Determination of Fecal Tri-iodothyronine and Cortisol as Physiological Proxies of Animal Welfare

Grantees: Michael Cherry, Joe Hediger

Institutions: Caesar Kleberg Wildlife Research Institute, Texas A&M University, University of Massachusetts Dartmouth, United States

Grant amount: $25,860

Grant type: Small grants

Focal species: White-tailed deer (Odocoileus virginianus)

Conservation status: Least concern

Disciplines: Physiology, animal welfare science

Research location: United States

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Project summary

Wildlife are susceptible to both direct and indirect effects of thermal stress imposed by a warming climate. Direct effects include increased energetic costs to maintain a healthy body temperature and immune system impairment, while indirect effects include changes in food availability and increases in disease transmission. White-tailed deer (Odocoileus virginianus) in southern Texas are on the front lines of this environmental challenge. This project aims to assess the reliability of fecal tri-iodothyronine (T3) and fecal glucocorticoids (FGC) as non-invasive physiological metrics for monitoring the health of white-tailed deer. The use of T3 as an indicator of wildlife health and welfare is relatively novel, and the researchers will attempt to refine the use of these metrics through controlled experiments, correlating the T3 measurements with a more widely used indicator in FGC. Both physiological indicators will also be validated against behavioral observations of the same deer that are thought to reflect their emotional state.

Why we funded this project

The study findings will help in understanding how wild animals cope with increasing temperatures and the impact of thermal stress on their welfare and health. Notably, previous work has suggested that T3 measurements in ungulates are especially sensitive to thermal stress, and so comparing T3 with other indicators based on different physiological pathways, such as glucocorticoids, could help researchers to diagnose the relative significance of different environmental stressors an animal is facing. The project’s behavioral metrics are also crucial for realizing that potential. A secondary reason for our interest in this project is that it has near-term policy implications, potentially highlighting the value of preserving or promoting specific landscape features for the ecosystem service they offer, in the form of shade, to wild ungulates.

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Validation and efficacy of faecal glucocorticoid metabolites as indicators of animal welfare

Grantee: Miriam Zemanova

Institution: University of Fribourg, Animalfree Research, Switzerland, Oxford Centre for Animal Ethics, United Kingdom

Grant amount: $28,500

Grant type: Small grants

Disciplines: Physiology, animal welfare science

Research location: Switzerland, United Kingdom

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Project summary

One of the central components of the stress response is the production of glucocorticoids (GC). The measurement of glucocorticoid levels in blood serum is therefore often used in animal welfare studies. However, it is not always apparent how valuable these measurements are for understanding stress reactions and their relationship to animal welfare. Moreover, blood sampling causes discomfort and is impossible without restraint or immobilization, which can be harmful to the animal and distort the experimental results. Fecal glucocorticoid metabolites (FGM) may be a suitable non-invasive alternative to blood analysis. While there have been several reviews written on fecal glucocorticoids, no systematic review of their validity as an animal welfare indicator is currently available. Therefore, through a systematic review process, this project seeks to provide an evidence base for the efficacy of non-invasive measurement of stress levels in wild animals using fecal glucocorticoids. Evidence will be compiled from studies that have assessed the correlation between fecal glucocorticoids and at least one other credible animal welfare indicator or factor that may be assumed to lead to impaired welfare. The project also aims to compare the performances of blood and fecal samples to assess stress levels. 

Why we funded this project

This study will review the evidence base for the validity and efficacy of non-invasively obtained glucocorticoid measurements and identify both challenges and best practices for working with fecal samples across multiple animal taxa. This is important because WAI is supporting multiple projects utilizing FGM as a physiological welfare indicator. Invasive measurements of stress, such as blood-based GCs, are disadvantageous because, without proper strategy and training, the stress induced by the sampling procedure can influence the measurement. Therefore, non-invasive measurement techniques are not only better for the animals, but may make the science less expensive and more reliable. Another reason we are supporting this study is that it will help to address a general need for better validation of the link between glucocorticoids and animal welfare, which is related to but conceptually distinct from physiological stress.

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Characterization of fecal oxytocin and immunoglobulin A in lions (Panthera leo): Exploring a multi-biomarker approach in animal welfare research

Grantee: Paula Serres Corral

Institution: Universitat Autònoma de Barcelona, Spain

Grant amount: $25,385

Grant type: Small grants

Focal species: Lion (Panthera leo)

Conservation status: Vulnerable

Disciplines: Physiology, animal welfare science

Research location: Spain

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Publications

Serres-Corral, P., et al. (2025). Exploring immunoglobulin A as a stress biomarker in lions (Panthera leo): Validation of an immunoassay for its measurement in feces. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 299. https://doi.org/10.1016/j.cbpa.2024.111762

