PHYSIOLOGY: BIRDS
Washburn B. E., Millspaugh J. J., Schulz J. H., Jones S. B., Mong T. (2003): Using fecal glucocorticoids for stress assessment in mourning doves. The Condor 105: 696-706.
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Fecal glucocorticoid assays provide a potentially useful, noninvasive means to study physiological responses of wildlife to various stressors. The objective of our study was to validate a method for measuring glucocorticoid metabolites in Mourning Dove (Zenaida macroura) feces. We validated the assay using standard procedures (e.g., parallelism, recovery of exogenous corticosterone) to demonstrate that the assay accurately and precisely measured glucocorticoid metabolites in Mourning Dove fecal extracts. We conducted adrenocorticotropin (ACTH) challenge experiments to validate the assay’s ability to determine biologically important changes in fecal glucocorticoids. Fecal glucocorticoid levels increased significantly approximately 2–3 hr after administration of ACTH at 50 IU per kg body mass to wild Mourning Doves held in captivity. In contrast, fecal glucocorticoid metabolites did not increase in control birds, birds that received saline injections, or a lower dose of ACTH (1 IU per kg body mass). Variation in overall fecal glucocorticoid metabolite levels may have been influenced by season and the length of time birds were held in captivity. Noninvasive fecal glucocorticoid metabolite analyses, in combination with demographic information, may have considerable utility for monitoring the effects of natural and anthropogenic disturbances on Mourning Dove populations.
Tempel D. J., Gutierrez R. J. (2004): Factors related to fecal corticosterone levels in California spotted owls: implications for assessing chronic stress. Conservation Biology 18: 538-547.
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The California Spotted Owl (Strix occidentalis occidentalis) is under consideration for federal protection and has stimulated ecosystem‐level management efforts in Sierra Nevada national forests. Because some populations are declining, we used a noninvasive fecal sampling method to estimate stress hormone (i.e., corticosterone) levels within a local population from April to August 2001. Fecal corticosterone levels were similar to those recorded in a previous study of Northern Spotted Owls (S. o. caurina) (x̄= 80.1 ng/g dry feces, SE = 75.8). We then used an information‐theoretic approach to identify factors that influence fecal corticosterone levels in Spotted Owls. Our best overall model indicated that nonbreeding owls had higher fecal corticosterone levels than breeding owls early in the breeding season and lower levels later in the breeding season. We collected few samples from breeding owls early in the breeding season, however, which may have influenced the results. Management‐related factors reflecting habitat condition and proximity to roads were not correlated with fecal corticosterone. However, factors such as field storage method and sample mass were correlated with the amount of measured fecal corticosterone and should be considered in future studies. Sample vials initially stored on ice had higher levels than those stored immediately in liquid N2 (βstorage= 0.269 ln[ng/g], 95% CI = 0.026, 0.512). Hormone metabolites were extracted from extremely small samples (0.01 g) by slightly modifying the assay protocol, but the amount of corticosterone detected increased as the sample mass decreased (βmass=−6.248 ln[ng/g], 95% CI =−8.877, −3.620). Corticosterone levels were significantly higher in 10 cecal samples collected simultaneously with fecal samples (paired difference = 74.7 ng/g, SE = 45.0, p = 0.001 for a paired t test), so care must be taken to avoid contaminating fecal samples with cecal material. Most of the variation was unexplained by our best model (R2= 0.24), and additional factors influencing fecal corticosterone levels need to be identified. Therefore, we recommend that well‐designed experiments be conducted under controlled conditions to better determine the effect of factors such as sample handling, partial sampling, and diet on fecal corticosterone levels in owls and other birds.
Baltic M., Jenni-Eiermann S., Arlettaz R., Palme R. (2005): A noninvasive technique to evaluate human-generated stress in the black grouse. Annals of the New York Academy of Sciences 1046: 81-95.
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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 3H‐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.
Goymann W. (2005): Noninvasive monitoring of hormones in bird droppings: physiological validation, sampling, extraction, sex differences, and the influence of diet on hormone metabolite levels. Annals of the New York Academy of Sciences 1046: 35-53.
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During the past several years, the noninvasive measurement of steroid metabolites from mammalian feces and bird droppings has become more and more popular. With an increasing acceptance of the method, investigators may become less aware of the need to validate their assays. It is shown why such validations are essential for each new species investigated and various ways to physiologically validate such noninvasive methods are described. Using the European stonechat (Saxicola torquata rubicola) as a model, it is explained why a validated method to measure androgen metabolites in males does not necessarily work in females. In addition the difficulties that may be neglected owing to the superficial ease of sampling and processing of excreta are investigated. Various issues that may arise during sampling, storage, and extraction of excreta are addressed. Finally, results suggesting that experimental manipulations of the diet may affect hormone metabolite levels in European stonechats are presented. So far, only a few studies have investigated the impact of diet on hormone metabolite levels, and these are the first data to report such an impact in birds. More studies are urgently needed to learn more about differences between the sexes, individuals, and populations and the impact of diet and energy metabolism on hormone metabolites.
