Feline Luteinizing Hormone Test
For the Detection of Serum
Luteinizing HormoneThe Witness LH Test provides an accurate,
semi-quantitative measurement of canine and feline luteinizing hormone (LH).
The test is simple to perform and rapidly provides information for the
veterinary clinician. This assay, when used in conjunction with in-hospital
progesterone testing, identifies the pre-ovulatory LH surge, and thus, the
time of ovulation. Witness LH
may also be used to distinguish between ovariectomized and sexually intact
bitches or queens.
Identification of the LH surge
provides the most accurate means of canine ovulation timing. It may be used
for routine breeding situations and is especially recommended in those
instances where there are factors present that could adversely affect
conception rates. These include: chilled/extended semen breedings, frozen
semen breedings, bitches with a history of infertility, breedings with stud
dogs with low semen quality, limited access to the stud dog.
Identification of bitches and
queens that have had their ovaries removed is possible through the detection
of luteinizing hormone. Serum concentrations of LH increase after
gonadectomy in dogs and cats. Because LH concentrations in serum are < 1ng/mL
in sexually intact bitches and queens, except during the LH peak at estrus,
it may be assumed that high values in bitches and queens not in estrus are
indicative that the ovaries have been removed.
Timing in the Bitch
Since the LH surge may occur
within a 24-hour period, it is crucial that daily serum samples are tested.
Blood should be drawn every day at approximately the same time beginning the
4th or 5th day of proestrus, or when vaginal cytology approaches 50%
cornification (Cornification is defined as cells with a roughened, angulated
border with or without a nucleus). A progesterone test should also be
performed on the first day of testing to confirm a baseline progesterone
level. If testing is started after the onset of estrus, it is likely that
the LH surge will have already occurred and cannot be identified in
Once an increase in LH is
identified, Synbiotics recommends that daily serum samples should be tested
for progesterone levels. If the detected increase In LH was indeed the
actual pre-ovulatory surge, progesterone levels should rise, usually
reaching a value above 2 ng/ml within 3 days, and then stay elevated. If
progesterone remains low, it indicates that a proestrus fluctuation in LH
was identified. Continue daily LH testing until the true LH surge occurs.
test and test interpretation is referenced against the OVUCHECK
Premate. Use of
other progesterone tests may require different interpretations to explain
is a luteinizing hormone assay that provides a convenient semi-quantitative
measurement of luteinizing hormone levels without special equipment. The
test is an immunochromatographic assay that uses gold-conjugated antibodies
to give a visual line in the presence of luteinizing hormone.
One test device is used for
each serum sample to be tested. A control line is included on each test
device. Color development of the control line is indicative of proper
The Witness LH luteinizing hormone assay
allows you to identify the occurrence of the LH surge. A positive result
occurs when a line appears in the test area which is of similar or greater
intensity than the control line. When this occurs, the LH level in the serum
sample is greater than 1 nanogram per milliliter.
REPRODUCTIVE PHYSIOLOGY IN THE BITCH
Unsuccessful breedings result
more often from improper ovulation timing than any other cause. While the
estrous cycle of the bitch typically lasts for several weeks, the true
fertile period is short (48-72 hours) and is difficult to identify without
the use of hormonal assays.
The common indicators of
estrus and breeding time such as vaginal cytology and receptive behavior,
are primarily controlled by changes in the hormone estrogen. Unfortunately,
these changes are only an approximation of ovulation. Using only these
parameters, ovulation could be missed by more than a week. Precise
identification of the fertile period is made by measurement of the LH surge
which actually triggers ovulation. This surge, which consists of a rapid
increase in LH level, occurs in many cases within a 24 hour period.
Ovulation occurs 2 days after the LH surge; the oocytes then require an
additional 2-3 days to mature, and will live for about 48-72 hours. Thus,
the fertile period of the bitch falls between days 4-7 after the LH surge
with the most fertile days being on days 5 and 6 post-LH surge.
Progesterone assays are useful
for ovulation timing. Before the LH surge, serum progesterone remains low,
generally between 0 and 1.0 ng/ml. At about the time of the LH surge,
progesterone levels will begin to rise, usually changing from a baseline of
0-1.0 ng/ml into a range of 1.5-2.0 ng/ml. Progesterone will then continue
to rise as the cycle progresses and will remain elevated for 2-3 months in
non-pregnant as well as pregnant bitches. It is important to appreciate that
the absolute progesterone values discussed above may vary by individual. The
key event progesterone assays seek to identify is the initial rise of
progesterone above the particular individual's baseline level. Once
identified, this initial rise in progesterone may be used as an estimate of
the LH surge. However, this first rise in progesterone may vary from the day
of the actual LH surge in many bitches. While progesterone is elevated
during the fertile period, the rate of rise varies from bitch to bitch, as
does the absolute value of progesterone which coincides with the LH surge or
with the optimal time to breed. As a result, progesterone measurements alone
are, at best, only an approximation of the LH surge. Given the fact that
good breeding management often plans for multiple breedings over a period of
several days relying on the information provided by progesterone assays
alone may be sufficient. Some breedings, however, will benefit from a more
exact identification of the bitch's fertile period. Identification of the LH
surge itself allows the most precise ovulation timing.
