D. Michael Fry, PhD; L. A. Addiego, MS
This paper is a short summary of the effects of stress on birds and the
role of corticosterone in the stress response. Following the summary is a description of a field
study of oil exposure to seabirds, the effects of an environmental stressor, and the results on
reproductive behavior and physiology. For this study we developed methods to monitor steroid
hormones in the droppings of wild birds, an extremely powerful technique which is also
applicable to studies of birds in captivity.
Seabirds, in general, are very active, easily disturbed birds that normally
have very little interaction with humans and are difficult to keep in captivity. Even in the
field it is difficult to work with some species, although there is much species variation in
tolerance to human disturbance. Gulls, guillemots, and puffins learn to accept non-threatening
human observations, while murres and cormorants may panic at the sight of a human even at a
distance. Burrow nesting alcids appear to be more tolerant of disturbance than surface or cliff
nesting species. Gradual habituation to non-threatening human presence has been possible at some
colonies where birds have become aware of the limits of human movements, and sensitive species
are left undisturbed. Most seabird species are very longlived (10-30 years, Clapp et al, '82)
and adults of unknown age do not generally adapt well to captivity. An understanding of the
factors contributing to stress and the physiological responses of birds to acute and chronic
stress may provide insight into minimizing stress in these active species.
Environmental stimuli that are perceived as imposing a threat, either real
or anticipated, result in a stress response. Fowler ('86) lists a large variety of stressors
toward captive wild animals, many of which may not be realized by the casual observer. Human eye
contact, noises (especially those of nearby confined predators), and confinement may be
significant constant stressors capable of disturbing captive birds and mammals. Unexpected
touches, restraint, and collecting blood samples are more intense, acute stressors which are
occasionally capable of causing death in sensitive individuals. Disease organisms, malnutrition,
and heat or cold are miscellaneous stressors that may occur chronically when proper diets or
environmental requirements are unknown or unavailable.
Stress Response in Birds
Adverse stressful conditions elicit a sequence of protective and
compensating responses in healthy animals. The excellent reviews of Munck et al. ('84) and
Harvey et al. ('84) examine in detail the role of adrenal hormones during stress, and have
provided a revaluation of the traditional view of glucocorticoid hormone function.
Acute stress (injury, exposure to toxicants, or fright) in birds evokes a
rapid defense reaction, with stimulation of the sympathetic nervous system and release of
catecholamines (dopamine, epinephrine and norepinephrine) from nerve terminals and from the
adrenal medulla, preparing the animal to respond to the emergency. The adrenergic response may
vary between species, depending upon the proportions of catecholamines released. The type of
defense reaction evoked may be complex, but is usually appropriate to the stress insult.
Mediators other than catecholamines are released in response to different stressors: lymphokines
(including interferons and several lymphocyte stimulating factors) are released in response to
infection; endorphins in response to pain; prostaglandins, bradykinin and histamine with
traumatic tissue damage; vasopressin in response to hemorrhage; and insulin following metabolic
disturbances. Each of these defense mediators is activated to restore homeostasis following
With acute stress a nearly simultaneous release of corticosterone from the
adrenal cortex accompanies the defense response. The release is mediated through activation of
the hypothalamic-pituitary-adrenal axis (HPA), and to a lesser extent, probably directly by the
release of adrenal catecholamines (Harvey et al '84). Activation of the HPA is rapid, with
maximal release of ACTH occurring 2.5- min after the onset of acute stressors in rats (De Souza
and van Loon, '82). A 10-fold or greater increase in plasma corticosterone may be measured
within a few minutes of capture and handling of a bird, illustrating the rapidity and intensity
of the process (Beuvingand Vonder '78, Harvey et al '80).
