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Use of fecal glucocorticoid metabolite measures in conservation biology research: considerations for application and interpretation
Abstract
Fecal glucocorticoid metabolite analyses are increasingly being used by a variety of scientists (e.g., conservation biologists, animal scientists) to examine glucocorticoid (i.e., stress hormone) secretion in domestic and wild vertebrates. Adrenocortical activity (i.e., stress response) is of interest to conservation biologists because stress can alter animal behavior, reduce resistance to disease, and affect population performance. The noninvasiveness of fecal-based assessments is attractive, particularly when studying endangered species, because samples can often be obtained without disturbing the animal. Despite such advantages, many confounding factors inhibit the utility of this technique in addressing conservation problems. In particular, interpretation of fecal glucocorticoid metabolite (FGM) measures may be confounded by the length of time animals are held in captivity, normal seasonal and daily rhythms, body condition, sample storage and treatment techniques, diet of the animal, assay selection, animal status (i.e., social ranking, reproductive status), sample age and condition, and sample mass. Further complicating interpretation and utility of these measures is the apparent species-specific response to these factors. The purpose of this paper is to discuss the factors that confound interpretation of FGM measures, summarize research that addresses these issues, and offer an agenda for future research and interpretation. We urge conservation biologists to carefully c…
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“Sample collection took place immediately after defecation to avoid bacterial and microbial degradation (Millspaugh and Washburn, 2004).”
From Effect of weather conditions and presence of visitors on adrenocortical activity in captive African penguins (Spheniscus demersus) (2017) · cites this paper as Millspaugh and Washburn, 2004
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“This non-invasive method permits the monitoring of the animal's endocrine status without the necessity to capture or handle the animals, which may compromise the accurate assessment of stress (Millspaugh and Washburn, 2004;Goymann, 2012).”
From Effect of weather conditions and presence of visitors on adrenocortical activity in captive African penguins (Spheniscus demersus) (2017) · cites this paper as Millspaugh and Washburn, 2004
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“Samples were gathered in cryovials and frozen immediately after collection at À20 °C (Millspaugh and Washburn, 2004;Palme et al., 2005;Sheriff et al., 2011).”
From Effect of weather conditions and presence of visitors on adrenocortical activity in captive African penguins (Spheniscus demersus) (2017) · cites this paper as Millspaugh and Washburn, 2004
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Li, Y., Yu, C., Liao, J., Jiang, Y., Liu, T., Luo, L., Yang, S., Yang, B., Xu, X., Deng, T., et al. (2026) · Conservation Physiology · doi:10.1093/conphys/coag019 · Cited by 1
Serres‐Corral, P., Almagro, V., Carbonell, L., Borragan, S., Martínez‐Nevado, E., Quevedo, M. Á., Fernández-Bellón, H., Carbajal, A., & López‐Béjar, M. (2025) · Animals · doi:10.3390/ani15162409 · Cited by 1
Chaudhry, E. A., Cheeseman, A. E., Cohen, J. B., Whipps, C. M., Perrotti, L., & Downs, C. J. (2025) · Journal of Experimental Zoology Part A Ecological and Integrative Physiology · doi:10.1002/jez.70010 · Cited by 1
Cinque, C., Bencini, C., Bartolommei, P., Bonacchi, A., Gasperini, S., Manzo, E., & Cozzolino, R. (2025) · Wildlife Biology · doi:10.1002/wlb3.01487 · Cited by 1
Shimamoto, T., Taki, T., Kumaki, A., Motohashi, A., Tamatani, H., Oshima, G., Tanaka, J., & Yamamoto, T. (2024) · Journal of Zoology · doi:10.1111/jzo.13213 · Cited by 1
Burger, M., Webster, A. B., Majelantle, T. L., Scheun, J., & Ganswindt, A. (2024) · MethodsX · doi:10.1016/j.mex.2024.102883 · Cited by 1
“As a prerequisite for non-invasive investigations of reproductive physiology, test systems quantifying reproductive hormone metabolites must first be validated for reliability in any species investigated for the first time [ 8 , 14 , 15 ].”
