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Domestic Animal Endocrinology 43 (2012) 317–324

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Circadian and circannual rhythms of cortisol, ACTH, and

␣-melanocyte-stimulating hormone in healthy horses
M. Corderoa, B.W. Brorsenb, D. McFarlanea,*
a
Department of Physiological Sciences, Center of Veterinary Health Sciences, Oklahoma State University, Stillwater, OK 74074, USA
b
Department of Agricultural Economics, Oklahoma State University, Stillwater, OK 74074, USA
Received 25 February 2012; received in revised form 13 May 2012; accepted 13 May 2012

Abstract

Cosinor analysis was used to evaluate whether pituitary and adrenal hormones exhibit circadian rhythmicity in horses. The
effect of season and animal age on their respective rhythms was also determined. In addition, the usefulness of evaluating cortisol
rhythmicity for the diagnosis of pituitary pars intermedia dysfunction (PPID) was assessed. Serum cortisol concentrations (P ⬍
0.01), but not plasma ACTH or ␣-melanocyte-stimulating hormone (␣-MSH), showed a significant circadian periodicity in horses.
An effect of season on hormone concentration was observed with plasma ACTH and ␣-MSH concentration greater in the fall and
cortisol concentration greater in the spring (P ⬍ 0.001). Age did not affect cortisol rhythm, but it did blunt the variation in cortisol
concentration in horses, similar to what has been previously reported to occur in aged people and dogs. In addition, our results
suggest that clinically and diagnostically normal, non–PPID-affected horses commonly have a loss of cortisol diurnal rhythm.
Therefore, measurement of circadian rhythm is not an appropriate diagnostic test for PPID.
© 2012 Elsevier Inc. All rights reserved.

Keywords: Cosinor analysis; Biorhythms; PPID; Season; Aging

1. Introduction sure to stressors, exercise, or generalized disease [7,8].

Circadian periodicity in circulating glucocorticoids
has been found in many species, including humans,
sheep, dogs, pigs, and horses [1–5]. However, little is
known about the diurnal patterns of plasma ACTH and
␣-melanocyte-stimulating hormone (␣-MSH) concen-
trations. In horses, the cortisol circadian rhythm peaks
in the early morning with the nadir occurring in the
evening hours [6,7]. This rhythm can be disrupted or
abolished by minor environmental perturbations, expo-
* Corresponding author. 264 McElroy Hall, Department of Physi-
ological Sciences, Center of Veterinary Health Sciences, Oklahoma
State University, Stillwater, OK 74074. Tel.: 405 744 2072; fax: 405
744 8263.
E-mail address: diannem@okstate.edu (D. McFarlane).
The cortisol rhythm also undergoes changes with nor-
mal aging [9] and with season [10]. Both ACTH and
␣-MSH are known to have circannual rhythms in
horses, with highest concentrations occurring in the fall
months [11,12]. However, little is known about the
diurnal patterns in the plasma concentrations of these
hormones in the spring and fall seasons.
Pituitary pars intermedia dysfunction (PPID) is a
naturally occurring, progressive neuroendocrine dis-
ease of aged horses and ponies. Affected horses are at
high risk of developing life-threatening complica-
tions, including laminitis, secondary infections, and
metabolic disturbances [13]; therefore, disease diag-
nosis and therapeutic intervention are important. The
measurement of endogenous plasma concentrations