Wasser S. K., Hunt K. E. (2005): Noninvasive measures of reproductive function and disturbance in the barred owl, great horned owl, and northern spotted owl. Annals of the New York Academy of Sciences 1046: 109.
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There is an urgent need for noninvasive methods to study reproduction and environmental stress in at-risk species such as the northern spotted owl (Strix occidentalis caurina). Two related owl species (barred owl and great horned owl) were used as surrogates to validate hormone assays for fecal metabolites of progesterone, 17beta-estradiol, testosterone, and corticosterone. Infusions of radiolabeled hormones showed that the owls excreted most hormone within 6 h. Feces and urine contained roughly equal amounts of hormone, and most fecal hormone metabolites were quite polar. The testosterone and corticosterone assays in this study bound to the major excreted metabolites of these hormones, but two progesterone assays did not appreciably bind to the major progesterone metabolites. All assays showed excellent parallelism with hydrolyzed and unhydrolyzed samples and with previously dried or undried fecal samples. Thus, samples do not require hydrolysis or prior drying. Samples from a female barred owl had significantly higher fecal estrogen, lower fecal testosterone, and higher fecal estrogen/testosterone ratio than samples from two male barred owls. The fecal estrogen/testosterone ratio was the most accurate predictor of owl gender, particularly if two or more samples are available from the same individual. Fecal corticosterone metabolites also demonstrated considerable utility for wild northern spotted owls. Fecal glucocorticoid levels varied by gender and breeding stage, being highest in male northern spotted owls early in the breeding season and highest in females when nestlings were fledging. Collectively, these studies show that noninvasive fecal hormone measurements show great promise for noninvasive assessment of reproduction and stress in wild owls.
Koch M., Möstl E., Steinmetz H. W., Clauss M., Masello J. F., Quillfeldt P. (2009): Non-invasive measurement of faecal glucocorticoid metabolites in Upland Geese Chloephaga picta. Polar Biology 32: 281-285.
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Glucocorticoid (GC) hormones rise in response to stressors, including natural events including weather or predator presence, and human activities, such as hunting, scientific research or recreational visits. However, because blood sampling itself causes stress and is dangerous or even impossible in some wildlife species, feedback-free methods for GC determination are needed to assess stress in these animals. Faecal GC analyses have thus gained interest. Here, we validate a non-invasive method to estimate the physiological stress in the Upland goose Chloephaga picta. An adrenocorticotropin hormone (ACTH) challenge was conducted in captive adults (female and male), and droppings were collected before, during and after the experiment. Corticosterone metabolite (CM) secretion in response to the ACTH challenge was measured with several enzyme immunoassays (EIA) to find the most appropriate test. We used CM levels during the periods before and after the experiment as control data. An EIA for 11-oxoetiocholanolone achieved the highest response to the ACTH challenge and also reflected a stress response to unfamiliar environment. Furthermore, CM concentrations of dry samples were highly correlated with the corresponding non-dried (frozen) samples. The data suggest that this method is appropriate to measure the stress in Upland geese, and that samples can be stored either frozen or dry form.
Gladbach A., Gladbach D. J., Koch M., Kuchar A., Möstl E., Quillfeldt P. (2011): Can faecal glucocorticoid metabolites be used to monitor body condition in wild Upland geese Chloephaga picta leucoptera?. Behavioral Ecology and Sociobiology 65: 1491-1498.
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The measurement of faecal glucocorticoid metabolites is used as a non-invasive technique to study stress in animal populations. They have been used most widely in mammals, and mammalian studies have also treated issues such as sample stability and storage methods. In birds, faecal corticosterone metabolite (CM) assays have been validated for a small number of species, and adequate storage under field conditions has not been addressed explicitly in previous studies. Furthermore, while it is well-established that baseline plasma corticosterone levels in birds rise with declining body condition, no study so far investigated if this relationship is also reflected in faecal samples. We here present data of a field study in wild Upland geese Chloephaga picta leucoptera on the Falkland Islands, testing different storage methods and investigating the relationship of faecal CM concentrations to body condition and reproductive parameters. We found that faecal CM measures are significantly repeatable within individuals, higher in individuals with lower body condition in both male and female wild Upland geese and higher in later breeding females with smaller broods. These results suggest that measuring faecal CM values may be a valuable non-invasive tool to monitor the relative condition or health of individuals and populations, especially in areas where there still is intense hunting practice.
