Measurement of Progesterone
in Plasma or Serum

Canine Cryobank uses an immunoenzymatic test which provides a reliable and precise masurement of progesterone in plasma. The test is based on the competitive binding of unlabelled progesterone present in the standard or sample, and a fixed quantity of progesterone labelled with the enzyme alkaline phosphatase (AP), to binding sites on a limited amount of specific progesterone antibodies.

The wells are pre-coated with antibodies, providing a solid phase for the convenient separation of the bound progesterone from the free progesterone in the sample. After incubation, all components other than those bound to the plate wells are washed away.

The amount of bound AP-labelled progesterone remaining on the wells is inversely propiortional to the concentration of the unlabelled progesterone present in the sample. The bound labelled progesterone is then measured by reacting the AP with its substrate during a second incubation.

The color produced is measured spectrophoto-metrically and the concentration of progesterone in the sample is determined from a standard curve.


So, if you have heard how wonderful progesterone testing is for timing the breeding, that is right!!! Progesterone testing is much better than doing a vaginal smear. But, be aware that the sampling techniques and progesterone test kits differ dramatically. Clients tell me they had progesterone tests done every day for five days and still missed the breeding last season.

Recently, clients tell me they had the LH measured AND progesterone tested and still missed the bitch. When questioned, many clients say they knew the bitch wasn’t ready because she showed no signs of standing, vulvular tilting, flagging, etc., but the progesterone test result said to breed, so they did. In the hundreds of bitches I have personally bred, I cannot remember a bitch that did not stand when ready, although some of my Shutzhund Rottweiler clients may growl protests. I hear stories of breeders tranquilizing bitches in order to breed them. I believe the reason we have never had to tranquilize any of the hundreds of bitches bred is simple: Do not try to breed until a reliable progesterone test shows she is ready!!!!

Note: The previous article appeared in the Jan/March 1997 Mastiff Reporter Volume V, No. 1-2. Here is the editor’s note:

Southport Mastiffs

Those of you who have been subscribers for a while may remember another article by Carol in Vol 1/5 on using Frozen/Cooled Semen. At that time we were using the progesterone method/kit indicated above on one of our bitches and conception did not occur. Our thoughts at the time were that the bitch may have stalled out and was inseminated too early. On subsequent season, the testing was performed again, using a second test to verify the rise in ng/ml and the stall was noted. This bitch took longer than normal to rise to the appropriate level, but the additional test showed that and she was bred at the appropriate time—a litter of 13 puppies resulted. Another bitch was also bred using this method, her rise was extremely fast by comparison, but again the progesterone testing was able to pinpoint the correct breeding date and a litter of 12 puppies resulted. Both were cooled semen shipments from out of state and inseminated by Carol using the intracervical A.I. method.).

Capsule Summaries of Presentations at the
First International Symposium on Canine and Feline Reproduction, July 1, 2 1988

In most bitches, ovulation occurs between 48-60 and 72 hours from onset of oestus. Ovulated eggs are not capable of being fertilized until 60 hours after ovulation. Eggs are receptive then for 48 hrs. Sperm is thought to be viable for 5 days in the reproduction tRact.


These researchers found that "neither vaginal cytology nor standing estrus are reliable indicators of the fertilization period per se.Although with natural mating or when using AI with fresh semen there may be enough biological leeway for pregnancy to follow insemination timed using these parameters alone. For the better timing reqired by frozen semen, the authors used vaginal scoyto detect the onset of shrinkage with angulation of the mucosal folds, an indication of the fertilization period. Serum progesterone levels of 6-ng/ml refers to the beginning fertilization period.


"Correct timing of the inseminations is therefore essential to obtain pregnancy with frozen semen." Thirty six bitches were inseminated with frozen semen from various sources: Twenty eight were inseminated with imported semen. Mean progressive post-thaw motility of semen was 60%. Overall conception rate was 67% with a mean litter size of 6.4. One insemination had a slightly lower conception rate than two inseminations. In a series of inseminations of 10 Labradors, bitches with imported semen of excellent quality – 80% motility post thaw, a conception rate of 80% with a mean litter size of 7.0 pups was obtained. Semen was deposited intrauterine with a specially designed catheter. Authors recommend inseminations with thawed semen at progesterone levels of 15-20 ng/ml.


All dogs in this study were privately owned. Inseminations were done even if the semen was poor quality and the bitches had been treated for fertility problems. (While these facts may negatively impact the study’s overall results, it is very much what may be expected in the frozen semen industry today because owners want to use a certain dog even though his semen may not be excellent and a certain bitch even if she’s not reproductively sound.- Carol’s note.) 470 bitches were inseminated; 405 with fresh semen into cranial vagina and 65 with frozen semen intrauterine. Pregnancy rate was 65% with frozen semen intrauterine. Pregnancy rate was 65% with fresh and 41.5% with frozen. When corrected for stage of estrus at time of insemination and for semen quality the pregnancy rate was 83.8% with fresh and 69.3% with frozen. Pregnancy rate increased with number of inseminations. Inseminations with fresh semen before ovulation were often successful. With frozen semen the progesterone at time of insemination was over 30nmol/1 in all but 1 bitch that became pregnant. Pregnancies occurred with semen of poorer quality in fresh inseminations although the litter size was smaller. No pregnancies occurred with frozen semen of poor quality. Litter size was 21.5% smaller with fresh than natural matings. Litter size in frozen litters were 23.3% smaller than fresh semen. There was a tendency to have a higher pregnancy rate and litter size when bitches were on antibiotics before estrus or early in the pregnancy. There was a tendency for breed differences in pregnancy rates also.

