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Mostl, E., Rettenbacher, S., & Palme, R. (2005). Measurement of corticosterone metabolites in birds' droppings: an analytical approach. Ann N Y Acad Sci, 1046, 17–34.
Abstract: Fecal steroid analyses are becoming increasingly popular among both field and laboratory scientists. The benefits associated with sampling procedures that do not require restraint, anesthesia, and blood collection include less risk to subject and investigator, as well as the potential to obtain endocrine profiles that are not influenced by the sampling procedure itself. In the feces, a species-specific pattern of metabolites is present, because glucocorticoids are extensively metabolized. Therefore, selection of adequate extraction procedures and immunoassays for measuring the relevant metabolites is a serious issue. In this review, emphasis is placed on the establishment and analytical validation of methods to measure glucocorticoid metabolites for a noninvasive evaluation of adrenocortical activity in droppings of birds.
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Li, C., Jiang, Z., Tang, S., & Zeng, Y. (2007). Influence of enclosure size and animal density on fecal cortisol concentration and aggression in Pere David's deer stags. Gen Comp Endocrinol, 151(2), 202–209.
Abstract: We investigated the impact of enclosure size and animal density on behavior and adrenocortical secretion in Pere David's deer in Dafeng Nature Reserve, China. From February 15 to April 16 in 2004, we conducted two experiments. First, we studied maintenance behavior and conflict behavior of Pere David's deer stags in a large enclosure (200 ha) with low animal density (0.66 deer/ha) and a small display pen (0.75 ha) with high animal density (25.33 deer/ha). The maintenance behavior we recorded included standing, locomotion, foraging and rest. During the behavioral observations, we collected fresh voided fecal samples from the stags periodically, and analyzed the fecal cortisol concentrations in those samples using radioimmunoassay technique. Second, we monitored the fecal cortisol concentrations of one group of stags (12 deer lived in an enclosure of 100 ha) before and after transferred into a small pen (0.5 ha). We found that in the first experiment: (1) there were significant differences in standing and rest whereas no significant differences of locomotion and foraging between the free-ranging group and the display group; (2) frequency of conflict behavior in the display group was significantly higher than those in the free-ranging group; and (3) fecal cortisol concentration of the display group (326.17+/-16.98 ng/g dry feces) was significantly higher than that of the free-ranging group (268.98+/-15.21 ng/g dry feces). In the second experiment, there was no significant difference of the fecal cortisol concentrations among sampling days, but the mean fecal cortisol concentration of the day after transferring (337.46+/-17.88 ng/g dry feces) was significantly higher than that of the day before transferring (248.44+/-7.99 ng/g dry feces). Comparison with published findings, our results indicated that enclosure size and animal density affect not only behaviors, but also adrenocortical secretion in Pere David's deer. Small living space with high animal density may impose physiological stress to captive Pere David's deer. Moreover, long-term physiological stress and increase of conflict behavior may subsequently affect survival and reproduction of the deer.
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Touma, C., & Palme, R. (2005). Measuring fecal glucocorticoid metabolites in mammals and birds: the importance of validation. Ann N Y Acad Sci, 1046, 54–74.
Abstract: In recent years, the noninvasive monitoring of steroid hormone metabolites in feces of mammals and droppings of birds has become an increasingly popular technique. It offers several advantages and has been applied to a variety of species under various settings. However, using this technique to reliably assess an animal's adrenocortical activity is not that simple and straightforward to apply. Because clear differences regarding the metabolism and excretion of glucocorticoid metabolites (GCMs) exist, a careful validation for each species and sex investigated is obligatory. In this review, general analytical issues regarding sample storage, extraction procedures, and immunoassays are briefly discussed, but the main focus lies on experiments and recommendations addressing the validation of fecal GCM measurements in mammals and birds. The crucial importance of scrutinizing the physiological and biological validity of fecal GCM analyses in a given species is stressed. In particular, the relevance of the technique to detect biologically meaningful alterations in adrenocortical activity must be shown. Furthermore, significant effects of the animals' sex, the time of day, season, and different life history stages are discussed, bringing about the necessity to seriously consider possible sex differences as well as diurnal and seasonal variations. Thus, comprehensive information on the animals' biology and stress physiology should be carefully taken into account. Together with an extensive physiological and biological validation, this will ensure that the measurement of fecal GCMs can be used as a powerful tool to assess adrenocortical activity in diverse investigations on laboratory, companion, farm, zoo, and wild animals.