Lattin C. R., Reed J. M., DesRochers D. W., Romero L. M. (2011): Elevated corticosterone in feathers correlates with corticosterone‐induced decreased feather quality: a validation study. Journal of Avian Biology 42: 247-252.
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The newly described technique of extracting corticosterone (CORT) from bird feathers may serve as a less invasive, more integrated measure of a bird’s stress response. Previous work indicated that elevated plasma CORT resulted in poorer quality feathers during molt. We tested the hypothesis that a direct link exists between plasma and feather CORT concentrations. We experimentally increased plasma CORT concentrations using implants and found that the corresponding rise in CORT could be detected in feathers grown during implantation. Furthermore, CORT levels in two feathers grown at the same time from the same bird were very consistent. These results provide evidence that elevated CORT is a causative factor in decreasing feather quality during molt. However, there remain technical details that suggest caution when interpreting data from CORT extracted from feathers. Different portions of a growing feather did not necessarily reflect changes in plasma CORT at the time different parts of the feather were forming, a standard pool of homogenized feathers indicated that sample mass affects measured feather CORT concentration, and different antibodies produced different measured CORT concentrations, leaving in doubt the exact steroid being assayed.
Bautista L. M., Silván G., Cáceres S., Martínez-Fernández L., Bravo C., Illera J. C., Alonso J. C., Blanco G. (2013): Faecal sexual steroids in sex typing and endocrine status of great bustards. European Journal of Wildlife Research 59: 815-822.
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Faecal sexual steroids have been used in field studies evaluating the relationships between gender and the multiple factors influencing endocrine status of individuals. The determination of faecal steroids has been also proposed as an alternative, non-invasive sexing method when other methods were deemed impractical or risky for the health of birds. In this study, we quantified sexual steroid hormones in faeces of the great bustard (Otis tarda), a large and sexually dimorphic polyginic bird species that it is threatened and subjected to intense wildlife management. We evaluated differences between captivity and wild conditions, flocks and sexes, and used faecal steroids to develop sex determination procedures. We found similar steroid levels in captive and wild bustards, no differences between unisexual wild flocks and clear between-sexes differences in testosterone but not estradiol. Faecal steroids accurately discriminated gender in both captive and wild known-sex great bustards. Total testosterone concentration was always higher than estradiol concentration in faecal samples from males, but estradiol was not always higher than testosterone in females. Faecal steroids failed to reveal the presence of young males in female flocks during winter, despite faecal testosterone levels increased with age in a small sample of captive males. Our results show that faecal steroid measurement for both sexing and characterizing the endocrine status of great bustards is feasible, and therefore it should be valuable in wildlife management, especially in combination with additional information obtained from faeces as diet.
Jenni‐Eiermann S., Helfenstein F., Vallat A., Glauser G., Jenni L. (2015): Corticosterone: effects on feather quality and deposition into feathers. Methods in Ecology and Evolution 6: 237-246.
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The concentration of the glucocorticoid hormone corticosterone (CORT) is increasingly used in ecology and conservation biology as an integrated measure of the historical record of an individual’s hypothalamo–pituitary–adrenal (HPA) activity during feather growth. However, where and how CORT is incorporated in feathers is incompletely known. We therefore examined whether CORT is reliably measured with an enzyme immunoassay, where CORT is incorporated in the feather and where it affects feather quality, and whether CORT incorporation is related to plasma CORT levels, feather growth rate and melanin pigmentation. During the regrowth of plucked tail feathers, we injected pigeons with tritium‐labelled CORT, and implanted a CORT‐releasing pellet to increase plasma CORT concentration for about 3 days. In feather segments, we measured labelled CORT (DPM3H) and we quantified CORT with an enzyme immunoassay EIA (CORTEIA) and double‐checked the results with ultra‐high performance liquid chromatography–tandem mass spectrometry (LCMS) (CORTMS). Administered CORT affected feather structure and colour at the very base of the feather (epidermal collar, ramogenic zone) and reduced growth rate. In contrast, incorporation of CORT into the feather happened mainly in the blood quill, as shown with all three methods (DPM3H, CORTEIA and CORTMS). Incorporation of CORT into feathers was only roughly proportional to plasma concentration, proportional to feather growth rate and increased with melanin pigmentation. Measuring CORT in feather is a way to reveal past events of increased stress during feather growth in birds.
Jerem P., Jenni-Eiermann S., Herborn K., McKeegan D., McCafferty D. J., Nager R. G. (2018): Eye region surface temperature reflects both energy reserves and circulating glucocorticoids in a wild bird. Scientific Reports 8: 1907.
