Fecal sample extraction (using 100% ethanol) and radioimmunoassay procedures were validated to permit accurate noninvasive assessments of fecal steroid metabolites in captive golden eagles (Aquila chrysaetos) and peregrine falcons (Falcoperegrinus). Furthermore, laboratory validations illustrated that serial dilutions of fecal extracts yielded displacement curves parallel to those of standard preparations for all assays. Recovery of known concentrations of unlabeled steroids in males and females for both species ranged from 97.3 9.8% (y = 1.03x – 0.63, R2= 0.98) to 119.4 5.3% (y = 1.04x + 4.11, R2 = 1.00). Additionally, in the golden eagles, HPLC analyses of fecal B in males and females illustrated that 2.7% and 2.5%, respectively, coeluted with [3HJB; however, a total of 7.5% and 11 .9% of total immunoreactivity coeluted with the reference tracer in the peregrine falcon males and females, respectively. Chromatographic profiles of E metabolites in golden eagle and peregrine falcon females revealed that 57.6% and 64.6% of the immunoreactivity coeluted with [3H1E2, respectively, as well as 26.9% and 4.1% aligned with [3H]E1, respectively. HPLC separation of the P metabolites in golden eagle and peregrine falcon females showed a large proportion of immunoreactivity (24.8% and 21.7%, respectively) coeluted with [4C]P4 reference tracer. Separation of fecal androgens in the golden eagle males revealed that 55.8% of the immunoreactivity coeluted with [4C]T reference tracer, whereas 63.7% of androgen metabolites coeluted with [4C]T in the peregrine falcon males. Additionally, physiological validation of these noninvasive hormone measures was demonstrated by intramuscular administration of exogenous hormones (adrenocorticotropin hormone (ACTH): 25 lU/kg; testosterone (T), estradiol-1713 (Es), and progesterone: 0.5-1.0 ~tg/bird) or saline to 6 birds of each species in a rotating split-plot design with repeated measures. We demonstrated a direct cause-and-effect relationship between ACTH administration and an increase in fecal corticosterone (B) metabolite excretion in golden eagles (P < 0.001) and peregrine falcons (P = 0.058). Fecal B excretion increased in both species after saline administration, which documented handling stress. Steroid excretion patterns were variable and lacked significant effects (F> 0.05); however, both species exhibited enterohepatic recycling in bi- and triphasic excretion patterns for fecal B, estrogens (E), progestagens (P), and T, but excretion lag times for fecal B metabolites were generally shorter (4-16 h post-treatment administration, 8.0 1.1 h) than for the E, P, and T metabolites (4-12 h post-treatment administration, 8.7 1.2 h).
Steroid excretion patterns of fecal B, E, P. and T metabolites were compared to breeding stages in the circannual reproductive lifecycle. During the breeding season, the peregrine falcons excreted higher concentrations of fecal E, P, and T than during the nonbreeding season (P < 0.05). A golden eagle female imprinted to humans excreted maximal B concentrations during incubation and the nonbreeding season and fecal E metabolites peaked during courtship then steadily declined, reaching minimal levels during parental care (P < 0.05). Progestagen metabolites peaked during copulation/egg laying and were lowest during parental care in the golden eagle female (P < 0.05). In the peregrine falcon females, fecal B concentrations were not different among stages.
Analysis of fecal E concentrations showed significant differences among breeding stages (P < 0.05), with peak concentrations during courtship (in pairs that did not copulate) or copulation/egg laying (in the pair that did copulate). In all females, E excretion was minimal during parental care and the nonbreeding season. Fecal P concentrations did not vary among stages in pairs that did not copulate. However, in the pair that exhibited copulatory behavior, fecal P concentrations were higher in all breeding season stages than during the nonbreeding season (P < 0.05). In peregrine falcon males, fecal B concentrations peaked during incubation, but there were no among-stage differences. However, fecal T varied according to breeding stage (F < 0.05), with peak concentrations during parental care for all pairs. The parental care stage was significantly higher during courtship in pairs with no copulation and was significantly higher during the nonbreeding season in the pair that copulated (P < 0.05).
Longitudinal excretion steroid patterns were analyzed for both breeding and nonbreeding birds. In breeding females of both species, fecal E and P excretion increased at or before egg laying; however, fecal E concentrations declined precipitously after egg laying, whereas fecal P levels remained elevated through parental care. Additionally, in the peregrine falcon females, mean ( SEM) fecal B and P were excreted in parallel throughout the nonbreeding season and courtship intervals, but diverged 2-wk before egg laying; furthermore, both metabolites were elevated during incubation and parental care. These data suggested that adrenal P4 production in falcons contributed, in part, to the overall fecal P excretion profile during the nonbreeding season. For breeding falcon males, T excretion increased during courtship and hatching. Furthermore, fecal B excretion in peregrine falcon males was highest during courtship and parental care.
Breeding and nonbreeding females excreted similar fecal B and P concentrations; however, the nonbreeders in both species (except one falcon) excreted lower concentrations of fecal E, suggesting that ovarian inactivity was influenced by cues other than changes in photoperiod. Excretory patterns of fecal T and B were similar in breeding and nonbreeding males, although T excretion was delayed in nonbreeders, suggesting that failure to breed may reflect missing social or environmental cues rather than endocrinological incompetence.
Overall, these data confirm the utility of noninvasive fecal steroid hormone monitoring for evaluating gonadal and adrenal status of these raptor species. These techniques can be applied in the laboratory or the field for conservation, biomedical, biological, and management investigations.