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Endocrine Chemical Pathology (Endocrinopathies)
Principles of Laboratory Testing for Hormone Anomalies When testing for any hormone deficiency in human medicine the gold-standard
method is to make use of a provocative or stimulation test. The theory being that
administration of an exogenous substance should induce a significant elevation in the
hormone been evaluated in a normal subject; whereas in a patient suffering from the
deficiency in question, the hormone elevation will be minimal or absent. And when
testing for a hormonal excess, a suppression test is the gold-standard method. The
principle of the test being that administration of an exogenous substance should
suppress the levels of the hormone in question to low levels or by a significant
proportion. When the patient is suffering from the hormone excess the administered
substance fails to suppress the hormone sufficiently or at all.
In veterinary medicine these principles are not always followed. In fact the ACTH-
stimulation test is commonly used to confirm the diagnosis of hyperadrenocorticism
in dogs. Although this is in contradiction to human medicine there is overwhelming
support for the use of this test in this context in the published literature. What is
vitally important is that such stimulation or suppression tests are not used in isolation;
in other words there must be strong clinical suspicion of the disease in question
before these tests are utilised. Using one of these tests without appropriate clinical
signs or when concurrent non-endocrine disease is present can result in misleading
results. It is important to note that the hormone normal ranges and diagnostic cut-off
values given below will differ slightly between laboratories and it is critical that the
tests are used only in cases with appropriate clinical presentation and that non-
endocrine factors that could influence the results are considered in the interpretation.
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Thyroid Function
Hypothyroidism in Dogs
Hypothyroidism denotes a deficiency of the thyroid hormones: thyroxine and tri-
iodothyronine. 95% of cases are considered primary, where there is impaired
production ofT4 or T3. Secondary or tertiary hypothyroidism involving depressed
thyroid hormone production due to hypothalamic or pituitary disease account for <5%
of cases. Commonly associated biochemical parameters include: non-regenerative
anaemia; hypercholesterolaemia; increased triglycerides; raised CK, ALT, ALP and
proteinuria. Beware of the ‘Euthyroid Sick Syndrome', non-thyroid factors falsely
suppressing serum values including hypoadrenocortism, hyperadrenocortism, chronic
renal failure, diabetes mellitus, hepatic disease and any chronic systemic illness.
Concurent drug administration, especially corticosteroids, can affect serum T4 and T3
values. For these reasons Free T4 and Canine TSH are the preferred assays as they are
unaffected by these factors.
FREE T4 DETERMINATIONS
Analogue assays for free T4 have been superseded by a modified equilibrium dialysis
method which is done on the dialysate which is free of autoantibodies, drugs and
proteins. Older analogue RIA assays are prone to giving false positive, low freeT4
values.
Protocol:
Free T4 by modified equilibrium dialysis sample collection: send
1ml of serum (not plasma). No cooling needed. Recommend
centrifuging and separating serum from blood. If samples are going
to be delayed in transport to the laboratory then freezing and
transport on ice are recommended.
Interpretation:
Assays of basal T3/T4 lack sufficient sensitivity and specificity to be reliable
indicators of hypothyroidism, and numerous factors other than hypothyroidism
can depress their values. A low freeT4 in the presence of strong clinical
evidence is highly suspicious for hypothyroidism.
CONDITION FREE T4
Euthyroid 10-45 pmol\l
Hypothyroid <10 pmol\l
CANINE TSH ASSAY Currently two validated canine TSH assays are commercially available. These assays
are only useful in conjunction with freeT4 and or a complete thyroid profile including
totalT4, thyroid autoantibody screen and totalT3 assays. The canine TSH is of no use
on its own.
Protocol:
A fasted serum sample is collected for RIA for endogenous TSH
(1.0ml). The analyte is stable and no special shipping requirements are
needed.
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Interpretation:
In primary hypothyroidism TSH is greater than 0.32 ng/ml. In normal dogs and also in
dogs with non-thyroidal disease the normal range can extend to 0.6 ng/ml. This
overlap can create difficulties in diagnosis and therefore clinical signs, history and
freeT4 are critical in making a diagnosis of primary hypothyroidism. Secondary and
tertiary hypothyroidism are rare but technically should have a depressed TSH less
then 0.03 ng/ml but the assays lack the sensitivity to detect the bottom end of the
normal range.
A raised TSH, indicating hypothyroidism, often precedes a drop in free T4 to
abnormally low levels and both tests should be repeated if clinical signs worsen.
Therefore, a low normal free T4 does not exclude hypothyroidism.
TOTAL T4 AND T3 ASSAYS
Total serum thyroxine is often used as an initial screening for canine hypothyroidism
when clinical symptoms are vague or not classic for hypothyroidism.
Protocol:
A fasted serum sample is collected for RIA or ELISA for serum
thyroxine. The analyte is stable and no special shipping requirements
are needed.
Interpretation:
Normal dogs have a range of totalT4 of 10 – 45 nmol/l. Non-
thyroidal illness and some drugs can suppress totalT4 to low-normal and even below
normal levels. Values greater than 25 nmol/l are highly unlikely to be associated with
hypothyroidism
.
* The clinician must correlate these results to the clinical signs, physical findings, clinical
pathology results and take note of concurrent systemic illness or drug treatments (tables below).
FACTORS AFFECTING THYROID HORMONES TOTAL T4, FREE T4, TSH RESULTS
Age less than 3 months Increased total T4
Age older than 6 years Decreased total T4
Body size small <10 kgs Increased total T4
Body size large > 30 kgs Decreased total T4
Breed differences : all sight-hound breeds Lower total and free T4 normal range. TSH unaffected
Weight-gain / obesity Increase all assays
Weight-loss / starvation Decreased total T4. Free T4 unaffected
Pregnancy Increased total T4
Surgery / anaesthesia Decreased total T4
Strenuous exercise / training Increased total T4. Decreased TSH. Free T4 unaffected
DRUGS AFFECTING THYROID ASSAYS THYROID ASSAY RESULTS
Carprofen Decreased all assays
Glucocorticoids Decreased T4 and free T4. TSH variable
Furosemide Decreased total T4. Free T4 unaffected
Methimazole Decreased T4 and free T4. Increased TSH
Phenobarbital Decreased T4 and free T4. Increased TSH
Phenylbutazone Decreased total T4
Progestagens Decreased T4 and free T4. Increased TSH
Propylthiouracil Decreased T4 and free T4. Increased TSH
Sulfonamides Decreased T4 and free T4. Increased TSH
TOTAL T4 INTERPRETATION
19 – 25 nmol/l Hypothyroidism less likely
12 – 19 nmol/l Equivocal result – non-diagnostic
6 – 12 nmol/l Hypothyroidism possible *
Less than 6 nmol/l Hypothyroidism likely *
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Other tests such as total T3, TSH stimulation, TRH stimulation, T3 suppression tests
add no extra value from a diagnostic point of view and increase costs unnecessarily.