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Project summary

Glucocorticoids (GCs) remain the most common physiological indicators of stress and, by extension, of animal welfare. However, welfare means more than stress, and therefore there is a need to develop complementary biomarkers to expand our knowledge of animals’ overall welfare states, both positive and negative. This project will consider oxytocin (OT) and secretory immunoglobulin A (IgA), markers of neuroendocrine and immune function respectively, which have been proposed as potential indicators of positive affective states. So far, both indicators have been reliably measured in the saliva and urine of a limited set of wild mammalian species. However, previous work on their analysis in fecal samples has been even more limited. This project aims to validate the measurement of OT and IgA in feces of lions (Panthera leo) as a model for social carnivores and evaluate their applicability as additional physiological indicators in wildlife welfare studies. The researchers will evaluate baseline levels for the species based on a small number of captive individuals, and assess how these biomarkers are correlated with GCs and behavior.

Why we funded this project

With a multi-biomarker approach, these potential biomarkers, in combination with GCs, will enable a more robust interpretation of findings in welfare assessments. We are especially interested in the potential for these physiological indicators to support identification of positive welfare states in wild animals, considering the crucial role of oxytocin, for example, in social bonding. Although the project itself is limited to a small number of captive individuals, we expect that this project will represent a significant step towards validating these putative indicators of positive welfare thanks to the detailed behavioral monitoring that the captive environment allows, including a Quantitative Behavioral Assessment (QBA) approach.

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Thermal imaging as a non-invasive welfare assessment tool for tracking the impact of environmental stressors across wild animal populations

Grantee: Ross MacLeod

Institution: Liverpool John Moores University, United Kingdom

Grant amount: $29,810

Grant type: Small grants

Focal species: Wild birds

Conservation status: Least concern

Disciplines: Physiology, animal welfare science, population ecology, ornithology

Research location: United Kingdom

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Project summary

This project aims to test and validate a standardized multi-species approach to monitoring physiological stress in wild birds by using thermal imaging cameras to measure the animals’ body surface temperature, which could enable tracking of chronic stress in wild populations facing different levels of environmental stressors. Building on pilot work, the project will focus on wild bird populations to develop a standardized thermal imaging methodology capable of monitoring surface temperature of a wide range of free-living wild animals. The methodology will be validated using bird communities visiting 54 standardized feeding and drinking stations spread across three urban-rural gradients in the UK, to quantify how changes in surface temperature are linked to starvation risk, predation risk, and human disturbance.

Why we funded this project

This approach to measuring wild animals’ physiological stress levels through thermal imaging analysis has great potential for scalable monitoring of large numbers of individuals and is usable for cross-species comparisons. We appreciated that this team was interested in addressing non-anthropogenic causes of suffering in starvation and predation, and in extending their method to other species that tend to be neglected (e.g., wild rodents). This work also relates to the project by Paul Jerem that we previously funded, creating a longer-term relationship between Wild Animal Initiative and project participants.

Find Ross’ other project, studying house sparrows, here.

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Developing loop-mediated isothermal amplification (LAMP) assays for detecting pathogens in wild animal populations

Grantee: Cameron Semper

Institution: University of Calgary, University of Lethbridge, Canada

Grant amount: $30,000

Grant type: Small grants

Focal species: Deer mice (Peromyscus sp.)

Conservation status: Least concern

Disciplines: Infectious disease, physiology, animal welfare science

Research location: Canada

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Project summary

Loop-mediated isothermal amplification (LAMP) is a low-cost technique that amplifies specific DNA to levels that can enable visual detection. LAMP has been extensively applied as a point-of-care diagnostic tool for human health, but its application in wild animal populations remains underexplored. This project aims to develop LAMP assays for rapid, in situ detection of representative viral and bacterial pathogens as well as parasitic worm infections in wild animals. This project will develop two methods for detecting viral and bacterial infections, respectively. The viral test will focus on viruses that have caused epidemics in wild frigatebird and sooty tern populations, while the bacterial test will focus on tick-borne illnesses and parasitic worms which infect deer mice. After developing the LAMP assays, the researchers will validate their potential contribution to monitoring wild animal welfare in a non-invasive manner by using them to test for pathogenic load in fecal samples from deer mice. 

Why we funded this project

This research will serve as a proof-of-concept for the applicability of LAMP for monitoring infectious disease, a key determinant of wild animal welfare. Additionally, the project is specifically targeting diseases that affect a large number of animals. LAMP is simple to perform, and results can be interpreted visually without the need for sophisticated equipment, reducing the cost in time and materials for assessing disease in wild animals. Because it can be carried out in the field, follow-up treatment or further study can be immediately given to the same animal.