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Body temperature of endotherms shows substantial within- and between-individual variation, but the sources of this variation are not fully understood in wild animals. Variation in body temperature can indicate how individuals cope with their environment via metabolic or stress-induced effects, both of which may relate to depletion of energy reserves. Body condition can reflect heat production through changes to metabolic rate made to protect energy reserves. Additionally, changes in metabolic processes may be mediated by stress-related glucocorticoid secretion, which is associated with altered blood-flow patterns that affect regional body temperatures. Accordingly, both body condition and glucocorticoid secretion should relate to body temperature. We used thermal imaging, a novel non-invasive method of temperature measurement, to investigate relationships between body condition, glucocorticoid secretion and body surface temperature in wild blue tits (Cyanistes caeruleus). Individuals with lower body condition had lower eye-region surface temperature in both non-breeding and breeding seasons. Eye-region surface temperature was also negatively correlated with baseline circulating glucocorticoid levels in non-breeding birds. Our results demonstrate that body surface temperature can integrate multiple aspects of physiological state. Consequently, remotely-measured body surface temperature could be used to assess such aspects of physiological state non-invasively in free-living animals at multiple life history stages.
Qasimi M. I., Mohibbi H., Nagaoka K., Watanabe G. (2018): Accumulation of steroid hormones in the eggshells of Japanese quail (Coturnix coturnix japonica). General and Comparative Endocrinology 259: 161-164.
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Oviparous mother transfer significant amounts of steroid to egg yolk during oviposition and the amounts may vary throughout the embryonic development. Eggshell may contain steroid hormones and the amounts could be different during embryonic development inside the egg. This study was designed to quantify the steroid concentrations in the eggshells of Japanese quail. We hypothesized that the steroids would be accumulated in the eggshells in a sex-dependent manner. Eggshells were obtained from three different stages (after laying, 15 days of incubation, and after hatching). The internal contents of the shells were carefully removed, completely dried and pulverized. The steroid contents of the eggshells were then measured by RIA. Physiologic variations in steroids were analyzed according to the amounts accumulated in the eggshells with the different embryonic stages. Results indicate that eggshell testosterone concentrations were high after laying. However, the concentrations were decreased during embryonic development and hatching and no difference was found in eggshell testosterone levels between male and female. However, eggshell estradiol concentrations were undetectable at laying time and the amounts were significantly increased at 15 days of incubation and slightly after hatching. Eggshell estradiol levels were significantly high in female eggshells than male during embryonic development. In contrast, eggshell corticosterone levels were significantly higher in males than in females after hatching. These results clearly demonstrated that eggshells accumulated steroid hormones, and the amounts varied during embryonic development concomitant with changes the internal contents of the eggs.
Bourne A. R., McKechnie A. E., Cunningham S. J., Ridley A. R., Woodborne S. M., Karasov W. H. (2019): Non‐invasive measurement of metabolic rates in wild, free‐living birds using doubly labelled water. Functional Ecology 33: 162-174.
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Doubly labelled water (DLW) is routinely used to measure energy expenditure and water turnover in free-ranging animals. Standard methods involve capture, blood sampling for baseline measurement, injection with isotopic tracers, captivity for an equilibration period, post-dose blood sampling, release and subsequent recapture for final blood sampling. Single sampling methods that minimise disturbance by reducing capture and handling time have been developed and tested. Sampling faeces rather than blood could further reduce disturbance to study animals in a range of species and study systems. However, the extent to which estimates of metabolic rate derived from blood and faecal samples diverge has not been investigated. We compared isotopic enrichment in blood and faecal samples taken concurrently from captive southern pied babblers Turdoides bicolor. Isotopic enrichment levels in faeces and in blood were used to calculate initial and final ratios of δ18O/δ2H for each individual. We then used these ratios to calculate daily energy expenditure (DEE) and directly compared measurements from blood samples with those from faecal samples within individuals. We found that faecal sampling resulted in estimates of DEE that agree with those based on blood sampling. Additionally, we field-tested a faecal sampling protocol with a habituated population of babblers in the southern Kalahari Desert. During the field test, study animals were not captured or handled for either dosing or sampling. Field-testing confirmed the practical feasibility of non-invasive dosing and sampling techniques in free-living animals, and we obtained measurements of DEE that we used to test an a priori prediction that DEE is inversely related to air temperature. Our data show decreasing DEE with increasing air temperature, a pattern consistent with studies testing similar predictions in birds using traditional DLW methods. We demonstrate that faecal samples can substitute for blood when measuring DEE using DLW and provide a method that will allow field-based researchers to obtain sound physiological measurements while minimising handling and removal of study animals from their natural environments.
