Endocrine Chemical Pathology (Endocrinopathies)

Chemical Pathology – Endocrine System

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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


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


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


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)


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:


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:


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


115

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.

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