The function of the stress-induced increase in glucocorticoids is not to
protect against the stress, but to prevent the normal defense reactions from overshooting and
threatening homeostasis (Munck et al '84). Glucocorticoids suppress the action, secretion or
synthesis of all of the above defense mediators. The mechanism of suppression is mediated
through receptors, and it appears that all glucocorticoid receptors are alike. Thus, while
corticosterone is the principal glucocorticoid of birds, the effects will be duplicated by both
synthetic and other natural corticosteroids, though with different potencies based upon receptor
binding affinities and metabolic clearance. Detailed explanations of the mechanisms and examples
of glucocorticoid effects on defense mediators are provided by Munck et a] ('84) and Harvey et
The magnitude of the stress response by an animal is conditioned by prior
experience and anticipation. Captive-raised animals are generally much less stressed by human
contact than wild caught individuals, and the reduced response to stressors may be a reflection
of acquired experience, especially as juveniles. Even with domesticated species, however, rough
handling or close confinement will elicit rapid corticosterone increases (Beuving and Vonder
Chronic Stress and Corticosterone Effects
Corticosterone release, in addition to reducing defense response
overshoot, also acts by negative feedback through the pituitary to suppress those
"nonessential" functions not immediately required for the "flight or fight"
reaction. Consequently, sustained stress may produce chronic high levels of circulating
corticosterone which will suppress several necessary physiological processes, including immune
system function, and reproduction. Chronic stress, therefore, may foster disease and disrupt
normal courtship and breeding behaviors through elevated corticosterone.
Birds under chronic stress require more than normal amounts of
corticosterone to prevent overstimulation of the catecholamine response. With wild or captive
animals of sensitive species the balance between appropriate defense response and maintenance of
homeostasis during stress may become critical. For example, chronic stress may lead to impaired
adrenal cortical responsiveness and an acute stressor may initiate a crisis. Shock (caused by
excessive pressor effects of catecholamines) or fear may occur during the flight or fight
syndrome and can result in death if exaggerated or maintained. Both shock and the behaviors
associated with fear are suppressed by corticosterone (Harvey et al '84), and insufficient
corticosterone release to restore homeostasis may be catastrophic.
Petroleum Exposure as an Example of Toxic Stress
Several acute and long-term toxicological studies with seabirds exposed to
petroleum products under captive conditions have described physiological changes in organ
systems and the relationships between adrenal function and stress (Holmes et al. '78, '79, '80,
Gorsline et al '81, '82' Cavanaugh et al '82, '83). Toxic oil causes hemolytic anemia (Leighton
et al '83, Fry et al '85, '87) and may directly disrupt salt balance, and intestinal function in
combination with adrenal mediated effects.
The complex physiological relationships make stress an important variable in
the toxicological assessment of birds. We have investigated these interrelationships in field
studies of wild, breeding Cassin's Auklets (Ptychoramphus aleuticus) to examine the
effects of petroleum exposure on the physiology and behavior of breeding birds. The studies
provide several useful insights into the reactions to stress by seabirds, and have provided new,
noninvasive, techniques for physiological assessment of wild or captive animals.
A four year field study with Cassin's Auklets was conducted on Southeast
Farallon Island (SEFI), 40 km west of San Francisco, CA to determine the long term effects of
oil exposure on seabird reproduction. Five hundred artificial nesting burrows installed on SEFI
were monitored for this study. The oil selected for study was artificially weathered Santa
Barbara crude oil (Monterey formation, a "sour crude") which did not penetrate the
plumage of the birds and cause loss of waterproofing. Birds were exposed to a single I ml
application of oil to the breast plumage either during courtship or on Day 14 or 15 of
incubation. The breeding success of exposed birds was monitored for 2-4 years.
Sample and Data Collection
Direct measurement of hormones in breeding populations of wild birds is
not a simple task, as severe disturbance usually leads to breeding disruption or abandonment of
nests. Auklets would not tolerate being disturbed for blood sampling in addition to periodic
nest box checks for data on mate fidelity, egg laying, incubation attentiveness and chick
growth. Most birds having only one blood sample taken abandoned their nest for the breeding
season. We therefore utilized excreta of Cassin's Auklets for hormone measurements without
increasing disturbance above that necessary to collect field data on breeding behavior.
The hormone method was based on the principle that steroid hormones
circulating in the blood are removed by the liver and kidneys and excreted in the feces and
urine. The types and amounts of steroids present are correlated with the sex and reproductive
state of the animal (Fry 1983, Czekela et al. '83). The technique of urinary steroid hormone
analysis has been used for investigations of reproductive status of mammals, but has not been
extensively applied to birds.
Auklets were banded during courtship in early spring, prior to egg laying in
19821984. Incubating birds were checked during the day only on the date of lay (day 0) and
days 1, 14, 15, 22, 23, 30, 31, 38, and 39 to identify both members of the pair and to collect
hormone samples. After hatching, boxes were checked on a continuing 8 day schedule during the
night when adults returned to feed chicks. Auklet droppings were collected when birds were
banded or when handled for identification during incubation and chick rearing periods. When a
bird was handled, its tail was placed in the opening of a plastic sample bag, and the bird was
held in that position during the reading of its band. Samples were kept frozen until analyzed.