During proestrus, small,
pulsatile fluctuations in LH may occur, while progesterone remains at low,
baseline levels. Progesterone rises, however, after the pre-ovulatory surge
in LH. By the third day post LH peak, the majority of bitches will evidence
a rise in progesterone levels above the 2 ng/ml level. Daily progesterone
testing after a positive Witness LH
test result allows simple differentiation between small fluctuating
increases in LH during proestrus and the true, pre-ovulatory LH surge. if
there is no confirmation of progesterone rise within 3 days of a positive
Witness LH test, it
may be assumed that the positive test result was due to a baseline LH
IV. WHEN TO
CONDUCT INSEMINATIONS IN THE BITCH
Witness LH test identifies the day of
the LH surge. This day is designated as day 0. Count forward to determine
the fertile period. Days 4-7 post-LH surge encompass the true fertile
period, with peak fertility on days 5 and 6. By properly planned breedings
or inseminations coinciding with this window of fertility, you will optimize
the probability of success.
Breeding or Fresh Artificial Insemination
Fresh semen of a normal
healthy stud may live up to 5 or more days in the bitch. Therefore, semen
inseminated a day or two before the fertile period should be viable at the
time of peak fertility. Synbiotics recommends breeding on days 2, 4 and 6
post LH surge to completely cover the fertile period, and to maximize viable
sperm numbers on the bitch's most fertile days. If only two breedings are
being performed, they should be accomplished between days 3 and 6 post-LH
Breeding with Compromised Semen Quality
At times, stud dogs produce
semen of compromised quality due to age, stress or disease. Breeding during
the true fertile period will increase the likelihood of limited sperm
numbers encountering mature eggs and will increase the chance of conception.
Synbiotics recommends breeding on days 4, 5, 6, and 7,
Insemination with Fresh Semen
Occasionally, it is desirable
to perform surgical insemination with fresh semen due to low fertility of
either the stud dog or the bitch. Synbiotics recommends that this procedure
be performed on either days 5 or 6 past-LH surge.
Extended Semen Breeding
Chilled extended semen will
usually live for 2 to 4 days post collection. One day of this time is
typically used in shipping. This reduced survival time requires the breeding
to be conducted closer to the short fertile period of the bitch. Synbiotics
recommends that 2 inseminations be conducted on days 4 and 6 or 3 and 5
post-LH surge. If a surgical insemination is desired, it should he performed
on days 5 or 6 post-LH surge.
Frozen semen survives less
than 24 hours after thawing, so timing is crucial. Inseminations must be
performed during the true fertile period. If one surgical insemination is
planned, it should be performed on day 5 or 6 post-LH surge. If multiple
vaginal or trans-cervical inseminations are to be conducted, they are
recommended on days 4, 5 and 6.
DISTINGUISHING BETWEEN OVARIECTOMIZED AND SEXUALLY INTACT BITCHES OR QUEENS
A single low LH test result
(i.e., lighter than the control line) confirms that the bitch is sexually
intact. Because brief (20 to 60 minutes) episodic surges in serum LH
concentrations may occur in sexually intact bitches, any positive test (i.e.
test line equal to or darker than the control line) should be repeated in 2
hours with a new sample.
requires only three drops of serum to run each test. Collect the blood
sample in a plain (red top) vacutainer or serum separator tube. Allow the
blood to clot and separate the serum by centrifugation. The sample should
not be hemolyzed or lipemic. Testing should be performed the same day as
sample collection. If this is not possible, refrigerate the serum for up to
24 hours. Serum may be frozen for prolonged storage. Do not thaw and
Store kit at room temperature.
Do Not Freeze. The foil pouch containing the test device and pipette should
not be opened prior to running the test. Witness LH
will remain stable until expiration date provided that the kit has been
Only use SERUM as a sample.
use, allow sample to come to room temperature (20° - 25° C; 68° - 77° F)
COLLECTION AND PREPARATION
the blood sample into a plain (red top) vacutainer or serum separator tube.