THYROID HORMONE AND THYROGLOBULIN AUTOANTIBODIES
These tests are often used in breeding programs to identify dogs that
may have the potential to develop hypothyroidism or in cases where
unexplained total T4 or TSH results have been detected. Positive
tests for these antibodies indicate possible lymphocytic thyroiditis
that may be asymptomatic with the potential to develop into primary
hypothyroidism. Dogs that are positive should be re-tested in at least
6 months. Not all dogs that are autoantibody positive will develop hypothyroidism;
some may test autoantibody negative subsequently with no signs of hypothyroidism,
while others may remain autoantibody positive but never show any changes in clinical
status, free T4 or TSH. Some dogs that initially test autoantibody positive become
autoantibody negative but develop hypothyroidism with changes in free T4 and TSH.
Minimum of 1ml per assay.
EUTHYROID-SICK SYNDROME
Ideally thyroid assays should not be conducted in dogs suffering from non-thyroidal
illness. The stress and possibly inflammatory cytokines will slowly suppress free T4
levels. In response the pituitary increases TSH secretion in order to maintain free T4
concentrations. Ultimately this increased TSH cannot keep free T4 above normal. If
the thyroid assays of free T4 and TSH are conducted at this point, the result of low
free T4 and elevated TSH mimics hypothyroidism, falsely.
MONITORING TREATMENT WITH LEVOTHYROXINE
Using a brand name synthetic Levothyroxine sodium product approved for animal
use, the starting dose is recommended to be 0.02 mg/kg, with a maximum of 0.8 mg.
Initial frequency of administration should be every 12 hours. The response to
treatment should be critically evaluated after 6 to 8 weeks.
Protocol:
Serum total T4 or free T4 and TSH (at least 0.5ml for each assay)
should be measured 4 to 6 hours after administration of
Levothyroxine.
Interpretation:
Total T4 or free T4 should be in the normal range or slightly
increased above normal. TSH concentration should be within the normal range.
Feline Hyperthyroidism Hyperthyroidism is considered the most common endocrine disorder of cats and one
of the major causes of disease in older animals. Beware of many factors that depress
serum T4 / T3 values to within normal range thereby creating false negatives.
Significant daily fluctuations, concurrent non-thyroidal illness and stress may result in
apparently normal serum T4 values in hyperthyroid cats.
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TOTAL SERUM T4 OR FREE T4 ASSAY
Assay of basal or free T4 remains the most accurate means of confirming a diagnosis
of hyperthyroidism in the cat. Free T4 may be more sensitive than basal T4.
Protocol:
Fasted serum samples of at least 1 ml needed for both RlA of basal
T4 or modified equilibrium dialysis for free T4.
Interpretation:
Norma1 values on a single assay do not rule out
hyperthyroidism.
*Results must be interpreted with clinical signs and it is important to take note of
concurrent clinical illness that may suppress total T4 into the normal range in
hyperthyroid cats. FreeT4 may often be elevated when totalT4 is within normal range.
On the other hand, certain non-thyroidal diseases can falsely elevate freeT4 above
normal leading to false positive hyperthyroidism diagnosis.
FREE T4
In the face of strong clinical evidence suggestive of hyperthyroidism,
a normal result should prompt a repeat assay after waiting at least a
week.
TOTAL T4 INTERPRETATION
Greater than 65 nmol/l Hyperthyroidism highly likely *
50 - 60 nmol/l Hyperthyroidism is likely *
39 – 50 nmol/l Hyperthyroidism is possible *
31 – 39 nmol/l Equivocal result non-diagnostic
25 – 30 nmol/l Hyperthyroidism is less likely
Less than 25 nmol/l Hyperthyroidism is unlikely
Hyperthyroidism Free T4 >15 pmol/l
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Hypothalamic-Pituitary-Adrenal Hormones
Canine Hyperadrenocorticism
A fairly common endocrine condition of the dog but considered rare in the cat. There
is now acceptance that at least two, and some authors suggest 3, hyperadrenocorticoid
‘syndromes’ occur in dogs - Typical or classic hyperadrenocorticism (HAC), Atypical
HAC and Alopecia X.
Some authors include Alopecia X in the atypical HAC category but it is a purely
dermatological condition with bilateral alopecia and hyperpigmentation of the
alopecic areas but without any of the biochemical or haematological abnormalities
seen in HAC. It is suspected by these authors that it is a mild form of pituitary-
dependant HAC, causing defects in steroidogenesis. Other authors do not consider
Alopecia X a pure endocrinopathy and suggest a genetic and/or intrinsic follicular
aetiology calling it a hair cycle arrest instead. It occurs in breeds that are double
coated and have a dense undercoat such as Pomeranians, Alaskan Malamutes, Chow
Chows, and Keeshonds. This problem has also been described in Miniature and Toy
Poodles. The breeders refer to this condition as “black skin disease” or “coat funk,”
depending on the breed affected; in the veterinary literature, it has been known by
many names including adult-onset growth hormone deficiency (hyposomatotropism),
growth hormone-responsive alopecia, castration-responsive alopecia, biopsy-
responsive alopecia, congenital adrenal hyperplasia-like syndrome, and, most
recently, Alopecia X.
Typical HAC is the most common presentation and most dogs are polydipsic and
polyuric. The majority have most of the haematological or biochemical abnormalities
listed below. It may occur concurrently with other endocrine disorders particularly
diabetes mellitus. Atypical HAC cases may have only some of the haematological or
biochemical abnormalities listed below; are not always PU/PD, and may not have or
may only have dermatological signs.
Classification: Pituitary-Dependant Hyperadrenocorticism(PDH)
or
Adrenal-Dependant Hyperadrenocorticism(ADH)
* A presumptive diagnosis should be based on physical examination and clinical
signs as well as haematological and biochemical changes observed in more than
90% of cases, which include:
Stress leukogram (neutrophilia; lymphopaenia; eosinopaenia with or without
monocytosis).
