Breeders Need to Know: All Progesterone Testing Is Not Reliable

"Three days! I have to stay here three more days?" exclaimed the unbelieving Great Dane breeder who had driven non-stop from Washington state to San Diego with her in-season bitch. Based on a "reproduction veterinary expert's" interpretation of a progesterone test, the breeder made a frantic 12 hour auto trip to the stud dog only to have the bitch ferociously try to take the stud's face off.

Breeding dogs is a serious commitment by conscientious dog owners. Normal life is suspended when a bitch comes into season. Vacation plans are put on hold, excuses are made to bosses, weddings and anniversary parties are changed, and even elective surgery is rescheduled. Such is the importance placed on breeding the bitch. So it is never a surprise when I get emergency calls from breeders.

The Great Dane stud owner has changed weekend show travel plans to accommodate this bitch when the Washington veterinarian said the bitch was ready NOW. Yet, the progesterone test I performed showed the bitch would not be ready for 60 hours or more. The same bitch, 58 hours later, standing steady, bred without any fuss. A fine litter resulted.

Unfortunately, this scenario is not infrequent at our Canine Cryobank laboratory now or in years past when vaginal smears were the only veterinary hint at timing the breeding. Years ago in our West Los Angeles office we were called in at 12 midnight because a veterinarian in Davis, California had done one vaginal smear that morning and insisted the bitch be bred immediately. The bitch was flown down by private jet and the stud greeted her happily but did not show any interest in breeding. Another vaginal smear showed the bitch had 100% intermediate cells, but no superficial (cornified) cells. One week later she, too, bred naturally.

Interpretation of vaginal smears relies on the technical staff's experience in properly preparing the specimens and most importantly, reading hundreds of vaginal smear series of bitches' seasons. Over and over in lectures to veterinarians and breeders, we emphasize that reading ONE smear is useless to time the breeding. When progesterone testing became available it was hoped that by removing the need for extensive experimental interpretive skill, the breeders would be better served. However, progesterone testing requires careful attention to laboratory techniques and awareness of current test kits' reliability.

Progesterone is a hormone measurable in the blood of the estrus bitch. Progesterone rises after the lutenizing hormone peaks and continues to rise whether the bitch is bred or not, pregnant or not for the next 60 days or so. Therefore, it is a wonderful tool for the breeder to determine breeding dates. Bitches are best bred when the progesterone is 9.5 through 18 ng/ml by our test. No more than three tests are usually needed for a normally cycling bitch.

Occasionally veterinary laboratories will offer progesterone testing by radio immune assay and the results will have a quantitative value, i.e. a numerical value. Several marketing companies have put progesterone kits on the market for in-house use; some are no longer made, some are marketed under different names. The kits use monocional antibody techniques to give a range as a result, i.e. below baseline; between 2 ng/ml to 5 ng/ml; over 7 ng/ml, for example. It was one of these $100 in-house kits that was responsible for the Great Dane's Washington to California trek. Only one in-house kit tested in our laboratory was reliable. This imported kit is used by Greyhound stud farms with incredibly reliable results for natural, cooled and frozen litters. (Camelot Farms, College Station, Texas).

Our laboratory uses an Enzyme Immune Assay kit of analytical laboratory quality, imported from France. The kit requires a spectrophotometer, laboratory quality pipettors, and printer for an investment of $10,000.

Reliability of the test results relies on proper sampling techniques and proper attention to test kit preparation. The kits must be at room temperature. The kits have a definite shelf life, which must be carefully monitored. The timed reading of results is very, very important. If the kit instructions say the results should be read in five minutes, any results later than that are suspicious.

In our laboratory, every effort is made to make the testing reliable. The kits are taken out of refrigeration at the same time every morning. The pipettors are quantified every morning. A time is used on every phase. The same amount of blood is always drawn in the same size syringe with the same amount of heparin used to rinse the syringe and needle. The blood is centrifuged immediately and clear plasma used for the test. Over the eight years and thousands of progesterone tests performed, the EIA is remarkably reliable. The recommendations we made to breeders are based on the experience with the testing method we use. Years ago, a researcher from Cornell announced that bitches ovulate at 4.77 ng/ml progesterone. Using that published data, we had terrible conception rates. Later, it was noted that the ovulation value was only a statistical determination. For our clients, pups are the only "statistical data" they desire. The kit we use was developed and tested on breeding canines, an important background fact when it is noted that several in-house progesterone kits were actually produced for the equine market.

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 sings 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!!

(Those of you who have been subscribers for a while may remember another article by Carol in Volume I/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 a 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 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 cervical A.I. method).