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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City living – assessing the welfare costs of urban living in spotted hyaena (Crocuta crocuta)

Grantee: Emma Stone

Institutions: Carnivore Research Malawi and University of the West of England, United Kingdom

Grant amount: $30,000

Grant type: Small grants

Focal species: Spotted hyaena (Crocuta crocuta)

Conservation status: Least concern

Disciplines: Human-wildlife conflict, physiology, animal welfare science, animal behavior, infections disease

Research location: Malawi

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Project summary

Lilongwe City (the capital of Malawi, central Africa) has a good network of green spaces and river corridors, supporting a population of urban spotted hyena (Crocuta crocuta). However, human-wildlife conflict (HWC) is frequent, often resulting in persecution of these animals. Using non-invasive techniques, this project will measure and compare the fecal and hair cortisol levels (as a proxy of stress), mean body condition, tooth ware, hematological parameters, and disease (toxoplasmosis and rabies) in spotted hyena between urban (Lilongwe district) and rural (Kasungu National Park) populations. Behavioral experiments (exposure to novel objects and threatening scents) will also be used to assess individual boldness in urban and rural populations as a risk factor, which may increase propensity for HWC and therefore mortality and stress. Results will be used to inform policies to reduce HWC and implement welfare considerations as part of the population’s conservation management plan.

Why we funded this project

Interactions between people and wildlife are increasing as urban areas expand. This is of particular concern with carnivores, as even small species can be perceived as threatening. Despite the fact that the developing world has the highest rate of urbanization, urban carnivore studies have generally focused on European and Asian cities, with research on African cities being relatively neglected. Although HWC is a traditional issue in conservation, we have encountered few projects that effectively address the more neglected question of how HWC interacts with non-anthropogenic welfare impacts. By considering a variety of welfare indicators/factors, including disease transmission and social dynamics, this project will help us understand that interaction, producing results that can be translated into more abundant urban species. Additionally, by funding at least some projects on endangered species, we increase the likelihood of our welfare-focused message reaching conservation practitioners, potentially leading to a greater emphasis on and interest in welfare within that community.

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Introducing a novel tool to compare stress levels in captive and wild Macaca fascicularis

Grantee: Vets4welfare

Institution: Vets4welfare Foundation, Netherlands

Grant amount: $20,000

Grant type: Small grants

Focal species: Crab-eating macaque (Macaca fascicularis)

Conservation status: Endangered

Disciplines: Physiology, wildlife rehabilitation, human-wildlife conflict

Research location: Indonesia, Netherlands

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Project summary

This project will compare cortisol concentrations in the hair of Indonesian monkeys (Macaca fascicularis) from three groups: a group whose members have spent their whole lives in the wild, a group that has been kept in captivity for the entertainment industry, and a group that are in the process of rehabilitation following rescue from the entertainment industry. Hair measurements can reflect welfare over a long period, as they integrate cortisol as they slowly grow. The concentration of cortisol in a segment of a hair is thought to reflect the activity of the animal’s physiological stress response at the time that segment was produced. On that premise, the project aims to compare the stress response of M. fascicularis under wild, poor-welfare captive (entertainment), and high-welfare captive (rehabilitation) environments. The hair cortisol data will be compared with veterinary and forensic records and other welfare-relevant observations (e.g., of infections or injuries) where possible. The researchers also intend to determine whether mistreatment in captivity leaves a signal in hair cortisol that could be detected after the fact and used to identify monkeys that have been illegally trafficked. 

Why we funded this project

Although this project focuses narrowly on anthropogenic harms in a particular threatened species, we funded it because their approach is interesting and very similar to one we have been interested in applying to study the welfare of stocked fish (i.e., fish raised in a hatchery and then released into the wild) using biological aging methods, another category of putative welfare indicators that integrate stress over time. Between these two funded studies, we are interested to see 1) how welfare indicators for wild and captive environments of varying quality compare and 2) whether hair cortisol from individuals whose environment changed is consistent (at the corresponding points in time) with that of individuals who were only exposed to one environment or the other. We anticipate that the relationship for the latter question is more complex than described, but investigating it will help us learn more about the validity of hair cortisol measurements and the importance of prior experiences in shaping animals’ stress responses.