In 1984, 1480 samples were collected for analysis of estradiol, testosterone, corticosterone and
creatinine. A single dropping was sufficient for measurement of three hormones and
Excreta were solubilzed in acetate buffer, extracted overnight and
centrifuged to remove solids. Aliquots of supernatant were incubated with Bglucuronidase and
aryl sulfatase to hydrolyze conjugates (Erb et al. 1982, Czekala et al. 1983), then extracted
with anhydrous ethyl ether to separate the steroids from interfering substances (Abraham et al.
1977). Antisera were obtained from G. D. Niswender, Colorado State University, Fort Collins,
Creatinine content of the droppings was used as an index of urine
production. Birds excrete both creatine and creatinine at relatively constant rates, and either
should be suitable as an index of urine output. Creatinine assays were based on a commercial kit
(Sigma, Catalog No. 555) with modifications to correct for non-specific absorbance of porphyrins
and crustacean pigments in the feces. Analysis was performed in microtiter plates on a Dynatech
#580 microtiter plate reader at 490 nm.
Hormone assays were processed with a modification of the NIH-RIA program of
Rodbard et al. (1980) which calculated the quantity of hormone in each sample, assay variance,
and limits of assay sensitivity. Field data for each bird were compiled for treatment group,
dosing date, laying date, mate, hatching success, fledging success, relaying date and breeding
success, then combined with hormone data and analyzed relative to dose date or date of egg
The Biomedical Data Program statistical package (BMDP) was used for an
analysis of variance and covariance with repeated measures comparisons between treatment groups
to examine the overall patterns of hormone excretion throughout the breeding season. A more
detailed description of the methods is given in Fry (87).
Effects of Oil Exposure on Breeding
A high proportion of auklets dosed externally with oil prior to egg laying
responded by abandoning the breeding season. Those birds remaining were delayed in egg laying by
more than 20 days in both 1982 and 1984 studies. The delay appeared to be a consequence of
disruption of egg formation and a probable delay in the initiation of growth of new ovarian
follicles. Auklets exposed externally to oil on day 14 or 15 of incubation exhibited a high
frequency of abandonment, low hatching success and low net breeding success. Eggs which became
oiled during incubation had lower hatching success than comparable controls, indicating the
possibility of direct embryo toxicity of oil transferred to eggs during incubation.
Oil exposure caused greater abandonment of pairs breeding for the first time
in nest boxes than for established pairs. Exposure to oil during incubation caused breeding
failure, but many established pairs remained together and laid a second egg. The net breeding
success of this group was higher than for new pairs, both as a result of less abandonment of the
first egg and because of a much higher relaying frequency.
Oil exposure resulted in a lower proportion of female auklets returning in
the year following exposure, but no change in the proportion of males returning. Breeding
failure resulted in many birds changing mates in the second year. Changing mates resulted in
lowered success in hatching and fledging the first egg, reduced relaying attempts and lowered
success in the year after dosing. The data strongly supports the hypothesis that breeding
failure of dosed birds as a result of oil application resulted in mate switching, and that mate
switching resulted in overall lowered breeding success.
Hormone Studies and Correlation with Breeding
The testosterone (T) values of control males are given in Figure 1 to show
the pattern throughout the breeding season. Levels were scattered, but generally high during
courtship prior to egg laying, with a peak at 22 days before lay. The T values dropped to a
plateau at the time of lay, remained at a relatively high level throughout most of incubation,
dropped to a minimum at about the time of chick hatching (38 days of incubation) and remained
low during the chick rearing period (chicks fledge at 38-42 days of age, 76-80 days post-lay).
An increase in T was observed at 70 and 80 days post-lay, in those birds preparing for a second
Corticosterone (C) values of males showed substantial variability, with high
values early in the season during courtship and nest box selection. The levels of C dropped
throughout incubation and the chick rearing period and remained low during the period preceding
the laying of a second clutch. Late in the season the C values were elevated in birds still
feeding and attempting to fledge chicks as other birds departed from the island Female controls
had very similar hormone patterns to males for both estradiol (E) and T throughout the season,
with a plateau and subsequent decline in late incubation and during the chick rearing period. A
peak of E occurred at 78 days post-lay, coincidental with the peak of T in males preceding the
laying of a second clutch egg. The amount of T excreted by females was substantial, about 4-8
fold higher than the amount of E excreted, and especially high during incubation. Corticosterone
pattern showed a high, variable excretion early in the season, followed by a plateau during
incubation and a minimum at 60-70 days postlay.