Imlay T. L., Angelier F., Hobson K. A., Mastromonaco G., Saldanha S., Leonard M. L. (2019): Multiple intrinsic markers identify carry-over effects from wintering to breeding sites for three Nearctic–Neotropical migrant swallows. The Auk 136: ukz053.
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Carry-over effects from one stage of the annual cycle to subsequent stages can have profound effects on individual fitness. In migratory birds, much research has been devoted to examining such effects from the nonbreeding to the breeding period. We investigated potential carry-over effects influencing spring body condition, breeding phenology, and performance for 3 species of sympatric, declining Nearctic–Neotropical migratory swallows: Bank Swallow (Riparia riparia), Barn Swallow (Hirundo rustica), and Cliff Swallow (Petrochelidon pyrrhonota). To examine carry-over effects, we used structural equation modeling and several intrinsic markers, including stable isotope (δ 2H, δ 13C, and δ 15N) and corticosterone (CORTf) values from winter molted-feathers, and changes in telomere length between breeding seasons. We found support for carry-over effects for all 3 species, however, the specific relationships varied between species and sexes. Effects leading to lower breeding performance were only observed in male Bank, female Barn, and female and male Cliff Swallows. In most cases, carry-over effects were attributed to differences in stable isotope values (most commonly with δ 2H) presumably related to differences in winter habitat use, but, for Cliff Swallows, negative carry-over effects were also linked to higher CORTf values and greater rates of telomere shortening. This work provides further support for the potential role of nonbreeding conditions on population declines, and indicates how multiple intrinsic markers can be used to provide information on ecological conditions throughout the annual cycle.
Jepsen E. M., Ganswindt A., Ngcamphalala C. A., Bourne A. R., Ridley A. R., McKechnie A. E. (2019): Non-invasive monitoring of physiological stress in an afrotropical arid-zone passerine bird, the southern pied babbler. General and Comparative Endocrinology 276: 60-68.
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Using faecal matter to monitor stress levels in animals non-invasively is a powerful technique for elucidating the effects of biotic and abiotic stressors on free-living animals. To validate the use of droppings for measuring stress in southern pied babblers (Turdoides bicolor) we performed an ACTH challenge on captive individuals and determined the effect of temporary separation from their social group on their faecal glucocorticoid metabolite (fGCM) concentration. Additionally, we compared fGCM concentrations of captive babblers to those of wild conspecifics and examined the effects of dominance rank on fGCM concentration. We found droppings to be a suitable matrix for measuring physiological stress in babblers and that individual separation from the group caused an increase in fGCM levels. In addition, babblers temporarily held in captivity had substantially higher fGCM concentrations than wild individuals, indicating that babblers kept in captivity experience high levels of stress. In wild, free-living individuals, dominant males showed the highest levels of stress, suggesting that being the dominant male of a highly territorial social group is stressful. Non-invasive sampling allows field-based researchers to reduce disturbance related to monitoring adrenocortical function, thereby avoiding artificially increasing circulating corticosterone concentration as it is not necessary to physically restrain study animals.
Krone O., Bailey L. D., Jaehnig S., Lauth T., Dehnhard M. (2019): Monitoring corticoid metabolites in urine of white-tailed sea eagles: Negative effects of road proximity on breeding pairs. General and Comparative Endocrinology 283: 113223.
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The white-tailed sea eagle (Haliaeetus albicilla) is known to be sensitive to disturbance. To better understand potential stressors, we measured corticosterone metabolite levels in H. albicilla excreta and recorded the nest success of breeding pairs. We tested the ability of four enzyme immunoassays (EIA) to measure urinary glucocorticoid metabolites (uGM) in the excreta of one adult female eagle subjected to a controlled physiological stress treatment. We identified corticosterone-21-HS to be the most sensitive EIA to changes in uGM concentration. To exclude a sex bias, we confirmed the assay’s applicability with samples collected from similar stress treatments in two juvenile males. We used the identified EIA to measure uGM in wild breeding pairs and tested effects of disturbance. Breeding pairs nesting closer to roads and paths had higher uGM concentrations (p = 0.02), which is likely an effect of human recreational activity and disturbance. There was no difference in uGM concentrations between failed and successful nests. Our results highlight the potential impact of road and path proximity on white-tailed sea eagles, with potential importance for species management and conservation, particularly with respect to nest protection zone legislation.
Lynch M. A., Youngflesh C., Agha N. H., Ottinger M. A., Lynch H. J. (2019): Tourism and stress hormone measures in Gentoo Penguins on the Antarctic Peninsula. Polar Biology 42: 1299-1306.
