J Comp Physiol B
DOI 10.1007/s00360-007-0241-9ORIGINAL PAPER
Annual pattern of fecal corticoid excretion in captive Red-tailed
parrots (Amazona brasiliensis)
Lucyenne G. Popp · Patrícia P. SeraWni ·
Angela L. S. Reghelin · Katherinne Maria Spercoski ·
James J. Roper · Rosana N. Morais
Received: 3 May 2007 / Revised: 10 December 2007 / Accepted: 12 December 2007
© Springer-Verlag 2007
Abstract Annual patterns of fecal corticoid excretion
were analyzed in the threatened Red-tailed parrot (Ama-
zona brasiliensis) in captivity. Corticoid concentration over
the 15 months of the study (mean § standard error,
12.6 § 0.32 ng g¡1, n = 585) was lowest around May (the
southern Fall), and greatest around September (late winter),
just prior to their normal breeding period. Corticoid excre-
tion follows a seasonal pattern best explained by reproduc-
tive cycles rather than climate, although climate may be
involved in the timing of corticoid excretion. Fecal cortic-
oids also show promise as a tool to measure stress levels.
We demonstrate that fecal corticoid measurement is a sim-
ple, yet eYcient method for monitoring adrenocortical
activity in captive, and perhaps wild, parrots. Monitoring
adrenocortical activity can inform researchers about
imposed stress in captivity, whether pair-bonds are forming
in captive birds, and of the timing of breeding both in cap-
tivity and in nature.
Keywords Red-tailed-parrot · Fecal corticoids ·
Reproduction · Seasonality · Conservation
Introduction
Parrots (Psittacidae) are among the most threatened birds in
the world, especially in the Neotropics. With more endan-
gered species than any other bird family, »30% of neotrop-
ical parrots were considered threatened by the early 1990s
(Collar et al. 1994). The Red-tailed parrot (Amazona brasil-
iensis) is threatened and endemic in southeastern Brazil.
Extensive poaching (for the national and international bird
trade) and habitat loss are its most serious threats (Wright
et al. 2001). Despite heavy trapping pressure in the early
1990s, the population is now believed to be relatively sta-
ble, and has consequently been downlisted from endan-
gered (Collar et al. 1994) to vulnerable by the IUCN (Bird
Life International 2004). Understanding the threatened sta-
tus of any species requires knowledge of population
dynamics, especially breeding cycles.
Reproduction in birds carries predictable and unpredict-
able physical, energetic and social demands that stimulate
behavioral and physiological responses that are mediated
mostly through the hypothalamic–pituitary–adrenal (HPA)
axis. Thus, monitoring HPA activity has great potential for
understanding reproductive cycles and stressful periods. In
birds, physiological responses to environmental challenges
involve the secretion of glucocorticoids, primarily cortico-
sterone (Oglesbee et al. 1997; Bentley 1998; Frigerio et al.
2004). For example, plasma corticosterone levels are asso-
ciated with behavior in tits and owls (Silverin 1997; Belt-
hoV and Dufty 1998) and with social interactions (e.g.,
courtship and parental care, Hirschenhauer et al. 2000;
Breuner and Orchinik 2000). Birds in captivity may face
Communicated by G. Heldmaier.
L. G. Popp
Curitiba Zoological Park, Curitiba, PR, Brazil
P. P. SeraWni
Society for Protection of Wildlife and Environmental
Education (SPVS), Curitiba, PR, Brazil
A. L. S. Reghelin · K. M. Spercoski · R. N. Morais (&)
Department of Physiology,
Federal University of Paraná, Sector of Biological Sciences,
PO Box 19.031, Curitiba, PR 81530-990, Brazil
e-mail: moraisrn@ufpr.br
J. J. Roper
Department of Zoology,
Federal University of Paraná, Curitiba, PR, Brazil123

J Comp Physiol Bunique challenges that force behavioral interactions, such
as limited space, poor diet and greater proximity of individ-
uals. Hence, stressful conditions peculiar to captivity may
arise and perhaps may explain the low breeding success of
many species in captivity. Since the Red-tailed parrot has
few reports of successful breeding in captivity (Low 2006)
understanding the factors that inXuence allostasis through
monitoring HPA activity may be a very useful tool. How-
ever, monitoring such activity must be non-invasive so as
to not introduce other stressful factors that may cause their
own reactions, further reducing chances of breeding in cap-
tivity.