Allow to clot and spin the cells down. Transfer serum to a clean glass or
polypropylene tube. Serum must be free of red cells, clots and visible
debris. DO NOT USE SEVERELY HEMOLYZED OR GROSSLY LIPEMIC SAMPLES. When in
doubt, collect a better quality sample. If sample will not be tested
immediately, store in refrigerator for up to 24 hours. Sample may be frozen
for longer storage.
REMEMBER: SAMPLE MUST BE AT
ROOM TEMPERATURE PRIOR TO TESTING.
LH test includes a pipet and a
test device. The device has a serum well (S) and a window with a control
area (C) and a test area (T).
Remove test device and pipet from foil pouch by tearing at the notch.
Place the device on a flat level surface at room temperature.
Draw some serum up into the provided pipet. Hold the pipet perpendicular
to the device and add 3 drops of serum to the Serum Well (S).
Allow the test device is sit undisturbed for 20 minutes.
INTERPRETATION OF RESULTS
the end of the test, a pink line should always appear in the area marked
C. This is your assurance that the test is complete.
If no line appears in the
area marked T, or if the line is less intense than the control line C,
the LH value is less than 1 nanogram per milliliter.
If you are trying to
determine the best time to breed, continue LH testing on a daily basis.
A negative result confirms
that the bitch or queen is intact.
If a line appears at the
area marked T which is of similar or greater intensity than the control
line C, the LH value is greater than 1 nanogram per milliliter.
For ovulation timing
purposes, in the bitch, the first time a positive result is observed is
the day of the LH peak. This day is counted as Day 0. Follow the
instructions under Section IV. WHEN TO CONDUCT
INSEMINATIONS. Note: It is recommended that you
perform a progesterone test in 3 to 4 days to confirm a rise in
A positive result in a
bitch or queen that is not in estrus indicates that the ovaries have
been removed. Repeat the test in 2 hours with a fresh sample to confirm
the results. A negative test result provides excellent evidence that the
bitch is intact.
COMMONLY ASKED QUESTIONS REGARDING OVULATION TIMING
Q How do I use
Utilizing both LH and progesterone assays improves ovulation timing
accuracy. You should run an initial progesterone test to establish a
baseline level during the first 5 days of the bitch's season. When
vaginal cytology indicates 50 percent cornification, you should begin
daily Witness LH
testing. When LH testing indicates a positive result, make preparations
to breed. Synbiotics recommends an additional progesterone test be run
72 hours post-LH surge to confirm a rise in progesterone.
Why do I need a confirming progesterone test after a positive
to the pulsatile nature of LH activity in the bitch, there is a small
chance that a positive Witness LH test result can be obtained due to identification of a baseline
fluctuation rather than the true LH surge. Confirming a rise in
progesterone before actually performing the insemination will easily
identify those few instances where a positive Witness
LH test result is due to
identification of LH baseline fluctuation.
When should I start LH testing?
Blood should be drawn the fourth or fifth day of proestrus or when
vaginal cytology approaches 50 percent cornification. Sampling should be
performed daily until the LH surge is detected. If sampling begins after
the onset of estrus, you may have missed the LH surge. Remember to
confirm a baseline progesterone level on the first day of testing.
Q How long will I have
Testing on 200 dogs has shown that, with daily testing beginning as
recommended, the LH surge will be detected in most dogs within 6 days.
There are six tests to a kit. Because the bitch may require additional
testing, you may wish to have another kit on hand to avoid missing a day
Q What if I miss a day
duration of the LH peak varies from dog to dog and lasts only 1 day in
approximately 40 percent of bitches. Samples should be
drawn and tested on a daily basis. Failure to do so may result in
improper timing. If you miss a day, you may continue to test, but you
should supplement your testing with progesterone assays in the event the
LH peak occurred on the day you did not test.
If at any point during
testing you suspect that you missed detection of the LH surge, a
progesterone assay should be performed. If progesterone is low (below 2
ng/ml), continue testing. If progesterone is high (above 2 ng/ml), call
Synbiotics for technical assistance.
TECHNIQUES = ACCURATE RESULTS
Do not use lipemic or
grossly hemolyzed samples. Such samples may interfere with the rate at
which the sample flows in the device or create a background color which
will make interpretation of the results difficult.
Store the kit at room
If samples cannot be
tested the same day, refrigerate for up to 24 hours. Samples may be
frozen for longer storage. Do not thaw and refreeze.
Do not use the kit beyond
its expiration date.
Do not open the foil pouch
until ready to perform the test.
Do not touch any of the
surfaces in the windows of the device.
Keep device flat during
testing. Do not disturb while test is being conducted.