in Plasma or Serum
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.
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
wasnt 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!!!!
previous article appeared in the Jan/March 1997 Mastiff Reporter Volume
V, No. 1-2. Here is the editors note:
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 timea 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.).
Summaries of Presentations at the
First International Symposium on Canine and Feline Reproduction, July 1,
PHYSIOLOGY AND TIMING OF OVULATION AND FERTILIZATION IN DOGS - T.TTSUTUI NIPPON VETERINARY COLLEGE TOKYO JAPAN
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.
DETECTION AND TIMING OF INSEMINATION USED ON HORMONE CONCENTATIONS, VAGINAL
CYTOLOGY, AND THE ENDOSCOPIC APPEARANCE OF THE VAGINAIN DOMESTIC BITCHES.
I.A. JEFFCOATE AND F.E.F.LINDSAY
UNIVERSITY OF GLASGOW, UNITED KINGDOM
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.
INFLUENCING THE SUCCESS RATE OF ARTIFICIAL
INSEMINATION WITH FROZEN SEMEN IN THE DOG.
W. FARSTAD AND K. ANDERSEN BERG
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.
IN DOGS IN RELATION TO SEMEN QUALITY AND THE TIME
AND SITE OF INSEMINATION WITH FRESH AND FROZEN SEMEN
C.LINDE-FORSBERG AND M.FORSBERG,
SWEDISH UNIVERSITY OF AGRICULTURAL SCIENCES,
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 studys 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 shes not reproductively
sound.- Carols 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
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
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!
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.
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.
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
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 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.
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.
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
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