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Griffin, B. (2002). The use of fecal markers to facilitate sample collection in group-housed cats. Contemp Top Lab Anim Sci, 41(2), 51–56.
Abstract: The provision of proper social housing is a priority when designing an experiment using domestic cats as laboratory animals. When animals are group-housed, studies requiring analysis of stool samples from individual subjects pose difficulty in sample collection and identification. In this study, commercially available concentrated food colorings (known as bakers pastes) were used as fecal markers in group-housed cats. Cats readily consumed 0.5 ml of bakers paste food coloring once daily in canned cat food. Colorings served as fecal markers by imparting a distinct color to each cat s feces, allowing identification in the litter box. In addition, colored glitter (1/8 teaspoon in canned food) was fed to cats and found to be a reliable fecal marker. Long-term feeding of colorings and glitter was found to be safe and effective at yielding readily identifiable stools.
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Thiel, D., Jenni-Eiermann, S., & Palme, R. (2005). Measuring corticosterone metabolites in droppings of capercaillies (Tetrao urogallus). Ann N Y Acad Sci, 1046, 96–108.
Abstract: The capercaillie (Tetrao urogallus), the largest grouse species in the world, is decreasing in numbers in major parts of its distribution range. Disturbances by human outdoor activities are discussed as a possible reason for this population decline. An indicator for disturbances is the increase of the glucocorticoid corticosterone, a stress hormone, which helps to cope with life-threatening situations. However, repeated disturbances might result in a long-term increase of the basal corticosterone concentration, which can result in detrimental effects like reduced fitness and survival of an animal. To measure corticosterone metabolites (CMs) noninvasively in the droppings of free-living capercaillies, first an enzyme immunoassay (EIA) in captive birds had to be selected and validated. Therefore, the excretion pattern of intravenously injected radiolabeled corticosterone was determined and 3H metabolites were characterized. High-performance liquid chromatography (HPLC) separations of the samples containing peak concentrations revealed that corticosterone was extensively metabolized. The HPLC fractions were tested in several EIAs for glucocorticoid metabolites. The physiological relevance of this method was proved after pharmacological stimulation of the adrenocortical activity. Only the recently established cortisone assay, measuring CMs with a 3,11-dione structure, detected an expressed increase of concentrations following ACTH stimulation. To set up a sampling protocol suited for the field, we examined the influence of various storage conditions and time of day on concentrations of CMs.
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Baltic, M., Jenni-Eiermann, S., Arlettaz, R., & Palme, R. (2005). A noninvasive technique to evaluate human-generated stress in the black grouse. Ann N Y Acad Sci, 1046, 81–95.
Abstract: The continuous development of tourism and related leisure activities is exerting an increasingly intense pressure on wildlife. In this study, a novel noninvasive method for measuring stress in the black grouse, an endangered, emblematic species of European ecosystems that is currently declining in several parts of its European range, is tested and physiologically validated. A radiometabolism study and an ACTH challenge test were performed on four captive black grouse (two of each sex) in order to get basic information about the metabolism and excretion of corticosterone and to find an appropriate enzyme-immunoassay (EIA) to measure its metabolites in the feces. Peak radioactivity in the droppings was detected within 1 to 2 hours. Injected (3)H-corticosterone was excreted as polar metabolites and by itself was almost absent. A cortisone-EIA was chosen from among seven tested EIAs for different groups of glucocorticoid metabolites, because it cross-reacted with some of the formed metabolites and best reflected the increase of excreted corticosterone metabolites, after the ACTH challenge test. Concentrations of the metabolites from fecal samples collected from snow burrows of free-ranging black grouse were within the same range as in captive birds. The noninvasive method described may be appropriate for evaluating the stress faced by free-living black grouse populations in the wild, particularly in mountain ecosystems where human disturbance, especially by winter sports, is of increasing conservation concern.
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