0739-7240/12/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.domaniend.2012.05.005
318 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324
of ␣-MSH and ACTH are useful in diagnosing PPID.
Consequently, it is important to characterize diurnal
variations in plasma concentrations of ACTH and
␣-MSH to select an ideal time for sample collection.
Loss of cortisol circadian rhythmicity occurs in horses
with PPID [14]. It has been suggested that evaluating
cortisol circadian rhythmicity can be useful in diagnosing
PPID [15], with a cutoff of less than a 30% difference
between morning and evening serum cortisol concentra-
tion being diagnostic for PPID. However, the performance
of this diagnostic test has not been evaluated, and, further-
more, the effect of normal aging on the cortisol rhythm in
the horse has not been determined. In dogs, age is asso-
ciated with blunted amplitude or even loss of cortisol
circadian rhythmicity [4]. In elderly persons, mean daily
concentrations of cortisol are increased and are accompa-
nied by a reduction in rhythm amplitude [16].
The aims of the present study were to determine the
circadian pattern of circulating cortisol, ACTH, and
␣-MSH concentrations and to determine the effect of
season on their respective circadian rhythms. In addition,
we sought to determine the effect of age on the cortisol
circadian rhythm as well as the usefulness of using corti-
sol rhythmicity to diagnose PPID. We hypothesize that the
equine cortisol circadian rhythm is not affected by season
but that its amplitude and Midline-Estimating Statistic of
Rhythm (MESOR) are affected by age. We also hypoth-
esize that plasma ACTH but not ␣-MSH would have
circadian periodicity similar to that of cortisol.
2. Materials and methods
2.1. Animals and experimental protocols
2.1.1. Experiment 1: circadian and circannual
rhythm of cortisol, ACTH, and ␣-MSH
Eighteen healthy horses (7 mares and 11 geldings),
ranging in age from 7 to 30 yr (mean ⫽ 18 ⫾ 7.4 yr),
were used in this study. Horses were maintained on
pasture at the Oklahoma State University Equine Re-
search Park (latitude 36°8= N, longitude 97°4= W), and
all samples were collected in accordance with the Okla-
homa State University Animal Care and Use Commit-
tee. One day, before sample collection, horses were
placed in individual box stalls in a barn with free access
to water and grass hay. To minimize stress, the study
was conducted in a barn where the horses were fre-
quently housed. Furthermore, care was taken to place
the horses in stalls to maximize positive social interac-
tions. Jugular catheters were inserted to aid in blood
collection. Sampling began 3 h after catheter insertion.
Sampling during hours of darkness was performed with
the use of low-intensity red light headlamps to prevent
an effect on the pineal gland. Blood samples were
collected into evacuated EDTA-coated or serum collec-
tion tubes every 2 h for 24 h. Plasma samples were
placed on ice until processed. Serum or plasma were
separated by centrifugation at 300g for 10 min at 4°C
within 3 h of collection. Samples were stored at ⫺80°C
until assayed. The sampling periods were May 2008,
September 2008, and May 2009. Eight horses were
sampled in May 2008 and again in September 2008. To
determine the effect of age on cortisol circadian rhythm
parameters, an additional 10 horses were sampled in
May 2009, and the resulting data added to the 8 horses
sampled in May 2008.
2.1.2. Experiment 2: assessment of cortisol
rhythmicity as diagnostic test for PPID
Fifty healthy horses from various farms in the Still-
water, OK, area were included in a second study de-
signed to evaluate the utility of assessment of loss of
cortisol rhythmicity as a diagnostic test for PPID.
Horses were mostly quarter horse type and included a
wide range of ages (2 to 29 yr; mean ⫽ 16 ⫾ 6.8 yr).
Sample collection and physical examinations were per-
formed during a single visit in January. Blood samples
were collected by jugular venipuncture before feeding
at 8:00 AM and 5:00 PM of the same day. The sampled
population consisted of healthy horses with no history
of hair coat abnormalities or other clinical signs of
PPID. Basal concentrations of ACTH and ␣-MSH were
confirmed to be within reference range (␣-MSH ⬍ 35
pmol/L; ACTH ⬍ 50 pg/mL) [17,18]. Serum cortisol
concentrations were determined in morning and eve-
ning samples, and a percentage difference was calcu-
lated for each horse with the use of the following
formula: percentage difference ⫽ [AM cortisol ⫺ PM
cortisol/AM cortisol] ⫻ 100%. A mean percentage dif-
ference for the group was calculated. Follow-up phys-
ical examinations were performed on the study popu-
lation 2 yr after sample collection, and any horse that
developed clinical signs of PPID was excluded.
2.2. Hormone assays
Cortisol (Coat-a-Count cortisol; Diagnostic Products
Corp., Los Angeles, CA), ␣-MSH (Euria ␣-MSH RIA;
American Laboratory Products Co., Windham, NH),
and ACTH (MP Biomedicals, Orangeburg, NY) were
measured with the use of commercially available RAI
previously validated for use in the horse with a limit of
detection of 0.2 ␮g/dL, 3.0 pmol/L, and 5.7 pg/mL,
respectively [18 –20].
 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324 319

