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Luteinizing Hormone Test
For the Detection of Serum Luteinizing Hormone
The 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 is also used to distinguish ovariectomized from 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
Determining 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.
Ovulation 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. Note: The WITNESS® LH test and test interpretation is referenced against the OVUCHECK® Premate®progesterone assay. Use of other progesterone tests may require different interpretations to explain test results.
II. TEST PRINCIPLES
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 testing
technique. The WITNESS® LH
luteinizing hormone assay allows identification 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
III. 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
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 fluctuation.
IV. WHEN TO CONDUCT INSEMINATIONS IN THE BITCH
The positive 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.
Properly planned breedings or inseminations coinciding with this window
of fertility will optimize the probability of success.
Natural 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
Natural 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 post-LH surge.
Surgical 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.
Chilled Extended Semen Breeding
Chilled extended semen will usually live for 2 to 4 days
after 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 be performed on days 5 or 6
Frozen Semen Breeding
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.
V. 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 or queen is sexually intact.
Because brief (20 to 60 minutes) episodic surges in serum LH
concentrations may occur in sexually intact bitches or queens, 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. If estrus is suspected and a positive result was obtained, repeat the test in 24 hours with a fresh sample.
VI. SAMPLE INFORMATION
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 refreeze.
VII. STORAGE AND STABILITY
Store kit at 2° to 25°C or 35° to 77°F. 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 stored properly.
WITNESS® LH Test Procedure
NOTE: Only use SERUM as a sample.
Prior to use, allow SAMPLE to come to room temperature (20° to 25°C; 70° to 77° F)
SAMPLE COLLECTION AND PREPARATION
- Collect the blood sample into a plain (red top) vacutainer or serum separator tube.
- Allow to clot and centrifuge sample.
- 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 serum sample will not be tested immediately, store in refrigerator for up to 24 hours or freeze for longer storage. REMEMBER: Sample must be at room temperature prior to testing.
The WITNESS® LH
Test includes a pipet and a test device. The device has a Serum Well
“1” and a window with a Test Area “2” and a Control Area “3”.
1. Remove test device from foil pouch by tearing at the notch. Place the device on a flat level surface at room temperature.
2. 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 “1”.
3. Allow the test device is sit undisturbed for 20 minutes.
4. Read results at 20 minutes.
Reading results prior to or beyond the 20 minute test interval will invalidate results.
At the end of the test, a pink line should always appear in the area marked “3”. This line assures that the test is complete.
FOR OVULATION TIMING
If no line appears in the area marked “2”, or if the
line is less intense than the control line “3”, the LH value is less
than 1 nanogram per milliliter.
Continue LH testing on a daily basis to determine the optimal time to breed.
If a line appears at the area marked “2” which is of
similar or greater intensity than the control line “3”, 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 progesterone.
FOR DISTINGUISHING OVARIECTOMIZED BITCHES/QUEENS
If no line appears in the area marked “2”, or if the
line is less intense than the control line “3”, this indicates that the
bitch or queen is intact.
A line appears at the area marked “2” which is of
similar or greater intensity than the control line “3”. This 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. However, if estrus is suspected, repeat the test in
24 hours with a fresh sample.
COMMONLY ASKED QUESTIONS REGARDING OVULATION TIMING
Q: How do I use WITNESS® LH with OVUCHECK® Premate®?
A: Utilizing both LH and
progesterone assays improves ovulation timing accuracy. Perform 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, 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.
Q: Why do I need a “confirming” progesterone test after a positive WITNESS® LH test result?
A: Due to the pulsatile nature of LH activity, 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.
Q: When should I start LH testing?
A: 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, the LH
surge may have been missed. Remember to confirm a baseline progesterone
level on the first day of testing.
Q: How long will I have to test?
A: 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 of testing.
Q: What if I miss a day of testing?
A: The 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 an LH test day is
missed, continue to test but supplement LH testing with progesterone
assays in the event the LH peak occurred on the missed day.
Perform a progesterone assay if it is suspected that the
LH surge was missed. If progesterone is low (below 2 ng/ml), continue
testing. If progesterone is high (above 2 ng/ml), call Synbiotics for
GOOD TECHNIQUES = ACCURATE RESULTS!
Lipemic or grossly hemolyzed 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.
If samples cannot be tested the same day, refrigerate for up to 24 hours or freeze 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.
FOR TECHNICAL ASSISTANCE CALL 1-800-228-4305
Corporate Headquarters: 12200 N. Ambassador Drive, Suite 101, Kansas City MO 64163
Manufacturing and Distribution: 16420 Via Esprillo, San Diego, CA 92127
2, rue Alexander Fleming, 69367 LYON Cedex 07 - France
+33 4 72 76 11 11
WITNESS® LH is a registered trademark of Synbiotics Corporation.
©2007 Synbiotics Corporation
NAC No.: 11150560