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Raman spectroscopy as a novel non-invasive technique to assess wildlife welfare

Grantee: Teresa Romero

Institution: University of Lincoln, University of Portsmouth, United Kingdom

Grant amount: $19,100

Grant type: Small grants

Focal species: Tufted capuchin monkey (Cebus apella)

Conservation status: Critically endangered

Disciplines: Physiology, animal welfare science

Research location: United Kingdom

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Project summary

Steroid hormones are routinely used as biomarkers of stress and can be measured in different biological matrices, such as serum, saliva, feces, and urine. However, commonly used collection methods in wildlife are challenging; the stress they induce in the animals may affect hormone levels and also presents ethical issues. More recently, analysis of hormones accumulated in the hair has emerged as a non-invasive tool for measuring chronic stress in wildlife, but current limitations of hair analyses include variation in cortisol concentrations depending on sample preparation and the amount of hair required for cortisol extraction. This project will test whether Raman spectroscopy, which is suitable for the field and does not require sample pre-treatment, is a better way to use hair samples to assess steroid hormones. The planned methodology will make use of samples of mammalian hair with a known cortisol content using a control technique — liquid chromatography mass spectrometry — for comparison. The samples will be subjected to analysis by Raman to validate the technique and establish a robust analytical methodology for the non-invasive analysis of welfare biomarkers in wildlife.

Why we funded this project

Although this project is focused on development of chemical methods, we expect that the method, if validated, could make glucocorticoid assessment in the hair of wild animals much easier, increasing the future quantity and quality of that type of data. Hair is an especially interesting medium for glucocorticoid analysis because it integrates glucocorticoid levels in the body over time, causing the measurements to be more stable over time and potentially more indicative of baseline welfare, since the values are less sensitive to an animal’s most recent activities.

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Novel epigenetic approaches to measure wild animal welfare and stress

Grantee: Dave Daversa

Institution: University of California, Los Angeles, United States

Grant amount: $30,000

Grant type: Small grants

Focal species: Western toad (Anaxyrus boreas)

Conservation status: Least concern

Disciplines: Physiology, herpetology, animal welfare science, population ecology, genetics/genomics

Research location: United States

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Project summary

A major challenge in wild animal welfare science lies in developing composite assays that consider the full breadth of factors collectively shaping the subjective experiences of animals. This project will apply epigenetic tools to develop minimally invasive and aggregate measures of wild animal welfare. Specifically, the researchers aim to demonstrate proof of concept for a DNA methylation (DNAm)-based model of wild animal welfare. DNAm joins other measurements such as telomere attrition as a biomarker of biological aging, because DNA becomes methylated as a function of both chronological age (time) and accumulated stress. This project will characterize DNAm in western toads (Anaxyrus boreas) from wild populations in southern California, testing the influence of infection with the fungal pathogen Batrachochytrium dendrobatidis (Bd) on DNAm, and its covariance with demographic factors such as age and sex, as well as with body condition, a common metric of physical health in wild animals. Captive-reared toads representing all life stages will be sampled to characterize DNAm under controlled conditions, providing a standardized profile of DNAm rates over toad life stages. The researchers will then sample toads from wild populations experiencing starkly different levels of environmental stress (Bd infection), with accelerated biological aging signaling elevated stress and impaired welfare. 

Project objectives

Objective 1: Determine the relationship between DNAm and basic risk factors linked to welfare: chronological age, sex, weight, and infection status.

Objective 2: Determine the effect of environmental stressors on epigenetic age.

Why we funded this project

We funded this project because we are interested in the potential for biological aging biomarkers to be used as very long-term, integrative metrics of animals’ lifetime welfare. DNAm is one such potential biomarker that has received relatively little attention in a welfare context. However, DNAm may potentially be easier to measure than more commonly discussed measures of biological age — or at least, be more familiar for mainstream ecological genetics researchers — because DNA methylation is already of interest for other reasons in biological science. We were particularly enthusiastic about this project because it focuses on a species belonging to a large species complex of amphibians, including both common and threatened species, offering broad transferability and potential impact. Amphibians are also relatively neglected in terms of welfare research. We appreciate that this project aims to establish baseline age-specific differences in biological aging rate in order to then assess age-specific differences attributable to different exposures (i.e., age-specific differences in welfare), which connects to the concept of “welfare expectancy” developed by Wild Animal Initiative researcher Luke Hecht.

Find Dave’s other project, studying western fence lizards, here.

