Progesterone Testing


Hypophyseal LH is the biological trigger for the cascade of events that culminate in
ovulation. Following a variable period (1 to 21 days) of estrogen level elevation, the LH
surge signals the transition from proestrus to estrus. LH stimulates follicular granulosa
cells to secrete progesterone. The LH surge is the key timing event dictating days of
peak fertility. Ovulation begins 2 days post-LH surge and continues for another day or
so. Ova mature and are capable of fertilization 2 days later. Mature ova live another 1-3
days. Optimal breeding times are day 4, 5, and 6 post-LH surge.

Pinpointing the day of the LH surge is cumbersome. The LH surge is short-lived,
typically lasting only 12 to 24 hours. Therefore, daily blood testing is required. LH is a
species specific hormone and assays are not currently available from commercial
laboratories in the US. A qualitative (test kit) assay is available for in-hospital use, but it
has a short shelf life (3 months maximum). LH assays are most often used in the timing
for breedings with frozen semen.


Progesterone's initial rise occurs concomitantly with the LH surge. At that time, baseline
progesterone levels (<1.5ng/ml) rise to 1.5-2.0 ng/ml. After the initial rise, progesterone
continues to rise and may reach levels of 10-15 ng/ml by the end of the fertile period.
Ovulation occurs when progesterone levels are between 4 and 10 ng/ml. Plan breedings
4 to 6 days after the initial rise, and 2 to 4 days after the onset of ovulation. Since
baseline and initial rise levels can vary from individual to individual, it is important to
start testing early enough to define the baseline progesterone level.

The gold standard for determining progesterone levels is quantitative measurement by
radioimmunoassay. Results are reported in ng/ml. Progesterone, like all steroid
hormones, is not species-specific and can be measured commercially by human and
veterinary laboratories alike.

Several semiquantitative ELISA progesterone kits are available, Status-Pro [Synbiotics],
Target [Biometallics], and PreMate [Camelot Farms], for example. Results are
intrepreted from a color change in the test well or membrane. Hemolyzed blood samples
will falsely lower the result. If test kit components are not warmed to room temperature,
then a falsely high result will be interpreted. The general consensus is that test kits are
usually adequate for most natural breedings, but are not accurate enough when planning a
fresh chilled or frozen breeding.

Recommendations for progesterone testing include starting to test around day 5 or 6 from
onset of sign of proestrus, followed by testing every other day until ovulation has been
confirmed. However, some females have very short seasons and require testing at the
first sign of proestrus. Others may not ovulate until after 21 or more days. When in
doubt, start testing early.


During proestrus, the vulva is swollen and a sersanguinous vulvar diacharge (of uterine
origin) is present. As proestrus transitions to estrus, vulvar edema diminishes and the
vulvar discharge becomes straw colored. Note however, that some normal bitches may
have a hemorrhagic discharge that persists throughout estrus. With the onset of diestrus,
vulvar edema subsides completely.

Excerpts taken from article written by:
Jane Barber DVM, MS, DACT

For the complete article "It's All In The Timing" go to:

Ovulation graph



The aim is to identify when the progesterone level reaches 2.5 ng/ml so the mating schedule can be set up, or the veterinarian and owner of the male dog can be notified that they should be prepared to collect and ship a semen sample. Depending upon the type of semen used, optimal times for natural or artificial insemination are:

1. Natural breeding should occur 3 days after the 2.5 ng/ml mark. Sperm in fresh semen survive 5-7 days after insemination.

2. Artificial insemination using fresh chilled semen should be used for a 1-time breeding. Insemination should take place 4 days after the progesterone reaches the 2.5 ng/ml mark or 48 hours (2 days) after the 5 ng/ml mark.

3. Sperm in chilled semen survive 48-72 hours after insemination. With artificial insemination, the semen should be deposited into the cervix to increase the chance of it being drawn into the uterus.