Fig. 1. Mean ⫾ SEM 24-h profile of serum cortisol concentrations from healthy horses measured in May (A; n ⫽ 18) and September (B; n ⫽
8) are shown. Red dashed lines denote the Midline-Estimating Statistic of Rhythm (MESOR). Arrows represent the circadian rhythm acrophase
(maximal value in the rhythm). Black bars on x-axis denote hours of darkness. Panel C shows the effect of age on the area under the curve (AUC)
and CV of the diurnal plasma cortisol concentrations from healthy horses. Significantly (*P ⬍ 0.05) greater CV values were observed in the old
horses than in younger horses.

2.3. Statistical analyses 3. Results

Diurnal hormone profiles were analyzed for circa-
dian rhythmicity by cosinor analysis [21]. The calcula-
tions were performed with the SAS (version 9.1; SAS
Institute, Cary, NC) NLMIXED procedure. Rhythm
detection was considered to be statistically significant
at P ⬍ 0.05. Circadian variables such as acrophase,
MESOR, and amplitude were extracted from the cosi-
nor analysis. The acrophase represents the clock time of
the maximum value of the curve. The MESOR is the
true mean of the oscillating variable over its entire
period. Amplitude is half of the difference between the
maximal and minimal value in the curve. Residual
standard deviations (RSDs) were estimated for each
rhythm, representing the variation around the individ-
ual curves. Horse standard deviations (HSDs) were also
estimated, representing the variation across horses. Cir-
cadian data are presented graphically as the mean ⫾
SEM.
In addition to cosinor analysis, the area under the
curve (AUC) and CV of individual horse raw data were
calculated for each hormone. Normality of the data was
tested with the Kolmogorov-Smirnov test. The Pearson
correlation was used to evaluate the association be-
tween cortisol and ACTH concentrations. The mean
AUC and CV of aged horses (ⱖ 20 yr) was compared
with that of adult horses (⬍ 20 yr), and the AUC and
CV in May were compared with that observed in Sep-
tember by using the Student t test. Results are reported
as mean ⫾ SEM, and values P ⬍ 0.05 were considered
significant. These analyses were conducted with the use
of statistical software program (GraphPad Prism 5.01;
GraphPad Software, San Diego, CA).
3.1. Circadian and circannual rhythms of cortisol,
ACTH, and ␣-MSH
The 24-h serum cortisol concentration profiles for
May and September are shown (Fig. 1). Cosinor anal-
ysis showed circadian periodicity for cortisol concen-
trations in both May and September (P ⬍ 0.001 and
P ⬍ 0.01, respectively). In May, the cortisol circadian
rhythm had a MESOR of 6.02 pg/mL, amplitude of
0.67 pg/mL, and acrophase at 6:40 AM. The RSD and
HSD were 1.27 and 1.38, respectively. The cortisol
rhythm from September had a MESOR of 5.12 pg/mL
with an amplitude of 0.78 pg/mL and acrophase occur-
ring at 6:27 AM. This rhythm had an RSD of 1.11 and
an HSD of 2.18. All circadian variables and their re-
spective SEs are summarized in Table 1. Test of cosi-
nor pooling found a significant seasonal difference with
a greater cortisol MESOR observed in May (P ⬎
0.001). The amplitude and acrophase were not signifi-
cantly different between seasons (Table 1). No differ-
ence in MESOR, amplitude, or acrophase was observed
between adult (⬍ 20 yr) and aged (ⱖ 20 yr) horses
(Table 2). Area under the curve for cortisol did not
differ by age; however, the CV was significantly
greater in the adult horses than in the aged horses
(22.81% ⫾ 2.01% vs 16.07% ⫾ 1.558%; P ⬍ 0.05;
Fig. 1).
The seasonal diurnal profiles of plasma ACTH are
shown (Fig. 2). No significant circadian rhythm was
detected in May (P ⫽ 0.307). However, a circadian
rhythm (P ⫽ 0.049) with low amplitude (3.03 ⫾ 1.21
pg/mL) was found in September with a MESOR of
320 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324