Fecal steroid analysis may oVer a useful non-invasive
method. Since avian steroids are metabolized by the liver
and excreted as conjugates via bile into the gut or by the
kidneys, non-invasive monitoring of adrenal function is
possible by extracting steroid metabolites from feces (Was-
ser et al. 1997; Touma and Palme 2005). This approach is
particularly useful for monitoring stress, since feces can be
easily collected without disturbing the birds. Monitoring
HPA can potentially measure adrenal response to a wide
array of stressors (Morais et al. 1997; Touma and Palme
2005). The inXuence of acute and chronic stress, seasonal-
ity, reproductive status, age, migration and social organiza-
tion on adrenal gland activity have all been monitored by
fecal corticosteroids analysis in a variety of bird species
(Wasser et al. 1997, 2000; Kortrschal et al. 1998, 2000;
Hirschenhauser et al. 2000, 2005; Carere et al. 2003; Dehn-
hard et al. 2003; Millspaugh and Washburn 2004; Scheiber
et al. 2005; Touma and Palme 2005).
In this study we examined fecal corticosteroids in a cap-
tive group of Red-tailed parrots to test that fecal corticoids
may be used to indicate HPA axis activity in response to
climate, reproduction and housing conditions. SpeciWcally,
we tested whether fecal corticosteroid variability indicates
a response to seasonality or reproduction in captive
parrots.
Methods
Study species and captivity
The Red-tailed parrot (or Red-tailed Amazon) occurs in the
narrow coastal region of the states of São Paulo, Paraná and
Santa Catarina (Scherer-Neto 1989; Cavalheiro 1999;
Sipinski 2003). The breeding season begins in late August,
with usually one nest per year. Sexual maturity and perma-
nent pair-bonds occur at 3–4 years of age. Parental care is
shared by both members of the pair (Scherer-Neto 1989;
Rupley 1997; Cavalheiro 1999; Sipinski 2003).
Thirteen captive-reared adult parrots (7 males, 6
females, 7–8 years of age) were studied during 2000–
2003. Parrots were kept at the Curitiba Zoological Park
(25°S, 49°W) after recovery from illegal captivity in the
southern Brazilian state of Paraná. In the zoo, all parrots
were housed together away from public exhibits. At
3 months prior to this study they were separated into pairs
(based on behavior) and each pair was placed in its own
enclosure. Enclosures (4.10 £ 2.5 £ 2.6 m) allowed Xight
and were mostly open-air, including a covered area for
shelter from rain and wind, and a breeding room
(1.2 £ 2.5 £ 2.6 m) containing a nest box. One solitary
male was housed separately (5.3 £ 2.5 £ 2.6 m). Adjacent
enclosures permitted birds to see, hear and interact only
with other parrots in the study. Daily care included feeding
and cleaning. Diet comprised fruit, nuts and grain, supple-
mented with commercial dry dog food. Water was pro-
vided ad libitum. Parrot health was monitored regularly by
the zoo veterinarian.
Fecal sampling
Feces were collected weekly twice from October 2003 to
December 2004. During warmer weather, pairs were sepa-
rated the evening prior to fecal sampling to determine the
source of the feces, and feces were collected in the morn-
ing. Females were held in the breeding room with the nest
box and males remained in the open-air part of the enclo-
sure. The door to the breeding room was opened after fecal
collection to allow the male and female free passage to all
parts of the enclosures. In colder weather (4 June–17
August) birds (3 pairs and 1 single male) were held in sepa-
rate cages overnight in the breeding room to separate and
collect fecal samples. Samples were stored at ¡20°C in
plastic bags until extraction and analysis. We assume that
this procedure caused little or no reaction in the birds, since
all were hand-reared, and they showed no adverse behav-
iors to the procedure.
Corticosteroid extraction and assay
Steroids were extracted following Schwarzenberger et al.
