Introduction

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 stress.

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 al ('84).

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 '78).

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 creatinine.

Hormone Assays

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, CO.

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 laying.

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 clutch.

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 oil.

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).

Conclusions

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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