Increased ALP (up to 40x), ALT, GGT and GLDH.
Hypercholesterolaemia.
Inconsistent findings include hyperglycaemia, increased amylase and lipase,
polycytaemia, thrombocytosis, lipaemia, decreased urea and creatinine,
hypophosphataemia, hypernatraemia and hypokalaemia.
Basal cortisol alone has no diagnostic significance and is considered a spurious test,
as most animals with HAC have baseline cortisols within normal ranges and secondly
secretion of cortisol is very episodic in dogs. In addition cortisol levels are affected by
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many other factors. The urine cortisol:creatinine ratio is an unreliable means of
screening for HAC as many false positives occur but its negative predictive value is
very high.
Screening Tests URINE CORTISOL:CREATININE RATIO This test was developed to offset the episodic nature of cortisol secretion and is very
sensitive but not specific.
Protocol:
Fresh urine should be collected at random by the owner (to reduce
stress), midstream free-flow sample, and tested for cortisol and
creatinine as quickly as possible.
Interpretation:
Due to the low specificity, it would be better to use a normal result
to rule out hyperadrenocorticism. An abnormal result must be viewed with caution.
Urine cortisol (mmol/l) or (mg/dl) divided by Urine creatinine (mmol/l) or (mg/dl) –
units must be the same. Result has no unit – it is convention to report 10 X 10-6
as
10 *
Normal < 13.5 x 10-6
or < 13.5*
Hyperadrenocorticoid 30 – 2100 x 10-6
or 30 – 2100*
Chronic illness or PU/PD due to other causes 10 – 150 x 10-6
or 10 – 150*
Note the wide overlap between hyperadrenocortiod and non-adrenal illness.
Confirmatory tests LOW DOSE DEXAMETHASONE SUPPRESSION TEST (LDDST) A useful screening test which can also be used to differentiate PDH from ADH; it
does not identify iatrogenic hyperadrenocorticoid cases though. Test results can be
affected by acute stress (concurrent illness) or corticosteroid and progestagen usage.
A single dose of methylprednisolone acetate (2.5 mg/kg) can suppress the adrenal
cortex of dogs for at least 5 weeks. A single dose of triamcinolone acetonide (0.22
mg/kg) can suppress the canine adrenal cortex for at least 4 weeks. Otic, ophthalmic
and topical preparations can produce comparable effects to parenteral corticosteroid
therapy.
Principle - In the normal dog dexamethasone should suppress ACTH secretion and
thus serum cortisol levels. In a case of PDH the dexamethasone may initially cause a
negative feedback affect, decreasing ACTH production by the pituitary but the excess
ACTH production overcomes the negative feedback and cortisol concentration rises
again. In a case of ADH the cortisol production is autonomous of ACTH stimulation
and the negative feedback has no effect on the cortisol levels. Remember, very
excitable or nervous dogs may have hyperplastic adrenals, less sensitive to
dexamethasone suppression leading to false positive results. In such dogs borderline
results should be checked with an ACT-stimulation test. Also anti-convulsant drugs
and exogenous corticosteroids will affect the outcome of this test.
Protocol:
The dog should be fasted for 12 hours and hospitalised
overnight prior to the test to reduce effects of stress. Serum
or heparin plasma samples (1.0ml) are collected before, at 4
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105
hours and 8 hours after administration of dexamethasone at 0.01 mg/kg IV for cortisol
determination.
Ideally, after collection samples should be centrifuged but this is not essential. Store
in the refrigerator (4°C) overnight before submitting to laboratory.
Interpretation: CASE BASAL CORTISOL CORTISOL 4 HOURS CORTISOL 8 HOURS
Healthy dogs Normal < 40 nmol/l < 40 nmol/l
PDH Normal < 50% basal * > 40 nmol/l
ADH ^^ High Normal > 40 nmol/l > 40 nmol/l
Non-diagnostic Low < 40 nmol/l < 40 nmol/l #
*In some cases of PDH, suppression of cortisol occurs at 4 hours but rebounds
again at 8 hours. In such cases the LDDS test provides differentiation between
ADH and PDH.
#When basal and both post-dexamethasone samples are low, previous
corticosteroid treatment must be ruled out.
^^Some PDH cases can present with this pattern of results.
One study has reported a so-called Inverse-LDDS pattern where true
hyperadrenocorticoid cases had 4-hour cortisols > 40 nmol/l but suppressed 8-hour
concentration. Therefore it is important that both 4- and 8-hour samples be evaluated.
In such inverse cases, an ACTH-stimulation test should be used to confirm the result.
ACTH STIMULATION TEST (ACTHS) This test is commonly used as a screening test because of its short duration and
relatively low cost. It is the test of choice for iatrogenic hyperadrenocorticism and for
monitoring adrenal reserve in dogs on therapy. The short duration of the test also
makes it suitable for use concurrently with insulin in diabetes mellitus. It has a
slightly lower sensitivity than the LDDST, especially for ADH, but has a better
specificity and is less affected by the stress response. It is also useful in cases of
atypical HAC, where steroid precursors can be measured on the samples
subsequently, if required.
Principle – Exogenous ACTH administration will induce an exaggerated release of
cortisol from hyperplastic adrenal glands (in PDH) or from the enlarged gland in
ADH. This explains the poorer sensitivity in ADH cases in which some adrenal
tumours are totally refractory to any ACTH stimulation.
Notes on Exogenous ACTH Preparations
Traditionally ACTH came in the form of gels usually of bovine origin but since BSE, bovine
ACTH is no longer available. Porcine ACTH gel can still be purchased commercially. ACTH
gels are cheaper but can be inconsistent in bioactivity. Synthetic ACTH preparations are
readily available but more expensive. Some synthetic products have recently been withdrawn
from the market but products that may be available include Tetracosactrin (Synacthen),
Cosyntropin and Cortrosyn.
The ACTH gels are given intramuscularly (IM) at 1.0 or 2.2 IU/kg. The latter dose is
recommended by most authors; maximum of 40 IU/dog. The synthetic ACTH
preparations come in vials that usually contain 0.25 mg. Synthetic ACTH should be
given intraveneously (IV). They can be given IM but can cause pain. Some authors
Basal Cortisol Dexamethasone Cortisol Cortisol
0 hours 0.01mg/kg IV 4 hours 8 hours
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recommend using one vial per dog, i.e. 0.25 mg/dog IV. Other authors recommend
dividing vials into half-doses into plastic syringes for dogs <5kgs, i.e. 0.125 mg/dog
<5kgs and 0.25 mg/dogs >5kgs. Some authors suggest doses as low as 5 – 10 ug/kg
are adequate to initiate the exaggerated response. Dividing vials into doses can be
done provided the ACTH is stored frozen and bacterial contamination does not occur.