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The impacts of tourism on wildlife have long been a concern in areas where ecotourism is a major industry. The issue is especially pressing in Antarctica, which has a rapidly growing tourism industry largely concentrated around penguin colonies on the Antarctic Peninsula. Guidelines developed by both the Committee for Environmental Protection and the International Association of Antarctica Tour Operators include measures to minimize wildlife impacts. In this study, we examined the relationship between physiologic stress in Gentoo Penguins (Pygoscelis papua) and tourism. Corticosterone is an adrenal glucocorticoid that has been shown in previous studies to increase in response to stressors such as low food availability, environmental conditions, as well as human visitation and proximity. Fecal glucocorticoids (FGM; primarily corticosterone and metabolites) were measured in Gentoo Penguin guano collected at 19 breeding colonies (n = 108, 3–10 samples per site) on the Antarctic Peninsula and the South Shetland Islands, representing a wide range of tourism visitation. We found a large degree of variation in FGM concentrations, and no relationship between FGM concentrations and number of tourists landed at that site. These results suggest that current tourism management guidelines on the Antarctic Peninsula are effective at preventing increased stress in Gentoo Penguins as measured by hormonal markers, and demonstrate the use of guano as a non-invasive, low-impact methodology for monitoring Gentoo Penguin stress.
Frongia G. N., Peric T., Leoni G., Satta V., Berlinguer F., Muzzeddu M., Prandi A., Naitana S., Comin A. (2020): Assessment of cortisol and DHEA concentrations in Griffon vulture (Gyps fulvus) feathers to evaluate its allostatic load. Annals of Animal Science 20: 85-96.
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The use of a non-invasive approach to collect biological samples from natural populations represents a great means of gathering information while avoiding handling animals. Even if corticosterone is the main glucocorticoid investigated in birds, there has been observed a proportional direct link between corticosterone and cortisol concentrations. Dehydroepiandrosterone (DHEA) can be produced by the adrenal cortex and should have prominent antiglucocorticoid properties also in birds. The aim of this study was to verify if there is any difference in the cortisol and DHEA feather concentrations between clinically normal and physiologically compromised Griffon vultures (Gyps fulvus) through the non-invasive approach of collecting moulted feathers without having to pluck them from the bird. The study was carried out using 8 physiologically compromised (PC) Griffons and 9 clinically normal Griffons considered as the control (CTRL) group that were necropsied or from the wildlife rehabilitation centre. Primary and secondary covert feathers were either collected directly from the birds’ cage floors, or, in the case of dead Griffons, they were plucked off the animals. The results, obtained by RIA, revealed that both cortisol (P<0.01) and DHEA (P<0.05) feather concentrations were higher in the PC than in the CTRL group. No difference was observed by comparing the cortisol/DHEA ratio between the two evaluated groups (P=0.15). Pearson’s correlation coefficients showed no correlation between feather hormone concentrations in the PC group (r=0.01, P=0.96) while a positive correlation in the CTRL group (r=0.65, P=0.006) was observed. In conclusion, our study reveals that moulted feathers can be a non-invasive and an interesting tool to evaluate the allostatic load of wild birds and they allowed better understanding the relationship between hormones of the hypothalamic–pituitary–adrenal axis and the physiological status of the birds.
Mohlman J. L., Navara K. J., Sheriff M. J., Terhune T. M., Martin J. A. (2020): Validation of a noninvasive technique to quantify stress in northern bobwhite (Colinus virginianus). Conservation Physiology 8: coaa026.
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Examination of the endocrine system through non-invasive fecal sampling may improve population management more than using demographic indicators alone. By addressing the physiological mechanisms that are influencing fitness, management actions can be proactively developed to alleviate stressors. Proactive determination of vulnerable populations is critical for species of concern, such as the northern bobwhite (Colinus virginianus), which have suffered decades of population decline. We validated an assay to noninvasively measure the adrenocortical response of captive reared bobwhite through fecal corticosterone metabolites (FCM). All individuals received three sequential 48-hour treatments in which samples were collected every 4 hours, including a reference period, an adrenocorticotropic hormone (ACTH) challenge and a biological stressor (exposure to a hunting dog). Reference FCM values had a mean concentration of 16.75 pg/mg (95% CrI: 13.68, 19.91) with adrenocortical activity increasing by 73% for the duration of the ACTH challenge (29.00 pg/mg; CrI: 25.01, 33.78). FCM concentrations remained similar to that of the reference levels during the biological stressor (16.56 pg/mg; CrI: 13.33, 19.92). Our study validates the use of feces to detect changes in FCM levels in our subject species but also demonstrates the complexity of FCM and the importance of both physiological and biological validation prior to field implementation.