Table 1 ated with PPID at second evaluation, 2 yr after sample

Summary of cortisol circadian rhythm variables obtained by collection. Horses that were euthanized before this date
cosinor analysis and season.
were assessed to be normal by postmortem histologic
Variable Value ⫾ SEM P for cosinoar pooling evaluation of the pituitary gland.
MESOR (pg/mL) ⬍0.001 Of the remaining 46 horses, 32 had a ⬍30% differ-
May 6.02 ⫾ 0.37
ence between morning and evening serum cortisol con-
September 5.12 ⫾ 0.78
Amplitude (pg/mL) 0.727 centrations. Therefore, using circadian cortisol rhythm,
May 0.67 ⫾ 0.13 70% of horses tested falsely positive for PPID.
September 0.88 ⫾ 0.23
Acrophase (rad) 0.863
May 1.76 ⫾ 0.20 4. Discussion
September 1.65 ⫾ 0.27
RSD (pg/mL) In this study, cosinor analysis was used to evaluate
May 1.62 whether pituitary and adrenal hormones exhibit circa-
September 1.27
HSD (pg/mL) dian rhythmicity in horses. Cosinor analysis is often
May 1.38 used to analyze biological data for the presence of a
September 1.52 cyclical rhythm. This analysis uses the least squares
Note: Significantly (P ⬍ 0.001) greater MESOR (Midline-Estimating method to fit a cosine wave to time series observations
Statistic of Rhythm) values in May (n ⫽ 18) than in September (n ⫽ and to determine the statistical significance of the fitted
8). No other significant seasonal differences were observed. Acro- curve [1]. For circadian rhythmometry, the cycle length
phase is the clocktime of maximal value in the curve and is repre-
sented in radians [time of day (h) ⫽ radians ⫻ (24/2␲)].
(period) is set at 24 h, and the fitted curve is used to
Abbreviations: RSD, residual standard deviation; HSD, horse stan- estimate rhythm variables such as MESOR, amplitude,
dard deviation. and acrophase. Pooling tests are conducted for each
variable separately; thus, t tests are used.
Results of our cosinor analysis found significant
49.32 ⫾ 3.80 pg/mL and an acrophase at 11:48 PM.
circadian periodicity for serum cortisol concentrations
There was an effect of season on ACTH daily mean
in horses. Circadian rhythmicity was present in both the
concentration with significantly higher concentrations
spring and fall with the daily peak occurring in the early
observed in September compared with May (49.3 ⫾ 3.8
morning between 6:00 AM and 7:00 AM. This is in
vs 27.1 ⫾ 2.2 pg/mL, respectively; P ⬍ 0.001; Fig. 2).
agreement with previous studies that observed circa-
A seasonal effect was also observed on the hormone
dian variation in equine cortisol concentrations with the
profile AUC and CV. Significantly greater AUCs were
acrophase occurring between 6:00 AM and 9:00 AM
seen in September, and significantly greater CVs were
[3,6,7]. In our study, however, we observed slightly
observed in May (Fig. 2). Daily plasma concentrations
lower amplitudes with approximately 20% difference
of ACTH were weakly but significantly correlated to
between rhythm peak and nadir. Several other studies
cortisol concentrations in both May and September
have reported an inconsistent or absent circadian
(P ⬍ 0.01, r ⫽ 0.31; P ⬍ 0.05, r ⫽ 0.25; respectively).
rhythm for cortisol [22,23]. The discrepancies in the
Diurnal plasma ␣-MSH profiles (Fig. 2) did not
show circadian periodicity in May (P ⫽ 0.075) or
September (P ⫽ 0.187), but there was a significant Table 2
seasonal effect on daily mean concentrations with higher Summary of cortisol circadian rhythms variables for adult (⬍20 yr)
and aged (ⱖ 20 yr) groups.
concentrations in September than in May (86.4 ⫾ 8.77 vs
9.86 ⫾ 0.91 pmol/L, respectively; P ⬍ 0.001; Fig. 2). The Variable Value ⫾ SME P for cosinor pooling
AUC and CV values were also significantly greater in MESOR (pg/mL) 0.099
September (Fig. 2). Adult 6.51 ⫾ 0.47
Aged 5.42 ⫾ 0.46
3.2. Assessment of cortisol rhythmicity as diagnostic Amplitude (pg/mL) 0.256
Adult 1.00 ⫾ 0.16
test for PPID Aged 0.63 ⫾ 0.10
Acrophase (rad) 0.564
Plasma ␣-MSH concentrations were within the ref- Adult 1.48 ⫾ 0.41
erence range for all 50 horses. However, 4 horses had Aged 1.68 ⫾ 0.20
ACTH concentrations above the reference range (53.5, Note: No age-related changes were observed in cortisol rhythm vari-
55.8, 81.7, 53.2 pg/mL). All horses remained healthy, ables.
with no hair coat abnormalities or other signs associ- Abbreviation: MESOR, Midline-Estimating Statistic of Rhythm.
 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324 321