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Developing a consensus profile of wild animal welfare: integrating non-invasive monitoring of the gut microbiome with stress physiology and behavior

Grantee: Sam Sonnega

Institution: University of Massachusetts Dartmouth, United States

Grant amount: $29,130

Grant type: Small grants

Focal species: White-footed mice (Peromyscus leucopus)

Conservation status: Least concern

Disciplines: Physiology, animal welfare science, animal behavior

Research location: United States

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Publications

Sonnega, S. and Sheriff, M.J. (2024). Harnessing the gut microbiome: a potential biomarker for wild animal welfare. Frontiers in Veterinary Science, 11. https://doi.org/10.3389/fvets.2024.1474028

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Project summary

This project will characterize the gut microbiome of wild white-footed mice (Peromyscus leucopus) and investigate its relationship with their stress physiology and behavior, including how it changes in response to ecological pressures. While a growing literature from lab-based studies has demonstrated the link between the gut microbiome and regulation of host physiology and behavior, the generality of these findings in ecological contexts remains largely untested. As part of this project, mice will be trapped during different seasons and fecal samples will be collected from which to measure both glucocorticoid concentrations and gut microbiome composition. Concurrently, open-field trials will be conducted to assess individual variation in cognitive bias towards optimism or pessimism (a well-established behavioral indicator of affective state). The mice’s perception of predation risk will also be experimentally manipulated by exposing free-living mice to playbacks of predator noises. By correlating the gut microbiome with both behavioral and endocrine metrics, a consensus profile of the mice’s welfare will be developed that reflects the complexity of their responses to both predictable and unpredictable environmental perturbations, and how those responses can scale up to population and ecosystem level changes via the demographic effects of stress in a so-called “landscape of fear.”

Why we funded this project

We are interested in the development and validation of the gut microbiome as a welfare indicator. Every additional indicator strengthens the interpretation of others, but understanding the gut microbiome may be especially important because it is part of the causal chain linking what is going on in the animal’s brain to fecal metabolites, which are often analyzed as a non-invasive and time-integrated record of physiological stress. This project also links and builds on other gut microbiome research we have funded (by Melissa Bateson, Pablo Capilla-Lasheras, and Davide Dominoni).

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Development of novel measures of welfare in juvenile European starlings exposed to nutritional stress

Grantee: Melissa Bateson

Institution: Newcastle University, United Kingdom

Grant amount: $60,000

Grant type: Challenge grants

Focal species: Starlings (Sturnidae sp.)

Conservation status: Least concern

Disciplines: Physiology, ornithology

Research location: United Kingdom

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Project summary

Juveniles of passerine species such as the European starling (Sturnus vulgaris) experience massive mortality, much of which is caused by direct or indirect effects of nutritional stress. Of birds that survive, many will bear the “scars” of early-life stress that have consequences for their welfare. The aim of this project is to identify the metabolic “fingerprint” of nutritional stress in starling nestlings and to validate sensitive and non-invasive molecular biomarkers that can be used to assess the welfare of wild starlings.

This group has been developing the hypothesis that biomarkers of biological age not only predict future morbidity and mortality, but also reflect the quality of an animal’s cumulative lifetime experience. Existing metrics of biological age, particularly telomere length, require invasive blood samples, and measurements are imprecise, meaning that large sample sizes are currently required to obtain significant effects in epidemiological studies. Estimating biological age based on measuring multiple age-related biomarkers (as is typical in the human aging literature) is likely to be more reliable than using telomere length alone. 