(1991) and Javorouski (2003), with modiWcations. Whole
feces homogenates were extracted (it was impossible to
separate feces and uric acid components). An aliquot of
»0.5 g of wet sample was transferred to a tube of 5.0 mL
90% ethanol in phosphate-buVered saline (PBS). After
overnight inverted-mixing, tubes were centrifuged
(1,500g/15 min) and extract diluted in PBS (1:1) and fro-
zen (¡20°C) until analysis. Fecal corticosteroid concen-
trations were measured in duplicate 100
L aliquots of
fecal extracts using a commercial double antibody 125I-
corticosterone radioimmunoassay (Corticosterone ICN
Biomedicals, USA). The antiserum description included
the following cross-reactivities: 100% corticosterone,123

J Comp Physiol B 0.34% desoxycorticosterone, 0.10% testosterone; 0.05%
cortisol, 0.03% aldosterone, 0.02% progesterone, 0.01%
androstenedione. The assay was validated for other bird
species (Wasser et al. 2000) and for the Red-tailed parrot
by demonstrating parallelism between dilutions (1:2; 1:4;
1:10; 1:20; 1:40; 1:80) of a pooled sample of fecal extracts
(n = 120) from all individuals and the standard curve
(r2 > 0.97 for both regression lines, slope ¡0.18 for stan-
dards and ¡0.15 for fecal extracts) and by the recovery of
exogenous corticosterone (12.5–500 ng mL¡1) added to
the extract pools (y = 0.94x ¡ 0.38, r2 = 0.99, P < 0.05).
Testing the physiological relevance of fecal glucocorti-
coids in the studied birds was not possible due to limita-
tions of working with few individuals of a threatened
species. However, it was tested in adult Amazona aestiva
(3 males, 3 females). Fecal corticoids increased (P < 0.01)
from the mean value of 5.8 § 0.3 ng g¡1 3 days before to
9.7 § 1.4 ng g¡1 on the day after a physical restraining for
blood collection. A total of 11 assays were used for valida-
tion and fecal extract analysis. Assay sensitivity, based on
89% maximum binding, was 12.5 ng mL¡1. The mean
inter-assay coeYcient of variation for high and low con-
trols (provided by the company) and for a fecal extract
control was 6.1% and the intra-assay coeYcient of varia-
tion was 3.2%. Fecal corticosteroid concentrations were
corrected for the dilution factor of the assay standards
(1:200) and are expressed as nanogram per gram¡1 of wet
feces.
Environment
Average daily temperature, relative humidity and rainfall
were obtained from the Meteorological System of Paraná
(SIMEPAR, http://www.simepar.br). Photoperiod was cal-
culated for this latitude as the interval between sunrise and
sunset. Partial correlation coeYcients were used to test for
climate and corticoid associations.
Data analysis
Fecal corticoid concentration was compared among sexes
and pairs over time by repeated measures analysis of vari-
ance. Analysis of Variance was used to examine seasonal
variation in overall fecal corticoids. Fecal corticoid con-
centrations were natural log transformed to Wt the assump-
tions of the analyses (normality of residuals and equality
of variances). Seasonality was also tested as an inXuence
on corticoids by correlation with the environmental vari-
ables. We followed Marques et al. (2004) to examine
climate by including lag-times of 30 and 60 days in the
analysis and monthly averages of daylength, temperature
and relative humidity. All tests used a signiWcance level
of 5%.
Results
Fecal corticoids
Fecal corticoid excretion varied over time (F14, 112 = 3.67,
P < 0.05). Also, pairs tended towards similar trends over
time (Repeated Measures ANOVA, P > 0.1 for sex-pair,
Fig. 3). May had the lowest corticoid levels
(6.7 § 0.4 ng g¡1) and August and September the highest
levels (12.4 § 0.6 and 13.2 § 0.6 ng g¡1, respectively).
Among seasons, winter had clearly the highest
(mean § 95% conWdence interval, 10.7 § 0.52 ng g¡1;
Tukey HSD, P < 0.05) concentration, and Fall the lowest
(7.6 § 0.51, Tukey HSD P < 0.05), while spring and sum-
mer were similar and intermediate (8.8 § 0.44; Fig. 2).
Corticoid levels were correlated with photoperiod after a
lag of 2 months (Fig. 1; partial r = 0.8, n = 13, P < 0.05).
Since photoperiod and temperature are so highly correlated,
the eVects of temperature and photoperiod cannot be sepa-
rated (Marques et al. 2004). Humidity apparently has no
additional eVect on corticoid levels (Fig. 2).