Chang, T.-K., Wang, C.-H., Lin, T., & Su, Y. (2024) · Journal of the American Association for Laboratory Animal Science · doi:10.30802/aalas-jaalas-24-000003 · Cited by 1
“Several studies 7,16,17,20,21,24 have reported the physiologic effect of housing density on mice, 17–19, 21 rats , 10 and Corticosterone levels are useful indicators for monitoring stress responses in animals.”
Lamb, C., Dubman, E., McNay, R. S., Giguere, L., Majchrzak, Y., Thacker, C., Slater, O., Macbeth, B., Owens-Beek, N., Muir, B., et al. (2024) · Canadian Journal of Zoology · doi:10.1139/cjz-2023-0032 · Cited by 1
Currier, S. A., Whitt, J. G., & Reyna, K. S. (2024) · Conservation Physiology · doi:10.1093/conphys/coae012 · Cited by 2
“Each quail was given a unique leg band and had their age, sex, and weight recorded. collected from the underlying waste pan trays 4–5 times daily (Millspaugh and Washburn, 2004; Mohlman et al., 2020).”
BackgroundEffect of Day Length on Growth and Gonadal Development in Meishan Male Pigs
Lu, N., Yuan, H., Jiang, X., Lei, H., Yao, W., Jia, P., & Xia, D. (2024) · Animals · doi:10.3390/ani14060876 · Cited by 1
“Notably, the fecal samples represent a cumulative measure of hormone release over a more extended period, rather than a ‘snapshot’ or point sample, as in plasma samples [33].”
BackgroundTampach, S., Lopez‐Olvera, J. R., Palme, R., Schwarzenberger, F., Baauw, A. H., Anderwald, P., & Albanell, E. (2024) · Global Ecology and Conservation · doi:10.1016/j.gecco.2024.e02832 · Cited by 2
Okita‐Ouma, B., Gort, G., Millspaugh, J., & Prins, H. H. T. (2023) · African Journal of Ecology · doi:10.1111/aje.13239 · Cited by 1
Self-citationOlsen, A., Smith, A., Hempstead, C., & Larson, S. (2023) · Animals · doi:10.3390/ani13132175 · Cited by 1
“Fecal metabolites may not be evenly distributed within the sample so dried samples were pulverized and mixed [13,29].”
BackgroundFlacke, G. L., Penfold, L. M., Schwarzenberger, F., Martin, G. B., Rosales-Nieto, C. A., & Paris, M. C. J. (2023) · General and Comparative Endocrinology · doi:10.1016/j.ygcen.2023.114338 · Cited by 1
Validation and effects of drying and prey hair on fecal hormone concentrations in spotted hyenas
Greenberg, J. R., Montgomery, T. M., Holekamp, K. E., & Beehner, J. C. (2021) · Journal of Mammalogy · doi:10.1093/jmammal/gyab168 · Cited by 1
Hickmott, A. J., Boose, K. J., Wakefield, M. L., Brand, C. M., Snodgrass, J. J., Ting, N., & White, F. J. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.01.04.474930 · Cited by 1
“quantified as proximate measures of stress in most mammals, including NHP [18], [29]–[31].”
BackgroundPatel, S., Biswas, S., Goswami, S., Bhatt, S., Pandav, B., & Mondol, S. (2020) · bioRxiv · doi:10.1101/2020.11.05.370635 · Cited by 1
“The influences of these major factors in physiological assessments and their technical solutions have been the major focus of research over years (Millspaugh and Washburn, 2004; Palme et al., 2013, 2019; Sopinka et al., 2015; Touma and Palme, 2005).”
BackgroundMarealle, W. N., Eggen, G. S., & Røskaft, E. (2020) · East African Journal of Forestry and Agroforestry · doi:10.37284/eajfa.2.1.142 · Cited by 2
“Faecal cortisol metabolites are a noninvasive method of measuring GCs that is particularly useful because samples can be easily obtained without disturbing the studied animals or interfering with the results (Millspaugh & Washburn, 2004; Möstl & Palme, 2002).”