Thawing and re-freezing of such doses is not recommended.
Protocol: Collect a baseline serum sample, administer dose of
ACTH, depending on formulation (see above) and then collect
second serum sample (1.0ml) 1 – 2 hours, later depending on ACTH
preparation for cortisol determination.
*Half this dose can be used in dogs <5kg (see above).
Interpretation:
It must be remembered that these results should not be assessed on their own, but
must have clinical signs and routine laboratory results consistent with HAC. CONDITION BASAL CORTISOL POST-ACTH CORTISOL
Healthy 15 – 150 nmol/l * 175 – 450 nmol/l *
Hyperadrenocorticoid 100 – 250 nmol/l > 650 nmol/l ^^
Iatrogenic hypercortisolaemia < 40 nmol/l < 40 nmol/l
*Very excitable or nervous, healthy dogs may have higher post-ACTH values.
^^Values between 550-650 nmol/l are equivocal, and if clinical suspicion is high, a
LDDST or re-testing in a month must be considered.
Note: 40 – 50% of ADH cases can have normal post-ACTH values!
SKIN BIOPSY
This can be most informative from a diagnostic point of view when used in
conjunction with the LDDS test. Areas of most advanced alopecia should be selected
for sampling. Histological features most commonly associated with HAC include:
folliculosebaceous, epidermal and dermal atrophy; calcinosis cutis; vascular ectasia;
follicular hyperkeratosis and decreased dermal elastin.
LIVER BIOPSY
A liver biopsy from a dog suffering from HAC, either spontaneous, ADH or PDH, or
iatrogenic HAC usually shows the typical, so-called ‘steroid hepatopathy’. This is a
vacuolar hepatopathy with centrilobular vacuolisation, intracellular glycogen
accumulation and focal centrilobular necrosis. Unfortunately this is not specific for
HAC and other differentials include diabetes mellitus, lipid metabolism defects, sex-
hormone excesses, chronic pancreatitis, IBD, severe hypothyroidism, severe systemic
disease, hepatocutaneous syndrome and even congestive heart failure.
ABDOMINAL ULTRASOUND
Only clinicians with high ultrasound expertise should consider this as a screening
method. In PDH cases persistently high ACTH production by the pituitary causes
bilateral adrenal hypertrophy, with most cases having adrenals greater than 7mm in
cross-section. With ADH cases, ideally the cancerous adrenal is enlarged and the
ACTH preparation ACTH dose Serum sample collected for cortisol assay
Synthetic ACTH (IV) 0.25mg/dog * Basal - 0 hours 60 – 90 mins post-ACTH
ACTH gel (IM) 2.2 IU/kg Basal - 0 hours 120 mins post-ACTH
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other atrophied but often the contra-lateral gland is normal in size. Bilateral adrenal
tumours can occur and make diagnosis difficult.
ATYPICAL HYPERADRENOCORTICISM
Cases usually have some clinical features (at least 3) consistent with HAC but ACTH-
stimulation and LDDS test results are normal or equivocal. Most cases have elevated
ALP concentration and many have raised ALT too. In fact unexplained liver enzyme
elevation is sometimes the only laboratory abnormality. Abdominal ultrasound may
show adrenals that are mildly enlarged. Liver biopsy and skin biopsy often have
pathology consistent with HAC. In some breeds an enzyme defect in the cortisol
production pathway from cholesterol results in a build-up of cortisol precursors,
which cause the clinical signs. The exact aetiology is not known in all breeds
however. In some cases of ADH, the tumour may secrete certain precursors such as
deoxycorticosterone, progesterone or 17-hydroxyprogesterone (17-OHP). The same
serum samples from an ACTH stimulation test can be used.
Protocol:
Collect a baseline serum sample (2.0ml), administer dose of ACTH,
depending on formulation (see ACTHST protocol above) and then
collect second serum sample (2.0ml) 1 – 2 hours, later depending on
ACTH preparation. Cortisol precursors that can be measured include
17-OHP, progesterone, oestradiol-2, androstenedione, dihydro-
epiandrostenedione and aldosterone. The latter three are not always available
and exact abnormal cut-off values have not been well defined.
Interpretation:
17-OHP is the most common analyte measured in these cases. In healthy dogs 17-
OHP increases to 2 – 3-fold baseline value, usually not exceeding 10 nmol/l. In
atypical HAC cases however, the post-ACTH 17-OHP concentration ranges from 8 –
40 nmol/l. Some authors suggest a cut-off of > 4 nmol/l being positive, but this leads
to too many false positives, lowering test specificity. CONDITION BASAL 17-OHP POST-ACTH 17-OHP
Healthy < 4 nmol/l < 10 nmol/l
Atypical HAC 6.5 nmol/l 8 - 40 nmol/l ^^
Non-diagnostic < 4 nmol/l 7 - 10 nmol/l
^^Many dogs suffering from non-adrenal illness can have 17-OHP concentrations
above normal. It is vital that such illnesses are excluded and that this test is run only
in cases of suspected atypical HAC with a high degree of clinical suspicion.
Discrimination Tests These tests should only be performed if a precise diagnosis is required. And only once
spontaneous hyperadrenocorticism has been confirmed. These assays aim to
distinguish between pituitary dependant HAC and adrenal dependant HAC.
Sophisticated diagnostic imaging methods such as CT and MRI may be more readily
available but are beyond the scope of this manual.
HIGH DOSE DEXAMETHASONE TEST A high dexamethasone dose is not expected to influence cortisol secretion by the
adrenocortical cells in ADH, but it may suppress ACTH and thus cortisol release in
PDH.
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Protocol:
A plasma or serum sample is collected before, at 4 and 8
hours after administration of 0.1 mg/kg dexamethasone IV for
cortisol determination.