This project will use untargeted metabolomics to identify multiple novel biomarkers of exposure to nutritional stress in nestling starlings. This “fingerprint” will be validated by testing whether it predicts gold-standard behavioral measures of adult affective experience in a cohort of laboratory-raised birds. Metabolomics measures thousands of small molecules in one biological sample and can be performed on a range of tissues including blood, hair, and urine. The aim will be to identify an applicable panel of metabolites that can be measured cheaply and easily from feathers and the uric acid component of guano.

Why we funded this project

This project should introduce a novel indicator of long-term welfare that is less invasive, requires fewer resources, and is potentially more reliable than similar existing methods. The PI is a world leader in the field of animal behavior and is the main originator of using biological aging to understand long-term animal welfare, especially in non-model species. For that reason, we are especially confident in this work being high-quality and having great academic reach and influence.

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Integrating individual-level juvenile welfare in dynamic habitats across time and space

Grantee: Tom Luhring

Institutions: Wichita State University, Texas State University, and Stephen F. Austin University, United States

Grant amount: $162,604

Grant type: Challenge grants

Focal species: Sirens (Siren intermedia)

Conservation status: Least concern

Disciplines: Herpetology, physiology, climate science

Research location: United States

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Project summary

The project will track individual juvenile lesser sirens (Siren intermedia) within and across years for four populations in Eastern Texas. The lack of a terrestrial life-stage and severely limited overland dispersal ability means that hydrologically isolated pools function as closed populations, facilitating recaptures and simplifying demographic estimates. Siren health is directly impacted by the effects of the environment (e.g., drought conditions) through the impacts of resource availability on body condition and growth rates. Furthermore, sirens show strong size-dependent and seasonal shifts in antagonistic behaviors such as biting which lead to acute injuries. 

Aquatic amphibians are especially well-suited for the collection of water-borne stress hormones (corticosterone), which offer the least invasive method of evaluating an integrated measure of corticosterone levels that are passively being released through the skin, gills, feces, and urine. This project will use water-borne corticosterone release rates to investigate changes in stress physiology as a function of changes in the environment experienced by the individual (population density, drought severity index, water temperature, pH, conductivity) across time and space to understand coping capacity. These data will also be used to investigate the welfare impact of an established marking technique compared to a novel approach based on pattern recognition by a machine-learning algorithm. 

Why we funded this project

Juvenile mortality is especially high in amphibians, and amphibian welfare in general is a neglected subject. This project should provide proof of concept for a cost-effective approach for assessing welfare at both an individual and population level. The waterborne measurements have the potential to integrate corticosterone over a longer period of time, increasing its reliability as a welfare indicator. Finally, this project will test a novel, non-invasive approach to mark-recapture studies, which could facilitate much better individual-level welfare research for amphibians and other (especially aquatic) animals in the future.

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Thermal imaging to investigate physiological state in altricial nestlings

Grantee: Paul Jerem

Institution: Tufts University, United States

Grant amount: $37,780

Grant type: Challenge grants

Focal species: House sparrow (Passer domesticus)

Conservation status: Least concern

Disciplines: Physiology, ornithology

Research location: United States

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Project summary

Animal stress responses evolved to increase survival, in part by stimulating behaviors that reduce exposure to challenging situations. However, young birds who are entirely reliant on their parents (“altricial”) are incapable of acting to change their circumstances, potentially exposing them to the damaging effects of chronic stress. Such species are known to suppress aspects of their stress physiology during development. However, it remains unclear if other parts of the system remain active and could serve as useful indicators for efforts to improve early life welfare. This project seeks to investigate this possibility in juvenile house sparrows (Passer domesticus) using a novel, non-invasive method for inferring internal state — thermal imaging of body surface temperatures.

Why we funded this project

We especially want to understand juvenile welfare, because in most species that is the most experienced life stage, and often the most vulnerable. However, developmental changes can make it difficult to compare welfare between juveniles and adults. This project will calibrate a non-invasive proxy of welfare that can be applied to both adult and altricial juvenile birds, enabling not only better welfare assessments, but more effective lifetime comparisons. A better understanding of the “shape” of lifetime welfare would in turn inform interventions that might disproportionately affect individuals of certain ages.

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