4. Artificial insemination using frozen semen should be performed 5 days following the 2.5 ng/ml mark or 72 hours (3 days) after the 5 ng/ml mark. Sperm in frozen semen survives less than 24 hours after insemination. Frozen semen is ideally deposited directly into the uterus through surgery to increase the chance of pregnancy.


The sperm require a period of approximately 7 hours after ejaculation before they are capable of fertilizing an egg. This period is referred to as the "capacitation time." The egg also needs time to mature after it is ovulated, generally 48 hours from ovulation until it can be fertilized. Fertilization occurs in the oviduct (Fallopian tubes) regardless of the method of insemination. The fertilized egg then travels into the uterus but does not implant until 17-18 days after ovulation. If there are problems with the lining of the uterus, the egg may not implant or the placenta may not grow or be maintained. A normal placenta grows into the lining of the uterus. If implantation does not occur or the placenta does not grow normally, the fetuses are resorbed.


Progesterone levels during pregnancy and whelping

After ovulation, progesterone concentrations continue to increase for 2-3 weeks, finally reaching 10-80 ng/ml. This level is necessary to maintain a pregnancy. In the dog, the progesterone level will remain at this level for about 60 days whether or not the dog is bred, and whether or not she is pregnant.

About 48 hours before whelping, the progesterone level drops to the 2 ng/ml range and within about 24 hours of whelping, the level drops to the 1 ng/ml range. This can help determine the proper timing of a c-section, especially if the progesterone level or LH level were not used to determine the ovulation date. By correctly determining the whelping time, it can prevent puppies from being taken by c-section too early and thereby decreasing their chance of survival.

Breeding diagram



1. NATURAL BREEDING: The 2 dogs are brought together and mated naturally.

2. REGULAR AI; Vaginal insemination involves inserting a rod loaded with semen into the bitches vagina and advancing it to the cervix opening. Called the os, the cervical opening is the ideal place to deposit semen. Once the rod is in place, the semen is deposited and the rod is withdrawn.

A less invasive and potentially faster surgical procedure, is a new technique becoming more prevalent. In laparoscopy, a small telescope inserted into the bitch's abdomen is used to locate and identify the uterus for semen injection.

Surgical insemination allows for direct insemination of the semen into the uterus. Similar to the technique used to spay a bitch. it involves injecting semen into the exposed uterus through a needle or catheter. Surgical conception rates using frozen semen closely match those of natural mating, Surgical insemination provides an opportunity to examine the bitches suspected of having uterine or ovarian diseases. In addition, giant and toy breeds, with historically poor conception rates, can benefit from surgical insemination. The technique also benefits males with low sperm-cell counts who have difficulty impregnating bitches in natural breeding.



Surgical Implants
Uterus is raised through a small incision and another small incisions is made in the uterus.
Semen is inserted through a catheter
directly into the horns of the uterus.


ICSB logo





Infertility is the inability to conceive or to carry a pregnancy to term. Infertility in bitches includes abnormalities of the heat cycle (estrus), failure of breeding (copulation), failure of becoming pregnant (conception), and pregnancy loss.

Errors in breeding management, anovulatory cycles and ovarian/uterine problems are frequently encountered as a cause of infertility in bitches of all ages, while poor semen quality is often a feature of adult to older male dogs. The most common factors influencing conception and the establishment of a normal canine pregnancy are listed below

Reproductive cycle disorders--Prolonged lack of heat or anestrus is sometimes observed especially in older bitches. Frequent cycling (3-4 heats/year) is also observed in bitches of all ages, and has been associated with infertility, although the mechanism involved is not yet clear. Anovulatory cycles occur fairly frequently (although there are no reported figures on their incidence) both at puberty as well as in the adult bitch. When a bitch experiences an anovulatory cycle, her reproductive behavior is generally absolutely normal, i.e., she attracts male dogs, has a normal vulvar discharge and accepts breeding. Failure to ovulate can only be diagnosed by finding a low serum progesterone concentration after breeding.