Interpretation:
Suppression is defined as a post-dexamethasone cortisol level less than 50% of the
basal cortisol or < 40 nmol/l. CONDITION 4 HOURS 8 HOURS
Healthy < 40 nmol/l < 40 nmol/l
PDH cases < 50% basal or < 40 nmol/l < 50% basal or < 40 nmol/l ^^
ADH cases No suppression No suppression
^^20 - 25% PDH cases will not undergo suppression – often macroadenomas in the
pituitary.
ENDOGENOUS PLASMA ACTH ASSAY Endogenous ACTH secretion from the pituitary is suppressed by the negative
feedback of high serum cortisol concentrations in ADH cases; whereas in PDH cases
it is high-normal to excessive. Stringent and meticulous sample handling is crucial
since ACTH activity in the plasma will reduce rapidly resulting in falsely low values
and incorrect interpretation. The endogenous ACTH assay used must be validated for
use in dogs. Two major indications for this assay include: 1- presence of an adrenal
mass with contralateral adrenomegaly; and 2- LDDST results suggesting PDH but
with suspicion of an adrenal mass.
Protocol: Requires thorough preparation with chilled blood-collection tubes and prior
arrangement with couriers and laboratory. A sample must be collected into
a chilled plastic EDTA tube, centrifuged immediately, and the plasma
separated into another plastic chilled tube that is placed on crushed-ice for
transport or frozen at -20°C. A protease inhibitor such as aprotonin can be
added to the sample to limit ACTH degradation.
Interpretation:
The endogenous ACTH is very low in ADH cases but elevated in most PDH cases but
some overlap with normal dogs, especially nervous dogs, can occur.
CONDITION PDH SUSPECT
PDH
NON-
DIAGNOSTIC
ADH
Endogogenous
ACTH
> 45 pg/ml
*
30 – 45 pg/ml
° 10 – 25 pg/ml
< 5 pg/ml
^^
*85 – 90% of PDH cases. °Some PDH cases but also some normal dogs have ACTH
levels up to 35 pg/ml. ^^Most ADH cases have almost undetectable levels but smaller
adrenal tumours may have results between 5 – 10 pg/ml.
Monitoring Response To Medical Therapy For Hyperadrenocorticism When monitoring treatment of hyperadrenocorticism with o,p' DDD (mitotane,
Lysodren), trilostane or ketoconazole the test of choice is the ACTH-stimulation test.
The former two regimens require an induction phase, which is complete when
reduction in appetite develops or the daily water intake falls to or below 60ml/kg. The
ACTH stimulation test is then used to confirm adequacy of medical control. The test
should be run as soon as these clinical changes occur, which is usually 7-1 0 days
after commencing induction therapy with mitotane, and 10-14 days after commencing
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ketoconazole therapy. Then once maintenance treatment starts, ACTHST are
recommended every 4 weeks until stable and then every 3 months providing clinical
status remains suitable. With trilostane treatment ACTHST are recommended after 1,
3, 6 and 13 weeks, and then 6 and 12 months. The aim of treatment is to achieve
clinical improvement without causing illness and a deficient or low normal
adrenocortical response to ACTH.
Protocol:
The same protocol applies as is used in diagnosis of HAC. (see above
pg 105 ). Samples should be collected 3 – 6 hours after dosing.
Interpretation:
Absolute values and not relative increases should be used for
interpretation. Ideally, the post-ACTH result should suggest a low-
normal response – i.e. cortisol below 135 nmol/l and preferably much lower, 40 - 60
nmol/l. If the post-ACTH result is that of a normal animal, medical control is usually
inadequate as such dogs tend to remain clinically hyperadrenocorticoid.
Feline Hyperadrenocorticism An uncommon disease in cats but can be PDH or ADH. There is a strong correlation
between feline hyperadrenocorticism and diabetes mellitus, particularly insulin-
resistant diabetes, with many similar clinical signs including pendulous abdomen,
bilateral alopecia and muscle atrophy. A few differences are apparent however: 1-
PU/PD is less severe and tends to occur later in disease progression; 2- diabetes
mellitus is more common in feline HAC; and, 3- the cutaneous lesions tend to be
more severe than in dogs. Cutaneous manifestations in feline HAC include bruising,
hyperpigmentation, thin skin with prominent subcutaneous vasculature, alopecia, and
most notably, severe fragility of the skin. Most of the common clinical signs
(polyuria, polydipsia, polyphagia) in feline hyperadrenocorticism are probably
manifestations of associated diabetes mellitus.
Screening tests Stress at the time of sampling, concurrent non-adrenal illness and use of progestagens
or topical or parenteral glucocorticoids may all affect cortisol values in stimulation
and suppression tests in cats. Biochemical parameters commonly encountered include
hyperglycaemia, hypercholesterolaemia, increased AST and ALP. Remember
glucosuria will elevate the USG and a normal result should not exclude HAC as a
differential diagnosis.
Confirmatory tests Both the LDDS and the ACTH stimulation tests have lower sensitivity and specificity
than their canine counterparts. Controversy exists amongst veterinary
endocrinologists as to the most reliable test. The ACTHST and LDDST used on their
own are not recommended in feline HAC. The high dose dexamethasone suppression
test (HDDST) may be the most reliable. Most authors do usually not recommend
combination tests but this may be one instance where it works well, combining HDDS
and ACTHS tests.
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HIGH DOSE DEXAMETHASONE SUPPRESSION TEST Difficult to interpret in the cat and lacks sensitivity, hence HDDS test results should
never be used as the sole evidence for a diagnosis.
Protocol:
Serum or plasma samples are collected before and at 4 and 8 hours
post IV administration of 0.1 mg/kg dexamethasone for cortisol
determination.
Interpretation: CONDITION 4 HOURS 8 HOURS
Healthy < 40 nmol/l < 40 nmol/l
HAC Cats > 40 nmol/l > 40 nmol/l ^^
^^Some PDH cases will suppress at the 4 hour.
COMBINED HDDST AND ACTH STIMULATION TEST This test is advocated by some authors and is aimed at improving the sensitivity and
specificity over a single test on its own.
Protocol:
Serum or plasma samples are collected for cortisol determination
before and at 4 hours after IV administration of 0.1 mg/kg
dexamethasone. Then synthetic ACTH is administered IV at 0.125
ug/cat (half vial). And then 60 minutes after ACTH administration
another serum sample is collected for cortisol measurement.