Behavior
Corticoid excretion in females and males of pair number 1
(n = 14), 2 (n = 15) and 5 (n = 15) pairs (circles) were cor-
related over time (all r > 0.58, P < 0.05, Fig. 3), suggesting
within-pair breeding synchrony. Corticoid excretion in the
unpaired male was also correlated with that of two females
(3, r = 0.58 and 5, r = 0.95, both P < 0.05). Since pairs
Fig. 1 Monthly averages for fecal corticoid concentrations, daily tem-
perature, and photoperiod during the 15 months of this study. Cortic-
oids were most strongly correlated with photoperiod after a lag time of
2 months, but photoperiod and temperature are strongly correlated as
well, so the relative importance of one over the other cannot be deter-
mined (see text)
5
10
15
20
25
Month
M
on
th
ly
M
ea
ns
Corticoids (ng/g)
Photoperiod (hours)
Temperature (oC)
2003 2004
O ON ND DJ F M A AM J J S123

J Comp Physiol Bvaried somewhat, combining pairs for an overall analysis
masks the trends shown by each pair.
During this study, only one female laid eggs that were
infertile and laid outside the nest box (female of pair num-
ber 2, Fig. 3). All individuals molted in early summer (3–12
December 2003, again 4–17 December 2004). One female
(pair number 4; kept in cages) plucked her own feathers
during winter (a common behavior for parrots in captivity,
Bauck 1997; Rupley 1997), besides which all individuals
seemed to exhibit normal behaviors of captive birds. There
was no indication in any analysis overall or by individual of
an inXuence due to routine care-taking and husbandry
practices.
Discussion
Red-tailed parrot fecal corticoid excretion patterns follow
the predicted hormonal cycle and demonstrate that this non-
invasive method can be useful for monitoring hormonal
cycles, including reproduction, in captive birds. Corticoid
levels rose after May, when courting behaviors may begin
(Sipinski 2003, Figs. 1, 3). Corticoid levels began to
decline after September, when this species usually has
occupied nest-sites and possibly begins egg-laying (Sipin-
ski 2003). Although these birds did not breed, we believe
that these results are promising for monitoring parrots. This
breeding cycle explains the correlation between photope-
riod and corticoid levels because they are both annual
cycles (e.g., Marques et al. 2004).
In this Wrst use of fecal corticoids to understand annual
cycles in the family Psittacidae, we Wnd the methods for
extraction and assay of the corticosterone metabolites to be
suYciently simple, cost-eVective and valuable for monitor-
ing adrenal activity in the Red-tailed, and probably other
parrots. We show that fecal corticoid excretion is seasonal
in which hormonal changes precede reproduction. We sug-
gest that temperature or photoperiod (or both) may trigger
the hormonal changes associated with reproduction, and
that completion of reproduction may trigger molting and
return to stasis. Thus, correlations with climate should not
be very strong or perhaps should be thought of as initiators
but not terminators of hormonal activity. Further study will
be required to determine the exact nature of the inXuence of
climate on hormonal activity, or whether photoperiod alone
is suYcient to initiate reproductive activity.
These subtle climate issues are not vital, however, for
monitoring annual cycles in parrots. Fecal corticoids
increased in the spring, when this species of parrot begins
its breeding season (Juniper and Parr 1998; Sipinski 2003).
Excretion reached a peak during the interval when parrots
initiate nests and declined when nests should have been in
use (Figs. 1, 3). Also, the slight rise in November may be in
anticipation of molt, suggesting a possible extra metabolic
cost of molting (Blem 2000). Prior to these changes and at
the beginning of the experiment, a time interval of variable
(especially for pairs 1, 4 and 6 in Fig. 3) excretion (Octo-
ber–May) may indicate acclimation of some birds to new
surroundings.
The very strong correlation between the unmated male
and female number 5 (r = 0.95), while unexplained, may
indicate pair formation that was not obvious to the animal
handlers. If so, a simple test could improve our identiWca-
tion of pair formation for captive birds. Continual monitor-
ing and comparing correlation coeYcients between
diVerent possible mating combinations could suggest pair
formation in birds that are not in the same cage. This could
then be tested by placing the birds together and analyzing
behavior. These behaviors and correlations oVer additional
tools for understanding mating behaviors.
Here, we suggest biological and behavioral implications
of fecal corticoid excretion in parrots. We recognize that
detailed analysis, beyond the scope of this paper, will be
required to test these possibilities, yet current understand-
ing of the physiology of reproduction supports our views.
For example, a reduction in basal activity of the HPA axis
of the Red-tailed parrot may occur during the non-breeding
season, as observed in greylag geese (Kotrschal et al.
1998). Thus, HPA axis activity may suggest two patterns—
homeostasis when not breeding, and reproductive during
the remainder of the year. The reproductive period perhaps
Fig. 2 Mean (§95% conWdence interval) overall corticoid concentra-
tions compared between seasons. Winter had clearly the highest con-
centration, and Fall the lowest, while spring and summer were similar.