BackgroundMethodWahyuni, R., Wahyuni, S., Azhar, A., & Gholib, G. (2018) · International Journal of Tropical Veterinary and Biomedical Research · doi:10.21157/ijtvbr.v3i2.13385 · Cited by 1
Monitoramento não invasivo do estresse em animais silvestres mantidos em cativeiro
Altino, V. S., Nogueira-Filho, S. L. G., & Nogueira, S. S. D. C. (2018) · Revista Brasileira de Zoociências · doi:10.34019/2596-3325.2018.v19.24760
“Adicionalmente, as excretas coletadas podem ser preservadas indefinidamente por congelamento (Wasser et al., 2000; Millspaugh & Washburn, 2004; Palme, 2005).”
BackgroundJachowski, D., Mong, T., Washburn, B., Schulz, J. H., & Millspaugh, J. (2014) · doi:10.2461/wbp.2014.10.5 · Cited by 1
Self-citationComing of age: steroid hormones of wild immature baboons (Papio cynocephalus)
Gesquiere, L., Altmann, J., Khan, M., Couret, J., Yu, J. C., Endres, C. S., Lynch, J., Ogola, P., Fox, E. A., Alberts, S., et al. (2005) · American Journal of Primatology · doi:10.1002/ajp.20171 · Cited by 1
Schmidt-Küntzel, A., Wultsch, C., Boast, L. K., Braun, B., der Weyde, L. K. V., Wachter, B., Brummer, R., Walker, E. H., Forsythe, K. J., & Marker, L. (2018) · doi:10.1016/b978-0-12-804088-1.00031-9 · Cited by 1
Flacke, G. L. (2017) · UWA Profiles and Research Repository (UWA) · doi:10.4225/23/5953425346527 · Cited by 1
Measuring Stress in Antarctic Seals
Hogg, C., & Rogers, T. (2009) · doi:10.1007/978-3-540-93923-8_15 · Cited by 1
“This is an evolving field, and recently other factors have been identified that can influence faecal analyses including body condition, diet of the animal, reproductive status, season, sample mass and sample storage and treatment techniques (Millspaugh and Washburn 2004).”
BackgroundSteroid Hormones Generated by CAFOs
Shore, L. (2009) · doi:10.1007/978-0-387-92834-0_3 · Cited by 1
Age, seasonality, and correlates of aggression in female Apennine chamois
Fattorini, N., Lovari, S., Brunetti, C., Baruzzi, C., Cotza, A., Macchi, E., Pagliarella, M. C., & Ferretti, F. (2018) · Behavioral Ecology and Sociobiology · doi:10.1007/s00265-018-2584-5 · Cited by 31
“The assessment of fecal hormone metabolites can provide unbiased results as it does not involve stressing the animal (Millspaugh and Washburn 2004; Sheriff et al. 2011).”
BackgroundInfluentialHutson, M., Ganswindt, A., & Webster, A. B. (2026) · Zoo Biology · doi:10.1002/zoo.70121
Scarlata, C. D., Fink, L. B., Suhn, M., & Wielebnowski, N. C. (2026) · Animals · doi:10.3390/ani16203171
“This helps to minimize acute and diurnal fluctuations and thus allows us to get a better picture of longer-term hormonal changes [10].”
Harper, E., Yarnell, K., Palme, R., Scott, D. M., Barber, I., & Yarnell, R. W. (2026) · Conservation Physiology · doi:10.1093/conphys/coag063
“…glucocorticoid metabolites in faeces needs to be both suitably sensitive to changes in stress response, and relevant—i.e. it reacts to FGMs produced in response to the stressor and can measure small changes in FGMs ( Millspaugh and Washburn, 2004; Touma and Palme, 2005; Edwards et al. , 2025).”
Lacomme, L., Fritz, H., Ganswindt, A., Guerbois, C., & Rey, B. (2026) · Journal of Zoology · doi:10.1111/jzo.70141
Kolářová, M. F., Kováčová, K., Součková, M., Svoboda, M., & Zeman, M. (2026) · Frontiers in Veterinary Science · doi:10.3389/fvets.2026.1859281
“Non-invasive approaches include the analysis of glucocorticoids across species from various biomaterials, e.g., feces (27, 28), fur (29, 30), feathers (31), shed snake skin (32), claws (33, 34), and earwax (35).”