Interpretation:
CONDITION Cortisol 4 HOURS (post-dex) Cortisol 5 HOURS (60 mins post-
ACTH)
Healthy < 40 nmol/l < 40 nmol/l
Borderline > 50% suppression of
basal 400 – 500 nmol/l
HAC Cats > 40 nmol/l > 500 nmol/l ^^
^^Very few non-adrenal illness cases will have post-ACTH > 500 nmol/l
Equine Pituitary-Dependant Hyperadrenocorticism or Pituitary
Pars Intermedia Adenoma / Dysfunction Equine Cushing’s disease is a common disease in horses older than 20 years but it
probably begins and goes undetected in middle-aged horses between 10 – 20 years.
Pituitary pars intermedia dysfunction (PPID) is suspected to be caused by
degeneration of dopaminergic neurons in the hypothalamus which results in decreased
dopamine secretion. The melanotropes in the pars intermedia of the pituitary are
normally under tonic dopaminergic inhibition, and when this inhibition is diminished
cell proilferation and hypertrophy of the pars intermedia follows. Increased hormone
secretion (ACTH and α-melanocyte-stimulating hormone [α-MSH]) subsequently
leads to hyperadrenocorticism.
Early PPID can be very difficult to diagnose. Clinical signs may only develop once
pars intermedia hyperplasia is advanced or microadenomas have developed. These
include delayed or incomplete shedding of winter coat, hirsutism, PU/PD, sometimes
hyperglycaemia, increased appetite to maintain weight, sway-back / potbelly and
laminitis. A stress leukogram may be seen in a full blood count. Mildly elevated ALP,
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AST and hyperglycaemia and hypercholesterolaemia often present in a biochemical
profile.
Screening Test ENDOGENOUS PLASMA ACTH The assay must be validated for use in horses. In PPID the pars intermedia of the
pituitary is refractory to negative feedback by hypercortisolaemia and ACTH
accumulates due to melanotrope hyperplasia / neoplasia.
Protocol: Requires thorough preparation with chilled blood-collection tubes and prior
arrangement with couriers and laboratory. A sample must be collected into
a chilled plastic EDTA tube, centrifuged immediately, and the plasma
separated into another plastic chilled tube which is placed on crushed-ice
for transport or frozen at -20°C. A protease inhibitor such as aprotonin can
be added to the sample to limit ACTH degradation.
Interpretation:
CONDITION ENDOGENOUS ACTH
Healthy < 25 pg/ml or 5.6 pmol/l
Borderline 25 - 35 pg/ml or 5.6 – 7.7 pmol/l
PPID > 45 pg/ml or > 10 pmol/l *
*At this concentration the confidence in the result can be high but importantly
seasonal variation in ACTH concentration occurs in horses with higher concentrations
detectable in autumn and early winter. This test should not be used in these months or
a positive result must be checked in spring or summer.
Confirmatory Tests OVER-NIGHT DEXAMETHASONE SUPPRESSION TEST (ODST) Used to be regarded as the test of choice but recent studies have detected some flaws
in its use. Nevertheless it can still be used to confirm PPID in cases where there is a
high degree of clinical suspicion.
Protocol:
Serum or plasma samples are collected for cortisol determination
before and at 19 or 24 hours after IV or IM administration of 40
ug/kg dexamethasone (20mg for a 500kg horse); usually at 5pm one
day and then at midday the next, or at 5pm again. In a healthy horse
this dose of dexamethasone suppresses serum cortisol for more than
24 hours.
Interpretation: CONDITION CORTISOL (POST DEX)
Healthy < 10 ng/ml or 1ug/dl or < 27 nmol/l
PPID > 10 ng/ml or 1ug/dl or > 27 nmol/l *
*A negative result in autumn and early winter can be accepted as accurate but
a positive result must be checked in spring or summer.
ORAL DOMPERIDONE ENDOGENOUS-ACTH TEST A recently devised test which may be the most reliable at detecting early PPID.
Domperidone is a D2-receptor blocker. The drug blocks the inhibitory effect of
dopamine on the pars intermedia melanotrophs resulting in increased ACTH secretion
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from them in horses suffering from PPID. In normal horses very little ACTH is
secreted from the pars intermedia.
Domperidone comes in a gel form that contains 110mg per millilitre of gel. The
correct dose is 5.5mg/kg, which equates to 2.75g/500kg horse, or 25ml of gel.
Protocol: The test should commence between 8 – 10am. One plasma sample is
collected for endogenous ACTH before domperidone administration. In
summer, spring and late winter a second plasma sample is collected 4 hours
post-domperidone. While if the test is conducted in autumn or early winter,
2 plasma samples are collected 2 and 4 hours post-domperidone.
Remember for endogenous ACTH the sample must be collected into a chilled plastic
EDTA tube, centrifuged immediately, and the plasma separated into another plastic
chilled tube that is placed on crushed-ice for transport or frozen at -20°C. A protease
inhibitor such as aprotonin can be added to the sample to limit ACTH degradation.
Interpretation:
CONDITION Basal Endogenous ACTH 4 hour post-domp ACTH
Healthy < 25 pg/ml or 5.6 pmol/l < 45 pg/ml or < 10 pmol/l
PPID > 45 pg/ml or > 10 pmol/l > Double the basal
concentration *
Non-diagnostic
autumn test 25 - 35 pg/ml or 5.6 – 7.7 pmol/l
2 hour sample < than 2-fold
increase but 4 hour sample > 2-
fold increase
*In autumn testing the response is exaggerated and an increase greater than double the
basal concentration in both the 2 and 4 hour post-domperidone samples is required for
a positive diagnosis.
A modification of this test is a single 4 hour (2-hour in autumn) post-
domperidone sample with endogenous ACTH concentration greater than 100
pg/ml or 22 pmol/l being consistent with PPID
Canine and Feline Hypoadrenocorticism Hypoadrenocorticism is an uncommon disorder in the dog and is rare in the cat.
Although all aged cats and dogs may be affected, hypoadrenocorticoid dogs are
typically young to middle-aged (average age 4 years) and female, and affected cats
are similarly young to middle-aged (average age 6 years). Primary (adrenal-
dependent) is more common than secondary (pituitary-dependent)
hypoadrenocorticism. Clinical signs in hypoadrenocorticism include lethargy,
anorexia, vomiting and weightloss. Biochemical parameters include non-regenerative
anaemia, hyperkalaemia, hyponatraemia ( Na:K < 23:1), pre-renal azotaemia,
hyperphosphataernia and hypochloridaemia. Normal Na:K ratios do not exclude
hypoadrenocorticism however.