Uterine disease--Uterine pathology is a common cause of failure to conceive both in bitches and queens. The bitch experiences 2 estrous cycles/year, with spontaneous ovulation, development of corpus luteum and progesterone secretion for about 2 months. The progesterone stimulation on the uterine lining (the endometrium) causes accumulation of secretion of endometrial glands with formation of cystic structures (cystic endometrial hyperplasia = CEH). Such cystic structures are very important for feeding the embryos, and if the female is not pregnant they normally regress towards the end the luteal phase (also called progestational phase or diestrus), leaving the endometrium free to regenerate and be ready for the next chance for a pregnancy. If the female is rarely if ever mated, these cystic structures will eventually persist, thereby making large sections of the endometrium unsuitable for the establishment of pregnancy. Uterine disease is not believed to be a problem in breeding establishments where bitches are bred and conceive on a regular basis, as pregnancy may have a protective effect on the endometrium. Unlike breeding bitches, the average intact bitch kept as a pet may experience problems in becoming pregnant if bred only as an adult dog, due to the deterioration of her uterine lining.

There have been reports of instances of suspected luteal failure, luteal insufficiency, or "hypoluteoidism" in which peripheral concentrations of progesterone were observed to be exceptionally low in bitches which were confirmed to have resorbed or aborted their litter. The extent to which the low progesterone was a cause versus a consequence of pregnancy failure has not been well established. Nevertheless, protocols have been suggested for progesterone supplementation in pregnant bitches in which progesterone fall below 5 ng/ml before day 55, or declines more rapidly than expected in mid-gestation. The application of such progesterone replacement in bitches which previously failed to become pregnant or which were documented to have resorbed litters has involved successfully pregnancies--but whether the "successes" have been the result of treatment, better breeding management, or unknown factors is not known. Some protocols for progesterone supplementation have involved administration of progesterone in amounts too low or too infrequent to result in a meaningful effect of circulating progesterone concentrations, and there have been no studies to determine the effects of specific supplementation protocols on circulating progesterone concentrations. Progesterone supplementation protocols have included the following: (1) progesterone in oil, i.m., 2 mg/kg, every 72 h. (2) altrenogest, daily, p.o., 0.088 mg/kg (0.2 cc/10 lb, using Regu-Mate©, Hoechst-Roussel). Medroxyprogesterone, megestrol and some other synthetics can cause masculinization of female fetuses. Recently, human products of micronized natural progesterone in capsules have been used, being given orally 1-2 times per day at doses of 5-10 mg/kg (i.e., 100 mg capsules in 10-20 kg dogs) and the efficacy monitored by progesterone assays. This has been successful in some clinics although it relies on owner compliance. Natural progesterone requires frequent administration but has he advantage that withdrawal can be done rapidly. Whether the successful pregnancies obtained in P-supplemented bitches have been incidental or the result of treatment is not known, and controlled study is lacking.

Any use of progesterone supplementation must be done with a plan to discontinue therapy in a manner that will allow for progesterone to decline below 2 ng/ml shortly before expected "term" and thus permit or promote normal whelping. Excessive exogenous progesterone or other progestin can compromise normal parturition and can result in dead puppies that must be recovered by C-section. Oral progestin therapy should be discontinued 2 days prior to the estimated day of parturition. Serial injections of synthetic preparations should be discontinued early enough for the progestin to decline to non-effect concentrations by the expected day of parturition.

Poor semen quality--Dogs ejaculate 500-2000 million spermatozoa diluted in 2-50 cc of seminal plasma. Quantity of spermatozoa as well as of seminal plasma varies according to body weight and testicular size, with Yorkshire terriers and Great Danes producing an average of 2-3 and 20-30 cc of ejaculate, respectively. Semen quality depends on quantity of spermatozoa present, their motility and morphology.

Failure to achieve a normal mating--Young stud dogs at their first attempts at breeding may sometime apparently look not capable of mounting properly, e.g., they may approach the female from her flank or from her head, or may spend a considerable amount of time pelvic thrusting without achieving an intromission. Although this should be considered part of the normal process of learning reproductive behaviour in young animals, it is considered abnormal when displayed by adult male dogs.