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A bird’s eye view to the five domains of welfare: a quantitative framework and proof-of-concept evaluation in a cetacean, Orcinus orca

Grantees: Saana Isojunno and Eve Jourdain

Institutions: University of St. Andrews, Scotland, and Norwegian Orca Survey, Norway

Grant amount: $28,707

Grant type: Small grants

Focal species: Orca (Orcinus orca)

Conservation status: Data deficient

Disciplines: Animal welfare science, bio/eco-informatics, physiology, animal behavior, marine biology

Research location: Scotland, Norway

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Project summary

This project will carry out body condition and welfare assessment for inshore-foraging killer whales (Orcinus orca) in the northeast Atlantic as an extension to ongoing research in Scotland and Norway. The researchers will use the Five Domains model to categorize likely factors influencing killer whale welfare, and then quantify some of those factors using aerial photography from drones. Specifically, the researchers intend to gather data on foraging time and feeding rates (nutrition), group composition and surface behavior (behavior), and body shape as a proxy for body condition and blubber reserves (health). Data will also be collected on reproductive success (e.g., calf loss). The researchers will then develop a proof-of-concept statistical model to infer latent motivational states (i.e., “true” welfare as a psychological state) beneath the observable data. The project will also utilize data from known cases of poor welfare (where individual social and nutritional needs are not met) to help define their welfare scale.

Why we funded this project

We funded this project because we see hidden state models as promising statistical tools for representing the relationship between disparate data types and welfare, and would like to see this project provide a proof of the concept. This project will also implement a cost-effective and non-invasive approach method based on photogrammetry to assess cetacean health and behavior. Finally, we wanted to fund this project because it spans multiple universities and a citizen science community, creating significant networking opportunities to promote welfare biology.

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Impacts of land-use on social networks in mixed-species bird flocks, with implications for the short-term and long-term welfare of Himalayan birds

Grantee: Akshay Bharadwaj

Institutions: Indian Institute of Science, India

Grant amount: $15,000

Grant type: Small grants

Focal species: Multi-species birds

Conservation status: Least concern

Disciplines: Ornithology, physiology, community ecology, infections disease, population ecology

Research location: India

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Project summary

This project will use mist-netting and bird-banding, followed by standardized observations of behavior and body condition, to examine the relationship between individual-level social behavior, bird health, and survival within mixed-species bird flocks (MSFs) in Eaglenest Wildlife Sanctuary, Arunachal Pradesh, India. Feather corticosterone level, ectoparasite load, and other morphological parameters will be used to measure the health of each banded individual. The researcher will also take advantage of a long-term bird-banding dataset, spanning 12 years, to examine the linkages between inter-individual differences in social behavior and the survival of each individual, comparing survival rates and reproductive success in primary and logged forest. 

Why we funded this project

Understanding the importance of multi-species flocking behavior in birds is relevant to our research priority of understanding indirect welfare effects in ecological systems, which this project approaches in a cost-effective way. We were especially impressed by the quality of the proposal for this project, particularly as it is led by a beginning graduate student in India. Funding this project serves to increase the geographic diversity of our grantee community and therefore of the nascent field of wild animal welfare research.

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Every breath you take, I‘ll be watching you: Automated measurement of breath rate from mobile phone videos as a severity assessment parameter in wild great tits

Grantee: Caroline Deimel

Institutions: Max Planck Institute, Germany

Grant amount: $19,200

Grant type: Small grants

Focal species: Great tits (Parus major)

Conservation status: Least concern

Disciplines: Animal behavior, animal welfare science, physiology

Research location: Germany

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Project summary

Breath rate (BR) is increasingly used as a non-invasive proxy of stress that is fast, cheap, and field-friendly. However, BR has not been evaluated in a bird welfare context, and it is unclear how it relates to established physiological proxies of stress, like glucocorticoid measurements in blood. Also, the currently used protocols to measure BR lack standardization, scalability, and validation. This project aims to provide a validated, non-invasive reference tool for standardizing BR measurements following capture and physical sampling protocols in wild birds by implementing computer vision capabilities and other improvements in software the researchers have recently developed to estimate BR metrics from mobile phone videos. This will allow the research community to gather objective, reproducible, and comparable data, and provide institutions tasked to oversee animal welfare with objective and feasible monitoring requirements. The project will also evaluate BR as a welfare indicator in great tits (Parus major), a songbird extensively used in wild animal research across Europe, by analyzing an existing, five-year dataset to test whether BR corresponds to simultaneous glucocorticoid measurements from free-living great tits. These data will provide reliable baselines and ranges for glucocorticoids and BR, and benchmarks for video recording lengths. 

Why we funded this project

We are generally interested in developing non-invasive ways of measuring indicators of wild animal welfare. One of the key advantages to non-invasive measurement, besides the obvious of not causing unnecessary fear or pain to animals, is that stress induced by the measurement process can obscure the animal’s baseline stress levels if the method is too invasive or not carried out properly. We also tentatively believe that instilling a norm of minimizing animal harm within welfare biology research will increase the likelihood that researchers act as scientist-advocates for implementation of wild animal welfare interventions. This project’s focus on making breath rate easy and inexpensive to measure in a consistent way also fits well with our desire for a greater volume and accessibility of welfare data collection.

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