Confirmatory Test Diagnosis of hypoadrenocorticism is based on clinical signs, classic electrolyte
disturbances and results of the ACTH stimulation test, which is the only endocrine
test that can be used in this condition.
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ACTH STIMULATION TEST In hypoadrenocorticoid animals, administration of exogenous ACTH should not result
in any increase in serum cortisol concentrations.
Protocol:
Serum or plasma samples are collected for cortisol determination
before and at 1 hour after IV administration of 250 ug/dog synthetic
ACTH for dogs > 5kgs; or 125 ug/dog < 5kgs. This latter dose is used
in cats but samples post-ACTH are collected at 30 and 60 minutes.
Interpretation: Hypoadrenocorticoid cats and dogs have a minimal or negligible response to ACTH
stimulation. CONDITION Basal Cortisol Cortisol post-ACTH
Healthy < 110nmol/l * 400 – 500 nmol/l
Hypoadrenocorticoid < 40 nmol/l < 40 nmol/l
in dogs
< 40 nmol/l at both 30 and 90 min in
cats
*One study has shown that a random basal cortisol that is greater than 55 nmol/l
makes hypoadrenocorticism in dogs highly unlikely.
Sex Hormone Anomalies and Investigations
Canine Hyperoestrogenism This appears to be an emerging disease entity in intact female dogs. In
hyperoestrogenism cases, oestradiol is the oestrogen that is in excess. ACTHS and
LDDS tests are usually normal for cortisol concentrations and thyroid function is
normal. Hepatopathies are frequent and typically include markedly elevated ALP,
hepatomegaly, steroid hepatopathy, hyperechoic liver on ultrasound. PU/PD is
commonly reported while panting may be present. Dermatological manifestations are
usually present and often the reason for patient submission to the veterinarian. Skin
biopsy results are non-specific but suggest an endocrinopathy. There are no alterations
in oestradiol concentration in response to ACTH stimulation tests. Resistance to
mitotane may occur and increase often occurs in response to trilostane treatment.
Protocol:
The assay for oestradiol-2 must be validated for use in dogs. A single
serum sample is collected and basal concentration determined.
Interpretation: CONDITION BASAL OESTRADIOL
Healthy 60 – 70 pg/ml
Hyperoestrinism > 70 pg/ml
Laboratory Pregnancy Determination Laboratory determination of pregnancy is not a full-proof method and should only be
used in conjunction with imaging methodologies and the case history and clinical
presentation. It can be useful in certain situations where testing for pregnancy or lack
thereof, is needed to make important management decisions in breeding programs on
studs.
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There are no laboratory tests for pregnancy in cats. Progesterone may be
analysed but this will only indicate if the cat is not pregnant - progesterone < 3
nmol/l.
PREGNANCY DETECTION IN THE BITCH Unfortunately in the intact bitch progesterone concentrations fluctuate from low levels
in pro-oestrus to high levels in metoestrus and therefore it is not reliable for
pregnancy testing. Relaxin is a serum hormone that is specific to pregnancy. The bulk
of relaxin is produced by the placenta but small amounts may be produced by
corporea lutea. Pseudopregnant bitches do not produce significant amounts of relaxin.
It can be used late in pregnancy to test for foetal death or resorption if suspected.
Protocol:
Relaxin is detectable in circulation by about day 28 of gestation. 0.5ml of
heparinised plasma must be submitted. Do not use EDTA.
Interpretation:
STATUS PLASMA RELAXIN
Non-pregnant < 1 ng/ml
Borderline 1 – 3 ng/ml *
Pregnant > 3 ng/ml
*Possibly early pregnancy, days 24-28 or small litter size.
PREGNANCY DETECTION IN THE MARE Endocrine control of the equine pregnancy is complex and not maintained by one
single hormone. Therefore, for the most reliable results at least two, if not three,
protocols are recommended. Progesterone, equine chorionic gonadotropin (eCG) and
oestrone sulphate all play important roles at different times. Progesterone initially
rises at day 18-40, followed by a slight decrease then rises to a peak at day 80, then
gradually declining to 1-2 ng/ml during mid-late gestation day 150. ECG is produced
by the endometrial cups beginning around day 37-42, peaking around day 60-80, after
which the endometrial cups start to decline, disappearing around day 120-
150. Oestrone sulphate can be tested for in almost any bodily fluid. In serum there is a
sharp rise after day 55-60, peak levels by 80-90 days. It is considered an indicator of
foetal viability after 45 days but before 60 days a false positive can be obtained due to
oestrus.
Protocol:
Serum samples are adequate but the serum must be separated from
the clot as soon as possible. At least 5-8ml are required if 2 analytes
are going to be requested.
Interpretation:
OESTRONE SULPHATE can be used as a very sensitive pregnancy test (after 70
days-post mating) and is the most specific test, demonstrating the presence of a live
foal; if the foal dies the oestrone sulphate decreases very rapidly. Oestrone Sulphate
starts to increase from about 70 - 80 days post-covering, increases to greater than 25
ng/ml and remains detectable until very close to term.
PROGESTERONE is assayed in a sample taken 18 - 25 days post-mating.
Progesterone levels greater than 13 nmol/l are indicative of a functional corpus
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luteum, which is highly suggestive of pregnancy at this time. Concentrations less
than3 nmol/l are likely to be non-pregnant. This is very useful for thoroughbred
mares, where time is critical and who need to be re-covered.
ECG is produced from the endometrial cups from about day 40 - 120 post-mating and
is a reasonable pregnancy test. ECG is prone to false positives as - if the foal dies, the
endometrial cups continue to produce ECG for some time. Values greater than 6
IU/ml are consistent with functional endometrial cups. STAGE GESTATION TEST SIGNIFICANCE
18 – 30 days Progesterone >13 nmol/l Functional CL; possibly pregnant
40 – 120 days ECG > 6 IU/ml Endometrial cups; possibly pregnant
90 - term Oestrone sulphate > 25ng/ml Pregnant = accurate
Laboratory Evaluation of Neutered Status
EQUINE CRYPTORCHIDISM OR RIG TEST Many so-called gelded horses that exhibit masculine or aggressive behaviour are
cryptorchid. Two tests are regularly used to identify the presence of retained testes.
OESTRONE SULPHATE SERUM CONCENTRATION
This test is only valid when used in horses older than 3 years. It is unreliable in
donkeys and mules.