The ANOVA was based on log-transformed concentrations, which
were then back-transformed for illustration purposes
Spring Summer Fall Winter
7
8
9
10
11
12
Season
Co
rti
co
id
Co
nc
en
tra
tio
n
(ng
/g)123

J Comp Physiol B would be preceded by a gradual rise associated with prepa-
ration for breeding. This “preparation” would be associated
with the various behaviors that precede actual egg-laying.
Pair-bonding increases, males feed females, nest-sites must
be located and modiWed; all of these behaviors may be
associated with increasing hormone levels. Thus, we expect
fecal corticoids to change prior to the actual onset of egg-
laying. Once egg-laying occurs, metabolic and behavioral
demands change, and so a concomitant decline in fecal cor-
ticoids is expected. We will require detailed studies of nest-
ing behaviors to conWrm these predictions.
Similar patterns have been found in other species where
hormones have been studied. In geese, increased hormonal
reactivity is linked to metabolic and behavioral needs of
reproduction, and confrontation with unknown individuals
caused increased fecal corticoids only in the spring, during
the breeding season (Kotrschal et al. 2000). Also in geese,
similar to patterns observed here, fecal corticoid excretion
is associated with breeding, with higher levels of corticoids
in males during mating and nesting, followed by a decline
during incubation of eggs (Hirschenhauser et al. 2000).
Finally, pair bond formation improves birds “well-being”
(von Holst 1998). In these parrots, while no reproduction
occurred, all individuals molted. Molt, in December, was
synchronous at the end of the breeding season and was
anticipated by increased excretion of fecal corticoids in
most individuals in November (Fig. 1, 3). The speciWc hor-
monal and metabolic changes associated with this event in
parrots are still unknown.
In summary, fecal corticoids can provide a useful, non-
invasive method for monitoring threatened and endangered
birds in captivity and, perhaps with modiWcation, in nature.
The energetically demanding reproductive and molt cycles
are especially important for monitoring, since they are
Fig. 3 Deviations from average
corticoid excretion for individu-
als, grouped by pair and sex over
the 15 months of this study (sol-
id vertical lines are winter and
summer solstices, dashed lines
are spring and autumnal equi-
noxes). Corticoid excretion in
females (open symbols) and
males (Wlled symbols) of pairs
number 1, 2 and 5 (circles) were
correlated over time (all
r > 0.58, P < 0.05), which sug-
gests breeding synchrony. The
female in pair number 2 laid
eggs in November 2004. The fe-
male in pair number 4 plucked
her own feathers, and her corti-
coid levels were most strongly
correlated with those of the un-
paired male, who is shown along
side her as a hatched-square.
Pairs identiWed by triangles
were not synchronous (uncorre-
lated), although pairs 3 and 4
(despite the stronger correlation
of the female with the unpaired
male) displayed apparent court-
ing behaviors. While the exact
pattern over time varied among
pairs, the magnitude (diVerence
from each individual mean) of
excretion was similar for all
pairs
-0.6
-0.3
0
0.3
0.6 1Spring Summer Fall Winter Spring
-0.6
-0.3
0
0.3
0.6
Laid eggs
2
-0.6
-0.3
0
0.3
0.6 3
-0.6
-0.3
0
0.3
0.6
D
iff
er
en
ce
fr
om
In
di
vid
ua
l A
ve
ra
ge
Square - Unmated Male
Female plucked
feathers
4
-0.6
-0.3
0
0.3
0.6 5
Nov 03 Jan 04 Mar 04 May 04 Jul 04 Sep 04 Nov 04
-0.6
-0.3
0
0.3
0.6
Sample Period
6123

J Comp Physiol B Wright TF, Toft CA, Enkerlin-HoeXich E, Gonzalez-Elizondo J,
Albornoz M, Rodrigues-Ferraro A, Rojas-Suarez F, Sanz V,
Trujillo A, Beissinger S, Berovides V, Gálvez XA, Brice AT,
Joyner K, Eberhard J, Martuscelli P, Gilardi J, Koening SE,
Stoleson S, Meyers M, Renton K, Rodriguez AA, Sosa-Asanza A,
Vilella FJ, Wiley JW (2001) Nest poaching in neotropical parrots.
Conserv Biol 15:710–720123