Research Grants 2003-2004

Measures to Reduce Stress Caused by Experimental Procedures

Bert van Zutphen, PhD and Vera Baumans, DVM
University of Utrecht, Utrecht, The Netherlands

Stress can be described as the inability of an individual to maintain homeostasis, which can occur as a reaction to a (negative) physical or mental stimulus. Laboratory animals are frequently subjected to routine experimental procedures such as injections or withdrawal of blood samples. Each of these treatments influences the horneostasis of the experimental animal to a certain extent, and can, therefore, have an effect on the well-being of the animals and, as a consequence, on the study results. The aim of the four-year PhD-project is to investigate the possibilities of environmental measures to reduce the degree of stress as experienced by the animals (mice) subjected to routine experimental procedures. It is anticipated that relative simple measures like cage enrichment, social housing and conditioning of the animals will have a substantial impact on the stress response of the animals. If so, the implementation of such measures would be a refinement in the use of laboratory animals.

In this research project, the following questions will be tackled:

What is the effect of the method of injection (e.g. subcutaneous vs. intraperitoneal) on the stress response (level and duration) of the animal?
What is the effect of different physical and social environmental factors such as cage enrichment or group housing, on the stress response (level and duration) in an experimental procedure?
What is the effect of the strain (genetic background) on the stress response of animals?
Can the stress response be reduced by conditioning the animals (by means of a stimulus like a noise, an air-puff or a light flash) on the experimental procedure and/or by combination of the procedure with a reward?
What is the effect of a stress response in an animal on its conspecifics; i.e. can stress in one animal induce stress in another animal that does not undergo any procedure but is present in the treatment room?

In the first year, an experiment has been performed that focused specifically on questions numbers two and three. In the second year, the effects of cage enrichment, handling, individual housing and strain will be investigated in more detail. Furthermore, we will focus on question four. It is hypothesized that an unpleasant, stressful procedure such as an injection will be less stressful when the animal can predict when it will happen (conditioning) and/or when it has a pleasant experience quickly afterwards (reward). In order to measure the stress experienced by the animals in this experiment, we will use radio-telemetry techniques for the stress-free measurement of heart rate and body temperature. Although radio-telemetry requires a surgical procedure to implant the transmitter, it is generally accepted as a refinement of animal experimentation. Furthermore, it reduces the number of animals used, as animals can serve as their own controls.

As far as we know, there has been no experience so far in using radio-telemetry to measure anticipation and/or stress during classic Pavlovian conditioning paradigms. Behavioral observations, however, have been extensively used to this purpose. We therefore want to support and validate the physiological parameters by behavioral observations using LABORAS, a device for the automatic registration of behavior of mice or rats. A combined device of LABORAS with the radio-telemetry system of DataSciences International is currently being developed and will be available in the near future.

As it will not be necessary to obtain organs for post-mortem examination after the conditioning experiments, the transmitter-implanted animals will not have to be euthanized. Therefore, the implanted animals can also be used in another experiment focusing on question number one. Experiments will be performed that compare different injection techniques to see if these techniques cause different levels of stress responses. If so, the less stressful methods can be advised to animal experimenters if both more stressful and less stressful methods are possible options.