How can stress in laboratory animals be measured in a non-invasive way? That is the main question in the research line of Dr. Hetty Boleij, lecturer at the department of Animals in Science and Society of the faculty of Veterinary Medicine at Utrecht University. She is studying ways of reliably measuring stress using an infrared (thermal) camera to record changes in body temperature. She was recently awarded a grant from the Utrecht University’s 3Rs Stimulus Fund to continue her research on this method. Application of an infrared camera has the potential to refine animal experiments, which results in better welfare and more reliable research results.
An increasing number of publications using infrared measurements on various animal species have recently been published. The results are obtained with an infrared camera, which measures the infrared radiation given off by animals, as well as by objects. So far, researchers using the infrared camera have demonstrated that in several species the temperature of the eyeball is related to changes in body temperature resulting from stress. The eyeball temperature can be measured remotely with an infrared camera. Boleij: “The temperature has to be measured as a change over time, so you can’t just measure once. You’re actually trying to see if the temperature has gone up, and if it goes back down when the stressor is removed.”
Non-invasive measurement technique
Very few of these studies were performed with mice and rats. But studies on other species suggest that using an infrared camera to measure the body temperature as an indicator of stress is a good alternative to, for instance, drawing blood for measuring an animal’s corticosterone values. Filming an animal is clearly less physically invasive than drawing blood with a needle and should therefore cause less stress, and consequently has also less influence on the outcome of the experiments. Moreover, using an infrared camera can be a non-invasive alternative for research on temperature regulation, where animals often carry transponders (a device that sends a signal, ed.) implanted in the body to measure temperature over time. These so-called telemetry devices require an invasive operation that will also have an effect on the animal’s physiology. So the idea of being able to measure body temperature in a non-invasive way becomes attractive.
Dr. Boleij has made some progress already: “We’ve finished a few small pilot studies with mice, and rats to a lesser extent, on the best way to apply a thermal camera. With the first project, we compared three different injection techniques as to their reliability and the amount of stress they cause. Besides testing their blood corticosterone levels, we also took measurements with the thermal camera in order to validate them as measurements of stress.”
Boleij and her colleagues weren’t able to draw firm conclusions about measuring stress with the thermal camera. They did find part of what was expected, which is an increase in eye temperature and a decrease in tail temperature after handling and injecting the mice. However, no difference in temperature between the control group where the mice were only placed in a test cage, and the group of mice getting an injection was observed. “By the way, simply handling the mice and placing them in a new environment are stressors in themselves, despite the fact that we had let them get used to the test cage for a few days,” says Boleij. “We also studied whether it made a difference if we let the animals get used to the test cage for a longer time. We placed the mice in test cages twice a day for a week. In the last session, we saw less increase in eye temperature than in the first one. This is favourable, because it indicates that the mice are probably less stressed after several days of testing. So we should be able to take this point further.”
Image of mice, made by the infrared camera
Current obstacles and future aims
Our mice live in group housing, as required by law. However, our measurements showed that the mice affect each other’s body temperatures, since they lie next to or on top of each other to keep warm. If you take the temperature of the tail that was warmed by a cage mate, your measurement isn’t meaningful. So we’d like to see if the measurements become more reliable when animals are individually housed.
Also, with home cage measurements, the cage cover needs to be taken off and the shelters need to be removed, since these can block the heat radiation making it difficult to get accurate measurements. So, we’d like to design a home cage made of material that doesn’t block the radiation. Then it would only be a matter of installing the camera in the cage, without interfering with the animals.
Dr. Boleij stresses the importance of this type of research. “I think it’s very important to always be looking out for innovative methods that refine research and improve animal welfare. It would of course be wonderful if we could demonstrate that, in particular with stress-related research, this method has less of an effect on welfare than traditional methods.”
So, it’s not only of interest for welfare research, but for other research directions as well, like thermoregulation studies, for example, studying hibernation in particular species. There are all kinds of other possible applications opportunities, even for research on people. But that would require validation of the method, which is envisaged in the near future.
Boleij and her colleagues have already tried out a new kind of cage for rats and the results look promising. It’s a double-decker cage with more space for the animals. Measurements with the camera haven’t worked up to now, because the camera can’t take in the entire cage, so you miss data once the animals move out of the frame. It would be best to make the top of the cage out of infrared-permeable material. The researchers have had contact with the supplier to ask if whether they can build a modified cage that is suitable for the thermal camera.
Boleij would love to hear from fellow researchers with ideas for using the thermal camera in research on temperature and stress. She’d also like to be in contact with people doing research using other ways of measuring temperature, to see if their results correlate with her measurements. Boleij is currently working only with rats and mice, but she also has some agreements with colleagues to use the camera with other animals. The research group ‘ecological determinants of behaviour’, led by Prof. Liesbeth Sterck, used the camera in a study on long-tailed macaques to look at temperature changes in the face in relation to emotional responses. The camera is also being used in an experiment on sleep deprivation in zebra finches. In the future Boleij would like to measure temperature in dogs and pigs. For the time being, this 3Rs project is focused specifically on rats and mice. ‘We only have one camera, so we’ll just have to be patient.’