Infection of the reproductive tract--Infectious diseases of the canine reproductive tract which can be responsible for infertility include bacterial infection such as brucellosis due to Brucella canis (rarely Brucella abortus or suis), infection due to salmonella species, streptococci and E. coli; viral diseases such as herpes virus, distemper, parvovirus 1 and 2, and to the parasites Toxoplasma gondii and Neospora caninum. Incidence may vary depending on the country and the area/s within each country. Brucella canis and herpes virus are highly contagious. Other bacterial infections (salmonella, streptococcus, E. coli) and toxoplasmosis are less contagious and tend to be a feature of the individual bitch. Not much is known about the role of Neospora caninum in canine abortion. Herpes virus has always been known as a cause of abortion.

Noninfectious causes of embryonic-fetal death--Endocrine diseases such as inadequate production of progesterone or thyroid hormones during pregnancy may cause abortion. Also, diabetes, adrenal insufficiency and other endocrine diseases may affect foetal viability. The improper use of drugs may also have an adverse effect on pregnancy. Chromosomal abnormalities are widely described as a cause of embryonic/fetal death in the dog.

Ovarian disease--Ovarian disease (ovarian cysts or ovarian tumors) is a rare cause of infertility in bitches due to the fact that incidence of ovarian cysts or tumors is higher is adult to older bitches (the older the bitch the less likely she is to be used for reproduction). The most common presenting complaint of ovarian cyst or tumor is prolonged heat due to a high estrogen production. Ovarian cysts and tumors have occasionally been reported in bitches as young as 2-3 years of age.




 Serologic testing for pregnancy in bitches is complicated because of female dogs' normal endocrine physiology. Bitches do not produce a pregnancy-specific hormone similar to human chorionic gonadotropin, the compound assayed in early pregnancy tests for women. In addition, progesterone is not a useful indicator of pregnancy because all bitches secrete progesterone for two months after heat, regardless of whether they were bred or not.

However, serum relaxin concentrations are increased in pregnant bitches three or four weeks after breeding. A test kit for canine relaxin is available, but I do not recomend it. A labratory testing is much more accurate. The ELISA may detect relaxin as early as day 20-23 and the Witness test often detects pregnancy relaxin as early as day 26-30. False positives occur in cases of recent resorption or with retained placental tissue. False negatives as late as day 30 or 34 have been observed anecdotally, and negative results should be followed by U/S or re-assay one week later. Ultrasound can detect pre-implantation vesicles by day 21 or earlier, embryonic masses by day 22-25, embryonic heart movement by day 24-27 using 5 or 7.5 MHz equipment.

Ruby pups


Pregnancy Time Table

Week One

  • Fertilization occurs
  • 2 cell embryos are in the oviduct
  • The embryo is fairly resistant to external interference in development
  • Possible morning sickness
  • Possible personality changes
  • Normal feeding
  • Normal exercise -- if she didn't normally get exercise before pregnancy, she should start getting moderate exercise every day
  • Check any and all medications with vet prior to administering
  • No insecticides (i.e., flea treatments)
  • No live vaccines
  • Put together pedigree on litter
  • Write contract
  • Contact AKC for litter registration application

Week Two (Days 8-14)


  • Embryo will be 4 cell at start of week and 64 cell by end of week
  • Embryo enters the uterus
  • Possible morning sickness
  • Continue as with Week One
  • Nothing special this week

Week Three (Days 15-21)

  • Day 19 -- Implantation of embryos in uterus
  • See above
  • See above
  • Nothing special this week

Week Four (Days 22-28)

  • Development of eyes and spinal cords
  • Faces take shape
  • Fetuses grow from 5-10 mm to 14-15 mm
  • Organogenesis begins-- Embryos are at their most susceptible to defects
  • Days 26 - 32 are the best days to palpitate (i.e.. feel for the puppies)
  • Possible clear vaginal discharge
  • Mammary development begins
  • After Day 26, palpitation may be possible to diagnose pregnancy
  • Limit strenuous activity (such as working, jumping, long runs)
  • Schedule ultrasound or palpitation with vet if desired

