Stress Tests

30 May 2019 - By: Alex Evans

Stress Tests

By Alex Evans

In an ever-changing world, our ability to sample and analyse the physiological state of wildlife is becoming more and more important—but at what cost to the animals themselves? One of the sessions taking place at the 2018 SEB Annual Meeting explored the fascinating recent advances being made in developing non-invasive methods of stress monitoring for both laboratory-bound and field-based research.


Amphibians are in the midst of a global decline and current research into the myriad of stressors currently faced by wild populations is crucial to conservation efforts. However, the processes involved in monitoring the manifestations of stress in these animals can themselves be stressful, creating an urgent need for more non-invasive techniques. Thankfully, a recent collaboration between amphibian behaviour researcher Lottie Hosie and endocrinologist Tessa Smith (both University of Chester, UK) set out to find this stress-less solution. “We got talking after a conference a few years ago and wanted to explore the possibility of non-invasive corticosterone measurements for aquatic amphibians,” they explain.

The key to their non-invasive methodology was the ability to sample the steroids in the water that the amphibians live in, taking an environmental sample of their body’s chemical output in response to stress. Similar approaches had recently been adopted for fish research, and the pair of researchers quickly recognised the potential that this technique held for amphibians of conservation concern. “There are many potential sources of stress in field-based conservation work which might have negative implications for successful establishment or maintenance of populations,” they said.

“These issues are largely unexplored for amphibians so we hoped that this project would be a first step in enabling further work in this important area.”

Starting with captive frogs (Xenopus laevis) in their laboratory, the team developed a novel method of extracting corticosterone (a hormone involved in stress responses) from tank water and used this to measure the levels of stress experienced by frogs during transportation and housing changes. Building on the success of this project, the team turned their attention to adapting the methods for use with amphibians in the field. “Our goals were to validate these techniques in a field setting and to ascertain whether they measure biologically meaningful changes in corticosterone.”

After analysing the data from their field trials, the Lottie and Tessa were pleased to find that their novel method proved successful in detecting these stress signals. One of their key findings was a clear interspecific difference between levels of waterborne corticosterone, with great crested newts (GCNs) excreting lower titres than palmate and smooth newts. “This was an interesting result since GCNs are perceived as being more sensitive in their habitat requirements than the other newts,” they explain. However, it wasn’t just species differences that affected the levels of steroid detected. “Our data also suggested differences in corticosterone across the sexes and in response to the trap they were caught in!”

Looking to develop and distribute these methods further, Lottie and Tessa are currently working with conservation partners to assess their application for monitoring the stress of amphibians at risk. “We are delighted that our work is helping to address this gap in amphibian welfare, both for captive lab species and those of conservation interest,” explain the pair. “Establishment of baselines of corticosterone levels and variation in the field is critical for reliable interpretation, so that will be our first step.”


The physiological stress experienced by animals manifests itself in many ways, some of which are easier to measure than others. Taking reliable measurements of stress is crucially important for assessing animal health and welfare, but, unfortunately, many of these methods require the trapping and handling of animals so that they can be fitted or implanted with biologgers or have blood samples taken. “These methods limit research to species that can be caught, interrupt natural behaviour, and can alter subsequent physiology, behaviour, and performance,” explains Dorothy McKeegan, an animal welfare researcher from the University of Glasgow, UK. This important issue spurred Dorothy to look for alternative methods that rely less on instrumentation and more on observation.
Blue thermal
Dorothy’s blue tits thermal image Photo :Dorothy McKeegan

Exposure to stressors is known to induce rapid changes in blood circulation that raise the core body temperature through stress-induced hyperthermia. “Measurement of this phenomenon is challenging and requires invasive methods such as the implantation of temperature loggers,” Dorothy says. “Our idea was to measure surface cooling of their bodies in a non-contact and continuous way using thermal imaging instead.” Infrared thermography has previously been used to investigate resting body temperatures in birds, but Dorothy hoped to validate the use of this technique as an effective and non-invasive assessment of welfare and physiological state too.

Working with two study species in two different research environments, namely captive laying hens and wild blue tits, Dorothy and her team assessed the surface temperature of a range of spatial locations in association with handling stress and the taking of hormonal measurements.

