levels reach 2.5 ng/ml and then matings, semen collection or shipping can be arranged. Depending upon the type of semen used, optimal times for natural or artificial insemination are:

  • Natural breeding should occur three days after the 2.5 ng/ml mark as fresh semen survives 5-7 days after mating.
  • Artificial insemination :
    • Fresh chilled semen should be used for once-off breeding, four days after 2.5 ng/ml progesterone levels are reached, as it can survive 48-72 hours post insemination. 
    • Ideally, semen should be deposited by a veterinarian into the cervix to improve chances of entry and transport through the uterus.
    • Frozen semen should be used five days following the 2.5 ng/ml progesterone level or 72 hours after the 5 ng/ml mark as the sperm only survives for less than 24 hours. Surgical administration of semen directly into the uterus is the best means of increasing the chances of pregnancy.

Who Will Use a Progesterone Test For Dogs?

The progesterone test can be used by breeders in combination with behavioral queue monitoring to determine the most appropriate time to either naturally mate or artificially inseminate their bitch. Ovulation detected by progesterone levels can help improve the success and fecundity of an estrous cycle. 

Veterinarians can also use progesterone levels to detect possible underlying reproductive issues. 

Other Uses For a Progesterone Test For Dogs

Progesterone Testing For Predicted Whelping 

Progesterone can be used to help predict when a bitch may whelp. The gestational length of a bitch is between 61-63 days. Progesterone levels drop to < 1ng/ml and this, in turn, affects a bitch’s core body temperature. So if owners measure progesterone as well as monitor body temperature two to three times daily, when the core temperature drops by 2°C, then parturition can be expected within 12-24 hours. 

Elective C – sections can also be planned by monitoring progesterone so that when the 61-day mark is passed then owners can wait until progesterone drops to avoid a premature delivery and potential mortalities.

If the progesterone is low and the bitch is not showing any signs of labor then it can help to indicate signs of primary uterine inertia as well.

Progesterone Testing For Potential Underlying Reproductive Issues 

Progesterone testing can be useful in a range of other reproductive conditions:

Split heats 

Young bitches can have a non-ovulatory, silent estrous that is not noticeable but then it results in a short inter estrous period and then a normal estrous cycle. To determine a non-ovulatory estrous cycle, progesterone levels will rise slightly and then drop off, so it is advised to then repeat progesterone measurements in the next 40 days after the silent heat.

Delayed puberty 

If a bitch does not show any signs of coming into season within two years progesterone tests are the first step in determining potential underlying conditions that are retarding estrous cycles. 

Hypoluteoidism
A condition that leads to the resorption or abortion of fetuses before gestational maturity can occur in certain breeds. Vets will monitor and possibly supplement progesterone if it is picked up early.  Breeds affected can include German Shepherd dogs or Old English Sheepdogs.

Signs That a Female Dog is Ready to Breed

Each stage has differing signs related to behavior, physical or clinical changes, hormonal changes, physiologic changes, and cytologic (vaginal smear) changes.

Here is a list of signs that your female dog is ready to breed:

  • The initial pink to red discharge during the proestrus period will change slowly to a tan color during estrus when she becomes fertile
  • The vulva (the fold of skin at the entrance of the vagina) will become swollen and soft, and if any pressure is applied above the base of the vulva it will move upwards
  • The bitch will also become receptive to the male dog advances and her tail will wag – also known as tail flagging which promotes her scent to an interested male.
  • Changes in habitus can also be noted such as moodiness, increased attention-seeking, whining, and appearing more sensitive than usual
  • Increased attention from male dogs and tolerating pressure on her back – standing to be mated 

Artificial Insemination in Dogs

Artificial insemination is a means to ensure breeding at the optimal timing with the most desirable sire. As with any breeding, there are risks of failure with AI but if done correctly by the correct specialist or experienced veterinarian then the desired outcomes are often achieved. Close monitoring and accurate record-keeping are the best advantages for AI success.

Progesterone testing in dogs is a very useful, inexpensive, and readily available tool in female canine reproduction. It can be used at different stages of the reproductive cycle and give information about the right time of mating and parturition as well as some abnormalities.