Voit M., Merle R., Baumgartner K., von Fersen L., Reese L., Ladwig-Wiegard M., Will H., Tallo-Parra O., Carbajal A., Lopez-Bejar M., Thöne-Reineke C. (2020): Validation of an alternative feather sampling method to measure corticosterone. Animals 10: 2054.
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The most common feather sampling method for feather corticosterone measurement is by plucking the feathers from the bird’s skin. This procedure performed on living, restrained birds is qualified as an animal experiment according to German/European legislation, which has to be applied for from the competent authorities. The Directive 2010/63/EU requires the full implementation of the 3-R Principle of Russel and Burch in animal experiments, which means not only to replace the use of animals, but also to reduce the number of animals used and to refine procedures whenever possible. In response to this issue, the aim of this study was to validate an alternative, less invasive sampling method by cutting feathers close to the skin in comparison to the gold standard of plucking them. For this proof-of-principle study, a conventional poultry husbandry with trial groups of geese (Anser anser domesticus) and ducks (Anas sterilis) was selected. All birds were kept under the same living conditions to standardize the influencing factors regarding husbandry, and thus, their stress levels. Feather samples were collected between the shoulders from 46 geese and 51 ducks, both by cutting as well as by plucking, directly after slaughter for meat production. Feather corticosterone levels were measured with Enzyme-Linked Immunosorbent Assay (ELISA). Results were compared using Bland–Altman plots and concordance correlation coefficients (CCC). It could be seen that concordance between corticosterone levels in cut and plucked feathers was rather poor: 0.38 for Anser, and 0.57 for Anas. However, comparing the mean corticosterone values in pg/mm of each species with their respective standard deviations, the differences between the methods were negligible. As the results showed that the differences between the individuals were markedly greater than the differences between the methods, the determination of corticosterone levels in cut feathers is valid compared to using plucked feathers. The validation tests of ELISA showed only acceptable repeatability and reliability. Hence, the results should be verified in further studies. In conclusion, it is recommended for future research to use cut instead of plucked feathers for corticosterone measurement.
Bouwer M., Ngcamphalala C. A., Ganswindt A., McKechnie A. E. (2021): Validation of a non-invasive technique for quantifying a stress-associated biomarker in a southern African hornbill. Journal of Ornithology 162: 615-619.
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Quantification of glucocorticoids and their metabolites as a central component of the physiological stress response has many potential applications in ornithological research, but require careful species-specific validations. We established a reliable non-invasive method to measure faecal glucocorticoid metabolites (fGCM) in the southern yellow-billed hornbill (Tockus leucomelas). Following an adrenocorticotropic hormone (ACTH) challenge, plasma corticosterone levels increased by 110%, with a tetrahydrocorticosterone enzyme immunoassay (EIA) revealing a ~ fourfold increase in fGCM concentrations approximately 3 h post-injection. These data reveal droppings are a suitable matrix for monitoring stress-associated biomarkers in this species and suggest the tetrahydrocorticosterone EIA may often be appropriate for wild birds.
Cinque C., Williams N. M., Bencini C., Cozzolino R. (2021): Adverse weather conditions reduce food availability and increase glucocorticoid metabolite levels in barn swallow nestlings. Wildlife Biology 2021: wlb.00747.
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Stimuli perceived as stressful by animals increase glucocorticoid secretion over basal levels. This is an adaptive response that by altering energy metabolism and animal behavior facilitates survival during acute stress. Secretion of corticosterone increases in adult insectivorous bird species after a short period of adverse weather conditions, which may determine a decreased availability of insect prey as well as thermoregulatory stress. Hormone response to stress in altricial nestlings is not yet clear. In some species (e.g. northern mockingbird, white-crowned sparrow) early age nestlings show a reduced or null response to stress stimulation, and the hypothalamus–pituitary–adrenal (HPA) axis seems to be fully active only at an age close to independence. In other species (e.g. barn swallow, canaries) the HPA axis seems to be active even in young nestlings. However, most of the data refer to experimentally induced stress while the physiological response to natural environmental perturbations has been scarcely investigated. The main aim of our study was to evaluate corticosterone secretion response to short periods of adverse weather conditions in two different age groups of barn swallow nestlings (7–14 days old and 15–21 days old). Furthermore, stress responses were compared between medium aged nestlings, late aged nestlings and adults, to understand whether HPA axis activity changed with age. A fecal non-invasive hormone assessment method was used. Our results showed that during adverse weather conditions, insect abundance decreased and corticosterone metabolites levels increased significantly and to a similar extent in droppings of medium and late aged barn swallow nestlings as well as in adults. Our data support previous findings, that the HPA axis is already fully functional in 7–14 days old nestlings. Further studies are required to verify the existence of a hyporesponsive period in younger barn swallow nestlings.