Protocol:
Serum samples are adequate but the serum must be separated from
the clot as soon as possible. At least 5ml is recommended.
Interpretation:
HUMAN CHORIONIC GONADOTROPHIN (HCG) DYNAMIC TEST
Serum testosterone concentration is measured before and after stimulation with HCG
(Chorulon).
Protocol:
Serum is collected before injecting 6000 IU HCG IV. A second
sample is collected between 30 and 120 minutes post-HCG. Clotted
blood samples are adequate but the serum must be separated from
the clot as soon as possible. At least 5ml is recommended for each
sample.
Interpretation:
Be sure to label tubes correctly, pre- and post-HCG, with times of collection.
Approximately 10% of tests will produce equivocal results. STATUS Basal Testosterone (nmol/l) Testosterone post-HCG (nmol/l)
Stallion 5 – 30 > 10
False rig or Gelding < 0.15 < 0.19
Cryptorchid or Rig 0.3 – 4.3 1.0 - 13
STATUS OESTRONE SULPHATE (ng/ml)
Stallion 10 – 50
False rig or Gelding < 0.02
Cryptorchid or Rig 0.1 - 10
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CANINE CRYPTORCHIDISM Serum testosterone concentration is measured before and after stimulation with HCG
(Chorulon).
Protocol:
Serum is collected before injecting 50 IU HCG IV. A second sample
is collected 60 minutes post-HCG. Clotted blood samples are
adequate but the serum must be separated from the clot as soon as
possible. At least 2ml is recommended for each sample.
Interpretation:
Be sure to label tubes correctly, pre- and post-HCG, with times of collection. STATUS Basal Testosterone (nmol/l) Testosterone post-HCG (nmol/l)
Castrated < 0.5 Little or no increase
Cryptorchid 3 – 30 Significant increase
DETECTION OF REMNANT OVARIAN TISSUE IN THE NEUTERED
BITCH A test to identify whether bitches have been neutered and to detect the presence of
residual ovarian tissue in spayed bitches showing feminisation. The test is based on
oestradiol response to pituitary-ovarian endocrine challenge using the synthetic
gonadotrophin releasing hormone (SGnRH), Buserelin (Receptal). This is available in
10ml vials containing 0.004 mg/ml (4 ug/ml). Dilution 1/10 with water for injection
may make the volumes more manageable.
Protocol:
Serum is collected for oestradiol concentration before injecting 0.32
ug/dog SGnRH IV. A second sample is collected 60 to 90 minutes
post-SGnRH. Clotted blood samples are adequate but the serum must
be separated from the clot as soon as possible. At least 2ml is
recommended for each sample.
Interpretation:
Be sure to label tubes correctly, pre- and post-SGnRH, with times of collection. Pre-
injection oestradiol levels >100 pmol/l would be indicative of pro-oestrus or oestrus
and would confirm the presence of active ovarian tissue STATUS Basal Oestradiol (pmol/l) Testosterone post-SGnRH (pmol/l)
Spayed < 18 Little or no increase
Remnant ovary tissue < 18 55 - 75
TRACKING THE BITCH OESTROUS CYCLE FOR OPTIMUM BREEDING
DATE
This investigation should not rely solely on the serum concentrations of hormones but
combine data from historical knowledge of the bitch’s cycle, clinical signs, behaviour,
vaginal cytology and laboratory measurement of progesterone, and possibly
luteinising hormone (LH) in problematic bitches. Unfortunately LH assays validated
in dogs are rare but one semi-quantitative ELISA assay kit for measuring LH in
canine serum has been marketed for ovulation timing (Status-LH, Synbiotics), which
has been used successfully. One concern is that it must be used daily to detect the day
of the LH surge
Basically at the beginning of pro-oestrus progesterone concentrations begin to rise but
slowly and often below detectable limits of most progesterone assays but on the day
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or just before the LH surge, which is reliably 2 days before ovulation, progesterone
increases steeply and changes can be detected by most assays. Therefore, once a bitch
is known to be beginning pro-oestrus she can be monitored every second day until
progesterone rises into detectable levels. Then daily until progesterone increases
above 5.5 – 6.5 nmol/l, typically the day of the LH surge, 2 days prior to ovulation.
TIME PERIOD Relative to LH Surge Relative to Ovulation
Possibe Fertile period -3 to +7 days -5 to +5 days
Reduced fertility -3 to –1 days -5 to –3 days
Period of high fertility for natural
mating 0 to +6 days -2 to +4 days
Preferred period for managed mating
or AI +2 to +6 days 0 to +4 days
Period of potential fertilization +4 to +6 days +2 to +4 days
Late mating with reduced fertility +7 to +9 days +5 to +7 days
Protocol:
Serum is collected for progesterone concentration. Clotted blood
samples are adequate but the serum must be separated from the clot as
soon as possible. At least 1ml is recommended for each sample.
Interpretation:
Day Day 7 before LH
surge Day of LH Surge
Days 4 - 5 after
LH surge, when
oocytes mature
Days 8 - 11 after
LH surge
Serum
progesterone
<1.6 nmol/l
(< 0.5 ng/ml)
3 – 9.5 nmol/l
(0.9 - 3.0 ng/ml)
11.1 – 38 nmol/l
(3.5 - 12 ng/ml)
25.4 – 80 nmol/l
(8 - 25 ng/ml)
Some authors suggest that the best breeding time is at 3-4 days after the rise of
progesterone above 6 nmol/l. Other authors however suggest greater success when
progesterone is greater than 25 nmol/l, when ovulation has occurred.
Semi-quantitative tests are available for in-clinic testing which may be more
convenient. These kits tend to give 3 possible readings: <2 nmol/l which is too early
for mating; >6 nmol/l indicating the likely LH surge; >10 nmol/l which is a good time
for mating.
TRACKING THE PREGNANT BITCH FOR PARTURITION DATE Again when assessing the most likely day for parturition the progesterone
concentration can be used but the time to start screening for the decline in
progesterone concentrations needs to be calculated by adding 64 ±1 days after the LH
surge but taking the previous pregnancy histories of the bitch into account (if
applicable).
Protocol:
Serum is collected for progesterone concentration. Clotted blood
samples are adequate but the serum must be separated from the clot as
soon as possible. At least 1ml is recommended for each sample.
Interpretation: 24 hours prior to parturition the progesterone concentration declines to <6.25 nmol/l.