Hormone Patterns of Auklets Dosed with Oil
Most birds dosed with a single I ml application of oil to the breast
plumage prior to egg laying abandoned the breeding season. Estrogen values of females were very
high at the time of dosing early in courtship, but quickly dropped to unmeasurable levels
within I day after oil exposure. The corticosterone patterns of the same birds remained high
after dosing, reflecting high levels of excretion of adrenal corticosteroids after exposure to
Data from treatment group pairs were examined for significant hormone
differences between treatment groups, between time periods throughout the breeding season, and
for interactions between time periods and treatment groups which would examine differences in
hormone patterns through the breeding season.
Highly significant differences were present between control and dosed
females with respect to both C and E, and between control and dosed males for all hormones.
Figure 2 demonstrates that dosed females excreted much more C immediately following dosing than
controls, a response consistent with elevated circulating levels of corticosterone as a response
to the stress of oil exposure.
Control and dosed females had significant differences in E at the time of
the second clutch. Control birds had higher levels of E consistent with the higher incidence of
laying and hatching second eggs. The highly significant correlations between oil exposure and
lower reproductive hormones is strong direct evidence that oil exposure suppressed reproduction.
The elevated C after dosing is consistent with a response to stress, with gonadal suppression at
the second clutch mediated, at least in part, by the negative feedback effect of corticosterone
on reproduction (Munck et al. 1984, Harvey et al. 1984).
Experienced breeding males also showed highly significant differences
between control and dosed groups. Males did not differ in levels of C during incubation, but had
a rise in C late in the season which was much more pronounced in dosed birds, probably
reflecting greater stress at the end of a traumatic breeding season.
Significant differences in corticosterone levels were also present between
two control groups: first-time breeding females compared to experienced females. Moderate levels
of C fluctuated throughout the duration of incubation with first time breeders having higher
values late in incubation and after hatching, perhaps reflecting increased stress in less
experienced birds. High levels of C at 70 days post-lay, coincidental with the time birds would
prepare to lay a second clutch, is consistent with failure of inexperienced birds to lay a
second clutch. Corticosterone differences between the two control male groups were also
consistent with the much higher relaying frequency of the more experienced birds that had lower
values of C at the time of second clutches. High circulating levels of C suppress gonadal
activity and the high values in inexperienced birds, both females and males, accompanied a much
lower frequency of relaying (experienced: 20 relays, first-breeding: 2 relays).
The hormone data developed in this study describe the natural hormone
patterns of seabirds throughout the breeding season, confirm the field data showing adverse
impacts of oil on breeding birds, and present possible physiological mechanisms for the adverse
effects of petroleum exposure.
The hormone patterns of auklets reflect generally the hormone cycles
observed for other species of birds during the breeding season (Farner and Gwinner 1980,
Wingfield '83). Testosterone increased early in the season in males as gonadal recrudescence
occurred in preparation for fertilization. Elevated T is characteristic of courtship and
necessary for expressing the aggressive behavior of territorial defense. Wingfield (1983) has
shown high levels of T in female birds that participate in nest defense, and the data presented
here indicate that Cassin's Auklet appears to fit as a species with territorial defense as
normal female breeding behavior. The maintenance of moderate to high levels of T throughout
incubation is particularly interesting, and perhaps reflects a need for burrow (nest box)
defense during incubation.
The hormonal changes in response to oil that have been shown here may have
been much more dramatic in individual birds than was demonstrated at the population level. The
hormonal patterns of birds that abandoned without laying showed immediately depressed E and
elevated C, but after these birds abandoned no further data were obtained. Only those birds that
remained, which, presumably, were less affected, provided continuing data.
This field and laboratory study of the effects of oil on auklets provided
the opportunity to investigate hormone patterns of a wild, free-living population of seabirds
throughout the breeding season. The data that were obtained support the hypothesis that
excretory hormone analysis of birds is a valid technique, and that important physiological data
can be obtained through collection of fecal samples. The collection of samples in a
noninvasive way enabled the development of data that could not have been obtained through
blood sampling. The correlation of hormonal data with field data on the petroleum toxicity to
seabirds has demonstrated an important new technique for avian biologists.
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