“In hens, we have shown that in the comb and wattle, stressor intensity predicts the extent of skin cooling and the occurrence of delayed skin warming, providing two opportunities to quantify stress,” says Dorothy. “Additionally, our findings from wild blue tits suggest that it may be possible to infer physiological state from just a snapshot of body surface temperature.” Dorothy’s successful validation of the technique provides welfare researchers with a powerful new tool for investigating physiological responses to stress in a non-invasive and continuous manner. “Thermography data can be collected from any terrestrial species that can be photographed or filmed, so this is an exciting approach with a wide variety of applications,” concludes Dorothy. “Furthermore, infrared technology is becoming increasingly cost-effective and portable, improving its availability.”


Stress hormone profiles can provide a detailed peek into the physiological state of an animal but procuring samples, especially from oceanic wildlife, can often be a difficult task. Whales are one such problematic group of animals, because the sampling of blood from free-swimming mammals of that size is almost impossible. However, by taking advantage of their regular need to surface and breathe, researchers are able to acquire biological samples without touching the whales. “We’ve known that hormones can be detected in whale blow for over 10 years, but for physiological assessments, it is quantifiable concentrations that really matter,” explains Elizabeth Burgess (New England Aquarium, USA).

With her sights set on improving this technique for assessing physiological stress in North Atlantic right whales, Elizabeth and her team hoped to overcome some of the major issues with accurately measuring the hormonal content of whale blow, such as dealing with unknown total volumes and samples contaminated with seawater. “We wanted to produce a methodology for accurately quantifying the hormone content of whale blow by identifying a compound to help normalise hormone concentrations,” says Elizabeth. “These could then be used to produce meaningful and comparable endocrine profiles for individual whales.” However, the hormone analysis was only one part of the investigation—first, they had to collect the samples. “Our sampling approach is actually rather low-tech,” says Elizabeth. “We used a boat with a long pole and a large petri dish at the end, allowing for samples to be taken without making contact with the animals.”
Sampling Whales
Sampling Whales. Photo: Anderson Cabot Center for Ocean Life at the New England Aquarium [Photo was taken under U.S. NOAA Permit #14233 and Canada DFO Permit under Species at Risk Act]

Thanks to significant contributions of North Atlantic right whale data from the Anderson Cabot Centre (at the New England Aquarium, USA), Elizabeth was also able to access valuable information about the individual whales she had sampled, such as sex, age, and calving history, in order to link their endocrine profiles to relevant life-history traits. Together, Elizabeth’s team created an effective method of non-invasive physiological assessment, and not a moment too soon. “There is an urgent need to pioneer new tools such as this that can provide evidence of the effects and impacts that human activity is having on our oceans before we see serious consequences,” she explains.

Improving our understanding of whale health and behaviour through their physiology is an increasingly important task thanks to the rapidly changing environment they inhabit. “Whales are a key part of ocean ecosystem health, since they travel the vast widths and depths of the world’s oceans,” Elizabeth says. “This method has the potential to broaden our perspective of large whale health and deliver insights into processes of conservation concern such as stress and reproduction.” Looking forward to future applications of this technique, Elizabeth hopes that other whale biologists will continue to develop and adapt this methodology towards a range of possible research streams beyond hormone analysis. “Our study delivers a great step forward for feasible research of this nature, but this is only the beginning! Analyses of respiratory vapour could open up a wealth of diverse biomarkers for understanding the physiological state and stress responses of whales!”

1. Tudorache C, Schaaf M, Slabbekoorn H. 2013. Covariation between behaviour and physiology indicators of coping style in zebrafish (Danio rerio). J Endocrinol, 219, 251–258.
2. Tudorache C, Slabbekoorn H, Robbers Y, et al. 2018. Biological clock function is linked to proactive and reactive personality types. BMC Biol, 16, 148.
3. Øverli Ø, Winberg S, Pottinger TG. 2005. Behavioral and neuroendocrine correlates of selection for stress responsiveness in rainbow trout—a review. Integr Comp Biol, 45, 463–474.
4. Teles MC, Dahlbom SJ, Winberg S, Oliveira RF. 2013. Social modulation of brain monoamine levels in zebrafish. Behav Brain Res, 253, 17–24.
5. Culbert BM, Gilmour KM, Balshine S. 2018. Stress axis regulation during social ascension in a group-living cichlid fish. Horm Behav, 103, 121–128.
Category: Animal Biology
Alex evans

Alex Evans

Alex Evans is a Research Postgraduate at the University of Leeds investigating the energetics of bird flight. In his spare time, Alex enjoys writing about the natural world, contributing to the Bird Brained Science blog and exploring other avenues of science communication.

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