Week Five (Days 29-35)

  • Development of toes, whisker buds, and claws
  • Fetuses look like dogs
  • Gender can be determined
  • Eyes (previously open) now close
  • Fetuses grow from 18 mm - 30 mm
  • Organogenesis ends -- embryos are now fairly resistant to interference with development
  • Swelling becomes noticeable
  • Loss of "tuck-up"
  • Weight will start to increase
  • Slightly increase amount of food and switch to puppy kibble. If you feed one meal a day, add a small extra meal. If you feed twice a day, slightly increase one of the meals.
  • Add supplements, a good multi vitamin and I use Raspberry tea leaves (in health food section of stores), it helps uterin muscles strengthen.
  • Palpitation no longer possible due to fluids in uterus
  • Nothing special this week

Week Six (Days 36-42)

  • Development of skin pigment
  • Fetuses should weigh around 6 grams and be 45 mm long
  • Fetal heartbeats can be heard with stethoscope
  • Nipples darken and enlarge
  •  Continues to enlarge
  • The same support
  • Make sure all your whelping supplies are in place
  • By this time you should be fairly sure that the bitch is pregnant.

Week Seven (Days 43-49)

  • Growth and development continues
  • Abdomen hair will start shedding
  • The bitch will start to look pregnant at this point
  • Slightly increase meals may have to spread them out into several smaller meals if she is large.
  • Do nopt let her jump on furniture, light exercise
  • Radiographs (X-rays) possible to determine number and size of puppies

Week Eight (Days 50-57)

  • Fetal movement can be detected when bitch is at rest
  • Puppies can safely be born from now on
  • Milk may be squeezed from nipples, but don't do it. 
  • The bitch will be very large.
  • Not much at this point 
  • Gather whelping equiptment
  • Prepare phone list for help/support. It should include your vet's phone number, the emergency clinic's phone number, the number of any friends who will be offering support during whelping, and anyone else you might need to contact before, during, or after whelping (like your office to let them know you won't be in!)
  • Make sure your car is gassed up and ready for a possible emergency trip to the vet's office.

Week Nine (Days 58-65)

  • Growth and Development continues
  • Nesting behavior may be seen
  • Bitch may become distressed (panting, pacing, acting uncomfortable)
  • Start taking rectal temperature. Temp should be around 100.2-100.8 degrees Farenheit
  • When temperature drops to around 98-97 degrees Farenheit, puppies should be born within 24 hours
  • Appetite may disappear as whelping approaches
  • Start taking temperature three times a day
  • Do not leave her out of your sight 
  • Notify vet or emergency clinic when temperature drops so that they will be ready if you have any problems
  • Keep detailed records on temperature and behavior of bitch
  • Double check that whelping supplies are ready

Post Partum

  • Make sure each puppy gets some of the bitch's colostrum (first thin milk) within first 24 hours. Colostrum is loaded with antibodies to protect the puppies from disease. It also helps to condition their tummies to accepting food.
  • Lochia (vaginal discharge) should be reddish to reddish-brown (green is okay on first day). If you see black discharge, contact your vet immediately!
  • Within 5-6 hours of last puppy's birth, take bitch and puppies to vet for check up. The vet will ensure that the bitch hasn't retained any puppies or placentas and that the puppies are in good health. Ask your vet to check the puppies for cleft palates and what options are available. This vet visit is something that should not be skipped. If the bitch has retained a placenta or puppy, she could quickly develop a serious infection which could prove deadly.
  • Discharge cold be black to green and placenta's left behind will pass. AFter all placenta mess is gone the discharge whould be red.  If you see a  tan to light brown discharge you may be dealing with an infection.  It is also very foul smelling.  Blood can discharge up to 10 weeks post whelp.