Scheun J., Miller R. J., Ganswindt A., Waller L. J., Pichegru L., Sherley R. B., Maneveldt G. W. (2021): Urofaecal glucocorticoid metabolite concentrations in African penguin (Spheniscus demersus) chick populations experiencing different levels of human disturbance. Conservation Physiology 9: coab078.
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Despite the importance of ecotourism in species conservation, little is known about the industry’s effects on wildlife. In South Africa, some African penguin (Spheniscus demersus) colonies have become tourist attractions. The species is globally endangered, with population sizes decreasing over the past 40 years. As African penguin chicks are altricial and unable to move away from anthropogenic stressors, it is important to evaluate the effect of tourist activities on baseline glucocorticoid levels as a measure of potential disturbance. Chicks at three study sites within two breeding colonies (Robben Island, Stony Point), with varying levels of exposure to tourism (low/moderate/high) were monitored. Urofaecal samples were collected to determine urofaecal glucocorticoid metabolite (ufGCM) concentrations as an indication of baseline stress physiology. Morphometric measurements were taken to compare body condition between sites. Penguin chicks experiencing low, infrequent human presence had significantly higher mean (± standard deviation) ufGCM levels [1.34 ± 1.70 μg/g dry weight (DW)] compared to chicks experiencing both medium (0.50 ± 0.40 μg/g DW, P = 0.001) and high levels of human presence (0.57 ± 0.47 μg/g DW, P = 0.003). There was no difference in chick body condition across sites. These results suggest that exposure to frequent human activity may induce habituation/desensitization in African penguin chicks. Acute, infrequent human presence was likely an important driver for comparatively higher ufGCM levels in chicks, though several other environmental stressors may also play an important role in driving adrenocortical activity. Nevertheless, as unhabituated chicks experiencing infrequent anthropogenic presence showed significantly higher ufGCM levels, managers and legislation should attempt to minimize all forms of activity around important breeding colonies that are not already exposed to regular tourism. Although the results of this study are crucial for developing enhanced conservation and management protocols, additional research on the long-term effect of anthropogenic activities on African penguin physiology is required.
Jakubas D., Wojczulanis-Jakubas K., Grissot A., Devogel M., Cendrowska M., Chastel O. (2022): Eye region surface temperature and corticosterone response to acute stress in a high-arctic seabird, the little auk. Animals 12: 499.
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Measuring changes in surface body temperature (specifically in eye-region) in vertebrates using infrared thermography is increasingly applied for detection of the stress reaction. Here we investigated the relationship between the eye-region temperature (TEYE; measured with infrared thermography), the corticosterone level in blood (CORT; stress indicator in birds), and some covariates (ambient temperature, humidity, and sex/body size) in a High-Arctic seabird, the Little Auk Alle alle. The birds responded to the capture-restrain protocol (blood sampling at the moment of capturing, and after 30 min of restrain) by a significant TEYE and CORT increase. However, the strength of the TEYE and CORT response to acute stress were not correlated. It confirms the results of a recent study on other species and all together indicates that infrared thermography is a useful, non-invasive measure of hypothalamic-pituitary-adrenal (HPA) axis reactivity under acute activation, but it might not be a suitable proxy for natural variation of circulating glucocorticoid levels.
Wang Y., Zhang G., Jiang H., Liu D., Hu X., Qian F. (2022): Dynamic changes in the hormones of black-necked cranes during reproduction. Conservation Physiology 10: coac040.
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Black-necked cranes (Grus nigricollis) are national first-level protected wild animals in China. Artificial breeding has been adopted by many zoos and reserves to achieve ex-situ conservation of black-necked cranes, but the breeding rate of the species in cages is low. This study used non-invasive methods combined with behavioural observations to investigate changes in sex hormones and glucocorticoid metabolites in the droppings of black-necked cranes during the breeding cycle, with the results showing that (i) levels of estradiol and testosterone in black-necked cranes increased significantly when they entered the breeding period, and these levels could be used as an important physiological indicator to effectively monitor the physiological status of females and males during the reproductive period, thus providing a theoretical basis for the timing of semen collection; (ii) the level of progesterone in the mid-reproduction stage was significantly higher than that in other stages in female black-necked cranes after successful mating, and this level could be an effective indicator of the mating status of female black-necked cranes; (iii) droppings’ glucocorticoid metabolites in the breeding period showed different dynamics between paired and singly caged black-necked cranes, indicating that the physiological phenomenon of reproduction could result in a certain amount of physiological burden on black-necked cranes. These results provide a theoretical basis for the selection of physiological parameters in the artificial breeding of black-necked cranes.