Fig. 2. Twenty-four-hour profile of mean (⫾ SEM) plasma ACTH concentration from healthy horses sampled in May (A; n ⫽ 8) and September
(B; n ⫽ 8) are shown. Daily mean concentration is represented by the red dashed line. Black bars on x-axis denote hours of darkness. Panel C
shows the effect of season on the area under the curve (AUC) and CV for the diurnal plasma ACTH concentrations from healthy horses. A
significantly (***P ⬍ 0.001) greater AUC was observed for daily concentrations of ACTH in September (n ⫽ 8) than in May (n ⫽ 8) and a
significantly (**P ⬍ 0.01) greater CV was observed in May. Twenty-four-hour profile on mean (⫾ SEM) plasma ␣-melanocyte-stimulating
hormone (␣-MSH) concentration from healthy horses sampled in May (D; n ⫽ 8) and September (E; n ⫽ 8) are shown. Daily mean concentration
is represented by the red line. Significantly (P ⬍ 0.001) greater mean daily values were observed in May (n ⫽ 8) than in September (n ⫽ 8). Black
bars on x-axis denote hours of darkness. The effect of season on the AUC and CV for the diurnal plasma ␣-MSH concentrations in healthy horses
is shown in Panel F. A significantly (***P ⬍ 0.001) greater AUC was observed for daily concentrations of ␣-MSH in September (n ⫽ 8) than
in May (n ⫽ 8). A significantly (***P ⬍ 0.001) greater CV was observed in September.