Olvera‐Maneu, S., Regidor, M., Serres‐Corral, P., Silvestre, A. M., & López‐Béjar, M. (2026) · Animals · doi:10.3390/ani16101485
“…has been shown to return to baseline concentrations within hours [31–33], and the integrative nature of fecal glucocorticoid metabolite measurements minimizes the potential confounding effect of transient acute stressors such as the initial transfer between enclosures performed in our study [34].”
Scheun, J., Labuschagne, K., Jansen, R., Ganswindt, A., & Long, C. (2026) · Discover Conservation · doi:10.1007/s44353-026-00090-2
Faecal inorganic matter affects androgen metabolite levels in seals before migration
Otsuki, M., Horimoto, T., Kobayashi, M., Ijiri, S., & Mitani, Y. (2026) · Journal of Experimental Marine Biology and Ecology · doi:10.1016/j.jembe.2026.152178
Цвей, А. Л., Loshchagina, J. A., & Naidenko, S. V. (2026) · Journal of Evolutionary Biochemistry and Physiology · doi:10.1134/s0022093026010138
“Thus, interpreting HPA axis activity solely based on metabolite con(cid:2) centrations (ng/g) may, in some cases, lead to mis(cid:2) leading conclusions, particularly when comparing animal groups differing in diet, digestion rate, energy expenditure, or other parameters [21, 47, 48].”
Blázquez-Castro, S., Barrientos, R., Reguera, S., Barja, I., & Megía-Palma, R. (2026) · Climatic Change · doi:10.1007/s10584-026-04119-2
“This physiological response can be adaptive in contexts of abundant food availability (Millspaugh and Washburn 2004; Cote et al. 2010; Racic et al. 2020).”
Zenuto, R. R., Brachetta, V., Carrizo, M. C., Fanjul, M. S., & Schleich, C. E. (2026) · Animals · doi:10.3390/ani16020234
“…https://doi.org/10.3390/ani16020234 not only to avoid contamination by urine, which significantly increases GC metabolites [19], but also to consider how metabolites change over time since defecation and after exposure to various environmental factors such as temperature and humidity [28,29].”
InfluentialLauderdale, L. K., Granger, D. A., Walsh, M. T., & Miller, L. J. (2025) · General and Comparative Endocrinology · doi:10.1016/j.ygcen.2025.114873
Boonstra, R., Castillo, J., Karels, T. J., Dobson, F. S., Palme, R., & Bosson, C. O. (2025) · General and Comparative Endocrinology · doi:10.1016/j.ygcen.2025.114856
Validating the underpinnings of water corticosterone measurement for aquatic amphibians
Smith, T. E., Holmes, A. M., Emmans, C. J., Coleman, R., & Hosie, C. A. (2025) · F1000Research · doi:10.12688/f1000research.157055.1
Land use influences the faecal glucocorticoid metabolites of multiple species across trophic levels
Chiu‐Werner, A., Fanson, K. V., Cameron, E. Z., & Jones, M. E. (2025) · Conservation Physiology · doi:10.1093/conphys/coae091
Close and frightening: predator presence affects cortisol levels in captive felines
Volobueva, K. A., & Naidenko, S. V. (2024) · Russian Journal of Theriology · doi:10.15298/rusjtheriol.23.2.08
“…metabolites (FGMs) measurement is a valuable tool to assess the in fl uence of different factors on adrenocortical activity in individuals of different species kept in captivity (Palme, 2019; Keay et al ., 2006) and living in the wild (Gerlinskaya et al ., 1993; Millspaugh & Washburn, 2004).”
Safavian, S., Briskie, J. V., Cameron, E. Z., & Chapman, H. (2024) · New Zealand Journal of Zoology · doi:10.1080/03014223.2024.2420800
“Secretion of glucocorticoids in birds is a key mechanism to deal with stressors (Millspaugh and Washburn 2004).”
