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Palme, R. (2019). Non-invasive measurement of glucocorticoids: Advances and problems. Physiol. Behav., 199, 229–243.
Abstract: Glucocorticoids (GCs; i.e. cortisol/corticosterone) are a central component of the stress response and thus their measurement is frequently used to evaluate the impact of stressful situations. Their metabolites from faeces of various animal species are more and more taken as a non-invasive aid to assess GC release and thus adrenocortical activity. The current literature review includes an extensive collection (1327 papers) and evaluation (see also Supplementary Tables) of the literature on faecal cortisol/corticosterone metabolite (FCM) analysis published to date. It aims at giving reference for researchers interested in implementing FCM analysis into their study or seeking to improve such methods by providing background knowledge on GC metabolism and excretion, conveying insights into methodological issues and stating caveats of FCM analysis and by highlighting prerequisites for and some examples of a successful application of such methods. Collecting faecal samples and analysing FCMs may appear simple and straightforward, but researchers have to select and apply methods correctly. They also need to be aware of the many pitfalls and potentially confounding factors and, last but not least, have to carefully interpret results. Applied properly, measurement of FCMs is a powerful non-invasive tool in a variety of research areas, such as (stress) biology, ethology, ecology, animal conservation and welfare, but also biomedicine.
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Yarnell, K., Hall, C., Royle, C., & Walker, S. L. (2015). Domesticated horses differ in their behavioural and physiological responses to isolated and group housing. Physiol.Behav., 143, 51–57.
Abstract: Abstract The predominant housing system used for domestic horses is individual stabling; however, housing that limits social interaction and requires the horse to live in semi-isolation has been reported to be a concern for equine welfare. The aim of the current study was to compare behavioural and physiological responses of domestic horses in different types of housing design that provided varying levels of social contact. Horses (n = 16) were divided equally into four groups and exposed to each of four housing treatments for a period of five days per treatment in a randomized block design. The four housing treatments used were single housed no physical contact (SHNC), single housed semi-contact (SHSC), paired housed full contact (PHFC) and group housed full contact (GHFC). During each housing treatment, adrenal activity was recorded using non-invasive faecal corticosterone metabolite analysis (fGC). Thermal images of the eye were captured and eye temperature was assessed as a non-invasive measure of the stress response. Behavioural analysis of time budget was carried out and an ease of handling score was assigned to each horse in each treatment using video footage. SHNC horses had significantly higher (p = 0.01) concentrations of fGC and were significantly (p = 0.003) more difficult to handle compared to the other housing types. GHFC horses, although not significantly different, had numerically lower concentrations of fGC and were more compliant to handling when compared to all other housing treatments. Eye temperature was significantly (p = 0.0001) lower in the group housed treatment when compared to all other treatments. These results indicate that based on physiological and behavioural measures incorporating social contact into the housing design of domestic horses could improve the standard of domestic equine welfare.
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Palme, R. (2012). Monitoring stress hormone metabolites as a useful, non-invasive tool for welfare assessment in farm animals. Animal Welfare, 21(3), 331–337.
Abstract: A multitude of endocrine mechanisms are involved in coping with challenges. Glucocorticoids, secreted by the adrenal glands, are in the front line of the battle to overcome stressful situations. They are usually measured in plasma samples as parameters of adrenal activity and thus of disturbance. Unfortunately, collecting blood samples itself can disturb an animal. Thus, non-invasive methods for the determination of glucocorticoids or their metabolites have become increasingly popular. The pros and cons of various non-invasive sample materials (saliva, excreta, milk, hair/feathers and eggs) for glucocorticoid determination are given. Above all, faecal samples offer the
advantage that they can be collected easily. In faecal samples, circulating hormone levels are integrated over a certain period of time and represent the cumulative secretion of hormones. Thus, the levels are less affected by short fluctuations or the pulse-like nature of hormone secretion. However, using this technique to assess an animal’s adrenocortical activity is not especially simple. Whether frequent sampling is necessary or single samples will suffice depends upon the study’s aim (whether one is examining the impact of acute or chronic stressors). Background knowledge of the metabolism and excretion of cortisol/corticosterone metabolites is required and a careful validation for each species and sex investigated is obligatory. The present review also addresses analytical issues regarding sample storage, extraction procedures and immunoassays and includes a comprehensive list of published studies (up to 2011) describing the use of such methods in farmed animals. Applied properly, non-invasive techniques to monitor glucocorticoid metabolites in faecal samples of various species are a useful tool for welfare assessment, especially as they are easily applied at farm or group level.
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Rizhova, L. Y., & Kulagin, D. A. (1994). The effects of corticosteroids on lateral bias in female rats. Behav. Brain. Res., 60(1), 51–54.
Abstract: In Experiment 1 female rats were given one trial per day for 8 days in a T-maze, and their initial direction of choice (left/right) was noted. Vaginal smears were also obtained daily. After this some animals were adrenalectomized and given Ringer's solution; others were adrenalectomozed and given hydrocortisone replacement; a third group was sham adrenalectomized, and a fourth group was an intact control. A week after surgery the animals were again tested for 8 days in the T-maze and vaginal smears were obtained. In Experiment 2 rats were subjected to the same surgical treatments as described above and were then tested for 8 days in the T-maze. In Expt. 1 there was no direction bias among the four groups prior to surgery. However, after surgery the Adrenalectomy + Ringer's group showed a significant increase in their rightward choices in the T-maze. This was also found in Expt. 2. Both adrenalectomized groups in Expt. 1 had a significant reduction in the duration of the estrus phase of their cycle. We conclude that corticosterone can affect lateral preference in a T-maze through a mechanism independent of the hormonal changes involved in the estrus cycle.