published literature have been attributed to the fragility
of the equine cortisol circadian rhythm which can be
affected by minor stressors, environmental factors, and
variations between experimental designs [7].
Season-related differences were found in the cortisol
rhythm’s MESOR with a higher concentration seen in
May. A previous study found no seasonal difference in
equine cortisol daily means but also observed no cir-
cadian rhythm for this hormone [22]. Seasonal changes
in the activity of the hypothalamic-pituitary-adrenal
axis have been reported in multiple species, including
humans [24], rodents [25], deer [10], sheep [26], and
goats [27]. In the ground squirrel, which is a highly
seasonal rodent, circannual changes in cortisol are as-
sociated with prehibernation and changes in body mass
[25]. Red deer also show seasonal changes in mean
cortisol concentrations and adrenal gland responsive-
ness with lower concentrations observed in autumn
when they are in reproductive quiescence. The physi-
ological significance of seasonal fluctuations in cortisol
concentrations is unclear and may differ with species.
Cortisol plays a significant role in energy metabolism
and stress response; therefore, these seasonal fluctua-
tions in horses could possibly occur in anticipation of
changes in food availability and energy demands that
take place in the fall and winter months. Serum cortisol
concentration also may vary in response to stressful
environmental conditions, such as excessive heat, cold,
or humid conditions [28]. However this was unlikely to
be a factor in the present study because the horses were
tested in May and September when the ambient envi-
ronment conditions were similar.
In Soay rams [26], a marked seasonal increase in
cortisol occurs in June, but it is accompanied by a
seasonal increase in plasma ACTH. Results from our
study, however, indicate a dysynchronous relation be-
tween equine cortisol and ACTH because cortisol con-
centrations were lowest in the fall when ACTH is at its
annual peak. The mechanisms responsible for the ob-
served dissociation are unknown but could be the result
322 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324
of alterations in adrenal responsiveness, changes in
cortisol metabolism, or seasonal variations in ACTH
bioactivity.
Aging is associated with a loss of complex variabil-
ity in biological functions as well as a loss of adapt-
ability to physiological stressors [29,30]. In humans, an
age-associated decline in adaptive capabilities has been
observed in cardiovascular output, muscle function,
immune response, and hormone secretion. In hormone
secretion, aging is associated with decreased rhythm
amplitude, loss of temporal synchrony, changes in daily
mean concentrations, and decreased variability or re-
sponsiveness [16]. The circadian rhythm of cortisol in
humans has been shown to undergo age-related phase
shifting as well as decreased amplitude [9,16]. In dogs,
aging is associated with a loss of cortisol circadian
rhythmicity [4]. In the present study no difference in
MESOR, amplitude, acrophase, or AUC was found
between adult and aged horses. However, CV was sig-
nificantly lower in the aged group. This decreased vari-
ability might be a result of decreased adrenal gland
responsiveness, which is similar to what has been re-
ported in humans. This change could also lead to blunt-
ing or complete loss of rhythm as reported in aged
dogs. The lower cortisol variability observed in aged
horses could also cause a decreased percentage differ-
ence between daily peak and nadir, therefore, compli-
cating the use of circadian rhythmicity as a diagnostic
test for PPID in an aged horse population.
The present study is the first to evaluate the daily
pattern of equine plasma ACTH concentrations from
consecutively drawn plasma samples. We found that
diurnal ACTH concentrations lack a well-defined
rhythm and show high variability within and among
sampled horses. Our findings are similar to a previous
study that evaluated the diurnal pattern in plasma
ACTH from samples collected 27 h apart [31]. Al-
though ACTH lacked a well-defined rhythm, it was
correlated with daily concentrations of cortisol. In the
present study, failure to detect a significant rhythm for
ACTH could be attributed to the high variation ob-
served around the fitted curve. High interanimal vari-
ability in daily peaks as well as hormone pulsatility
could potentially mask an underlying circadian rhythm
of low amplitude. Another possible explanation for the
lack of ACTH rhythmicity in the presence of cortisol
rhythm is that adrenal receptiveness or sensitivity to
ACTH might exhibit diurnal fluctuations.
As expected, we found a significant seasonal differ-
ence in the mean daily ACTH concentrations and AUC,