Breeder Share Some Important Tips and Success Stories
Another SIX YEAR OLD BITCH is successfully pregnant. The Bichon is owned by Marty Astor and was bred on day 21!!! Marty was ecstatic and admits she was doubtful but since the bitch missed last seasons without our progesterone testing, is now 'A Believer'. Congratulations. (Now wasn't it worth all those trips up to see us, Dan?) REMEMBER, Never vaccinate an estrus bitch. Never worm an estrus bitch, Never flea dip an estrus bitch!!!!! Vaccinations and other systemic insults may interfer with ovulation. We discovered this phenomena years ago when we first started progesterone testing!!!!

Internationally known Siberian Husky breeder and judge, Miki Polemini had a progesterone done on her pregnant bitch on day 61, knowing she had a singleton litter and the weekend was approaching, Miki needed to plan as best she could for a possible c-section. The progesterone was 1 ng. at 1 p.m. and we could predict that the bitch would whelp within 24 hours. Miki called the next morning to report a wonderful bitch pup was born during the early morning hours. Congratulations Miki and Frank!

Debi Morrow arrived with her Briard bitch progesterone tested by her Arizona vet. We confirmed with our high tech test and the bitch was ready to breed three days and go home!!! This is much different from Debbi's last Briard bitch visit. Debi sent her mother over here with an untested bitch and her mother had to stay for almost a week before the bitch was ready to breed. So this trip was much faster and less expensive and less anxiety driven.

Gail also reports her bitch which she brought down to breed to Lisa Peltzer super stud (he had just sired 10 pups) is pregnant. Gail had to take some cooled semen home because the progesterone showed the bitch would be ready the day that Gail was expected back "on the job"  in Northern Cal.  So we did the best we could; bred her and then sent cooled semen with Gail to inseminate more timely.  Congratulations, Gail and Lisa.

The following scenario explains why a shipped brood bitch may stand, breed successfully, be shipped home, and still not have pups.

"Hi Carol and staff,
Spice is really getting big. She stood for the stud a week before her eggs descended. Ron drove clear to Virginia to breed her so that we didn't have to fly her. If we had not used progesterone testing we would have missed her! Ron was anxious to get going since he had been away a month, but stayed until the time was right! Yes, yes yes puppies are due June 6-9. I will keep you posted on puppies' arrival date, etc. This is her last litter. Thanks so much for your help. You guys are the greatest! So happy that we had a chance to get to know you. Spice really looks big. We'll keep you updated on her progress. Thanks a bunch, Linda Waggoner

Carol's note: This will make three successive litters with progesterone testing after failures without progesterone testing for owners Ron and Linda Waggoner and Spice, their champion Whippet bitch pictured on our progesterone page.

Progesterone story #2 of the week….
The lady on the phone sounded stressed as she explained that she was in San Diego from the Bay area to breed to Enzo, Rod Thompkins' famous German Shepherd Schutzhund competitor. Her Bay area vet had done a progesterone test Thursday and told her to breed in two days. The breeding never happened Saturday and the owners were certain the bitch had a circular stricture because Enzo could not penetrate. I asked what the progesterone test result was on Thursday and she said around 2ng. I replied that the bitch would be ready in six days from Thursday and to re-evaluate the stricture situation then. The lady was not at all happy with my explanation as she insisted her vet said to breed!! The lady called on Thursday to say the breeding went very easily the night before (six days after the 2ng/ml) and she now was asking about her other bitch who last season, never stood and had to be muzzled, force bred, and never conceived. What do you think the reason could be??? I hope we hear that her bitch conceived this time. Enzo's semen evaluations are usually in the two billion range, including one done just days after her bitch was bred. So Enzo did his part in the breeding equation.

German Shepherd breeder, Rhonda Long had failed to get one of her bitches bred successfully. The next season, she brought her in for a progesterone test thinking she was early. However, the progesterone test said BREED NOW. Nice litter resulted. She was bred too late the seasons before, so it seems. I think she had 8 pups.

Progesterone Story: Great Dane bitch missed last time. Carla Akamini's Great Dane bitch missed last time even though she was bred naturally several times during her season. This time two days after she stood and bred, Carla brought her in for a progesterone test. Her progesterone was only 2.5 ng/ml, two days after the breeding! If Carla had stopped breeding, the sperm would need to last SIX DAYS OR MORE! Carla bred her again when the progesterone was 9.9ng/ml. Carla reported bitch whelped pups last week! Please note, even this bitch with a history of missing her last season, only had three progesterone tests.

ALL PROGESTERONE TESTING IS NOT THE SAME!!!! "My vet told me the progesterone test said to breed now, so I called the stud owner back East, and she flew out here (Palm Desert) with the stud but he will not breed her. Did we miss her?!!" worried the anxious bitch owner. A progesterone test done in our lab showed the visiting stud and owner would need to stay at least 5-6 days before the bitch is ready to breed!!! (True story) Bitch, by the way, had a nice litter. And even more remarkable the two owners are still good friends after the extended "vacation" of the stud owner and dog. If you would like more information fax or call us for the information sheet on ‘Progesterone Testing for Breeders’.