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Koolhaas, J. M., Korte, S. M., De Boer, S. F., Van Der Vegt, B. J., Van Reenen, C. G., Hopster, H., et al. (1999). Coping styles in animals: current status in behavior and stress-physiology. Neuroscience & Biobehavioral Reviews, 23(7), 925–935.
Abstract: This paper summarizes the current views on coping styles as a useful concept in understanding individual adaptive capacity and vulnerability to stress-related disease. Studies in feral populations indicate the existence of a proactive and a reactive coping style. These coping styles seem to play a role in the population ecology of the species. Despite domestication, genetic selection and inbreeding, the same coping styles can, to some extent, also be observed in laboratory and farm animals. Coping styles are characterized by consistent behavioral and neuroendocrine characteristics, some of which seem to be causally linked to each other. Evidence is accumulating that the two coping styles might explain a differential vulnerability to stress mediated disease due to the differential adaptive value of the two coping styles and the accompanying neuroendocrine differentiation.
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Kralj-Fiser, S., Scheiber, I. B. R., Blejec, A., Moestl, E., & Kotrschal, K. (2007). Individualities in a flock of free-roaming greylag geese: behavioral and physiological consistency over time and across situations. Horm Behav, 51(2), 239–248.
Abstract: The concept of personality implies individual differences in behavior and physiology that show some degree of repeatability/consistency over time and across contexts. Most studies of animal personality, particularly studies of individuals' variation in physiological mechanisms, have been conducted on selected individuals in controlled conditions. We attempted to detect consistent behaviors as well as physiological patterns in greylag ganders (Anser anser) from a free-roaming flock living in semi-natural conditions. We tested 10 individuals repeatedly, in a handling trial, resembling tests for characterization of “temperaments” in captive animals. We recorded the behavior of the same 10 individuals during four situations in the socially intact flock: (1) a “low density feeding condition”, (2) a “high density feeding condition”, (3) a “low density post-feeding situation” and (4) while the geese rested. We collected fecal samples for determination of excreted immuno-reactive corticosterone (BM) and testosterone metabolites (TM) after handling trials, as well as the “low density feeding” and the “high density feeding” conditions. BM levels were very highly consistent over the repeats of handling trials, and the “low density feeding condition” and tended to be consistent over the first two repeats of the “high density feeding condition”. Also, BM responses tended to be consistent across contexts. Despite seasonal variation, there tended to be inter-test consistency of TM, which pointed to some individual differences in TM as well. Aggressiveness turned out to be a highly repeatable trait, which was consistent across social situations, and tended to correlate with an individual's resistance during handling trials. Also, “proximity to the female partner” and “sociability” – the average number of neighboring geese in a close distance while resting – were consistent. We conclude that aggressiveness, “affiliative tendencies” and levels of excreted corticosterone and testosterone metabolites may be crucial factors of personality in geese.
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Touma, C., Sachser, N., Mostl, E., & Palme, R. (2003). Effects of sex and time of day on metabolism and excretion of corticosterone in urine and feces of mice. Gen Comp Endocrinol, 130(3), 267–278.
Abstract: Non-invasive techniques to monitor stress hormones in small animals like mice offer several advantages and are highly demanded in laboratory as well as in field research. Since knowledge about the species-specific metabolism and excretion of glucocorticoids is essential to develop such a technique, we conducted radiometabolism studies in mice (Mus musculus f. domesticus, strain C57BL/6J). Each mouse was injected intraperitoneally with 740 kBq of 3H-labelled corticosterone and all voided urine and fecal samples were collected for five days. In a first experiment 16 animals (eight of each sex) received the injection at 9 a.m., while eight mice (four of each sex) were injected at 9 p.m. in a second experiment. In both experiments radioactive metabolites were recovered predominantly in the feces, although males excreted significantly higher proportions via the feces (about 73%) than females (about 53%). Peak radioactivity in the urine was detected within about 2h after injection, while in the feces peak concentrations were observed later (depending on the time of injection: about 10h postinjection in experiment 1 and about 4h postinjection in experiment 2, thus proving an effect of the time of day). The number and relative abundance of fecal [3H]corticosterone metabolites was determined by high performance liquid chromatography (HPLC). The HPLC separations revealed that corticosterone was extensively metabolized mainly to more polar substances. Regarding the types of metabolites formed, significant differences were found between males and females, but not between the experiments. Additionally, the immunoreactivity of these metabolites was assessed by screening the HPLC fractions with four enzyme immunoassays (EIA). However, only a newly established EIA for 5alpha-pregnane-3beta,11beta,21-triol-20-one (measuring corticosterone metabolites with a 5alpha-3beta,11beta-diol structure) detected several peaks of radioactive metabolites with high intensity in both sexes, while the other EIAs showed only minor immunoreactivity. Thus, our study for the first time provides substantial information about metabolism and excretion of corticosterone in urine and feces of mice and is the first demonstrating a significant impact of the animals' sex and the time of day. Based on these data it should be possible to monitor adrenocortical activity non-invasively in this species by measuring fecal corticosterone metabolites with the newly developed EIA. Since mice are extensively used in research world-wide, this could open new perspectives in various fields from ecology to behavioral endocrinology.
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Touma, C., Palme, R., & Sachser, N. (2004). Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm Behav, 45(1), 10–22.
Abstract: In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science.
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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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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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