Sutton, N. M., Suski, C. D., Payne, K., & O’Dwyer, J. P. (2024) · Conservation Physiology · doi:10.1093/conphys/coae062
“Faecal hormone sampling and analysis can serve as a non-invasive and relatively low cost method for measuring GC levels in wildlife (Millspaugh and Washburn, 2004; Romano et al ., 2010; Ralph and Tilbrook, 2016), and can enable longer-term and more widespread hormone sampling from populations if…”
BackgroundCurrier, S. A., Whitt, J. G., & Reyna, K. S. (2023) · bioRxiv · doi:10.1101/2023.10.26.564168
“Steroid hormone concentrations can be affected by storage duration (Millspaugh and Washburn, 2004) and bacteria (Lexen et al. , 2008).”
BackgroundVerteramo, C. A. C., Lucio, J. A. R., Tessaro, S. A. G., & Sánchez, B. A. (2021) · doi:10.46925//rdluz.32.02
Brown, M. E., Torkelson, M. R., Olsen, G. H., Krisp, A., & Hartup, B. (2020) · Zoo Biology · doi:10.1002/zoo.21541
Chen, C., Chang, A., Tsai, M.-S., Huang, Y., Pei, K. J., & Li, Y.-C. (2021) · Research Square · doi:10.21203/rs.3.rs-294025/v1
Endocrinology and Behavior: Methods
Ayres, K. L. (2019) · Encyclopedia of Animal Behavior · doi:10.1016/b978-0-08-045337-8.00354-5
Davidian, C.-E. (2019) · Refubium (Universitätsbibliothek der Freien Universität Berlin) · doi:10.17169/refubium-25494
Biological consequences of rapid environmental change in the American pika Ochotona princeps
Waterhouse, M. D. (2017) · cIRcle (University of British Columbia) · doi:10.14288/1.0355850
“Interestingly, this same pattern was reported in fecal samples for both mourning doves, Zenaida macroura (Millspaugh & Washburn 2004) and California spotted owls, Strix occidentalis (Tempel & Gutierrez 2004), reinforcing the need to correct for extraction efficiency.”
BackgroundAtwood, E. M. (2012) · cIRcle (University of British Columbia) · doi:10.14288/1.0105190
Metadata
- Authors
- Millspaugh, J. J., & Washburn, B. E.
- Affiliations
- University of Missouri (US)
- Animal and Plant Health Inspection Service (US)
- Corresponding author
- Millspaugh, Joshua J.
- Venue
- General and Comparative Endocrinology
- Publisher
- Elsevier BV
- Published
- 2004-09
- Volume
- 138
- Issue
- 3
- Pages
- 189-199
- ISSN
- 0016-6480 (ISSN-L)
- OpenAlex
- W2168394255
- Cited by
- 553
- References
- 0
- Citations by year
- 2026: 19 · 2025: 20 · 2024: 24 · 2023: 25 · 2022: 18 · 2021: 43 · 2020: 25 · 2019: 25 · 2018: 32 · 2017: 25 · 2016: 30 · 2015: 25 · 2014: 43 · 2013: 37 · 2012: 31
- FWCI
- 3.53
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- General and Comparative Endocrinology (publishedVersion)
- PubMed (publishedVersion)
- Lincoln (University of Nebraska) (submittedVersion)
- Funders
- University of Missouri
- Topics
- Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Behavior and Welfare Studies
- Physical Sciences › Environmental Science › Ecology › Wildlife Ecology and Conservation
- Life Sciences › Neuroscience › Behavioral Neuroscience › Stress Responses and Cortisol
- Keywords
- fecal glucocorticoid metabolites, stress hormones, glucocorticoid secretion, adrenocortical function, conservation biology, seasonal rhythms, body condition, assay selection, social ranking, reproductive status, threatened species, wildlife disturbance, surrogate species
- MeSH terms
- Age Factors, Animals, Conservation of Natural Resources, Ecology, Feces, Female, Glucocorticoids, Male, Reproduction, Seasons, Sex Factors, Specimen Handling, Zoology, Reproducibility of Results, Clinical Laboratory Techniques
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