with higher values observed in September. This circan-
nual rhythm in ACTH secretion has been previously
documented [11,32], and our observed concentrations
were similar to those reports. The reason for the sea-
sonal increase in ACTH concentrations is unclear but
has been associated with changes in metabolism and
reproductive function [2].
Circadian periodicity in plasma ␣-MSH has been
reported in cats [33] and rats [34], and pulsatile secre-
tion has been observed in dogs [35]. In the present
study, plasma concentrations of ␣-MSH in horses had
no circadian periodicity. These results support the find-
ings of a previous study that found no significant dif-
ferences among ␣-MSH concentrations from plasma
samples collected at 8:00 AM, 12:00 PM, and 4:00 PM
[20].
In agreement with previous reports [20], we ob-
served a significant seasonal difference in mean daily
␣-MSH concentrations and AUC, with higher concen-
trations observed in September. The CV was also sig-
nificantly greater in September. The difference in vari-
ability is in part, associated with the seasonal increase
in ␣-MSH that coincides with that time of the year. The
rise in plasma ␣-MSH that occurs in the fall potentially
modifies various physiological processes in anticipa-
tion of the winter months. Although work in seasonal
rodents suggests that melanocortin tone is not respon-
sible for maintaining the decreased appetite and low
body weight that occurs in short days, ␣-MSH may
have a role in resetting metabolic tone from one of
increased consumption during long days to one of de-
creased intake in short days when food is anticipated to
be scarce [36,37]. Therefore, the greater variability in
␣-MSH concentrations observed in the autumn could
be associated with interanimal variation in pars inter-
media responsiveness to seasonal cues as well as re-
sulting thriftiness in the winter months.
Results of our 8:00 AM and 5:00 PM cortisol sam-
pling study found that 32 of the 46 sampled horses had
⬍30% difference between the morning and evening
cortisol concentrations, with an overall mean difference
of 28% between peak and nadir. On the basis of the
report suggesting the use of this method as a diagnostic
test [15], 70% of sampled horses would be false posi-
tive for PPID. Cortisol secretion can be affected by
minor perturbations, including stress response from
sampling, food anticipation, exercise, and environmen-
tal change. In addition, the cortisol circadian rhythm
has low amplitude; therefore, slight alterations in adre-
nocortical steroidogenesis can result in masking or
blunting of the underlying endogenous rhythm. Our
results suggest that blunted cortisol circadian rhythm
 M. Cordero et al. / Domestic Animal Endocrinology 43 (2012) 317–324
commonly occurs in clinically and diagnostically non–
PPID-affected horses. Because of the poor specificity,
measuring cortisol circadian rhythm cannot be consid-
ered an appropriate diagnostic or screening test for
PPID; therefore, further work to quantify the sensitivity
of the test was not pursued.
Diagnosis of PPID is difficult, because all avail-
able tests are plagued by false-negative results early
in the course of disease. Therefore, it is possible that
some horses included in this study were in a preclin-
ical stage of PPID despite all horses having endog-
enous plasma concentrations of ACTH and ␣-MSH
within reference range. In the present study, horses
were re-evaluated by physical examination 2 yr after
initial sample collection. Re-evaluation of horses
years after diagnostic testing is likely the most rigorous
method of ensuring the absence of preclinical cases
within a study. In our study, live horses remained free
of hair coat abnormalities or other clinical signs of
PPID, and those that died were determined free of PPID
by postmortem examination.
In summary, our results indicate that cortisol has a
circadian rhythm of secretion in horses. This rhythm is
of low amplitude and shows a seasonal variation in
daily mean. These findings emphasize that monitoring
the cortisol circadian rhythm for the diagnosis of PPID
is inadequate. Blunted amplitude occurs commonly in
healthy horses; therefore, a decreased difference be-
tween morning and afternoon cortisol is not specific for
PPID. This test is further confounded by the decreased
cortisol variability observed in aged horses. Our results
indicate the absence of a circadian rhythm of plasma
ACTH and ␣-MSH concentrations as well as a lack of
correlation between immunonoreactive ACTH and cor-
tisol concentrations in the fall. Further work is required
to determine the mechanisms responsible for the ob-
served dyssynchrony between ACTH secretion and its
downstream effects on adrenal cortisol release.
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