Session B3: New Developments in Monitoring Animal Physiology and Behavior

Chairs: Bernward Garthoff (Germany) and Rosemarie Einstein (Australia)

B3: Implanted Telemetry Transmitters Alter the Noradrenergic Response in Vas Deferens from Mice
R. Einstein and R.L. Billing. Department of Pharmacology, University of Sydney, NSW 2006, Australia. einstein@med.usyd.edu.au.

Implantable telemetry devices are widely used in experimental animals. We investigated the potential for such implants to induce stress in mice. Changes in body weight and post-mortem responses of the vas deferens to norepinephrine (as a measure of sympathetic activation) were measured 28 days after transmitter implantation. We examined the influence of the anaesthetic used during implantation (pentobarbitone or Hypnorm/midazolam), the strain (balb/c and CBA) and the body weight at the time of implantation (small = 19-25g; large = 27–30 g). A sham-implantation procedure did not significantly affect the responses to norepinephrine. When telemetry transmitters were implanted in small mice of both strains, there was a significant increase in the maximum response to norepinephrine compared to that obtained in tissues from large mice. The anaesthetic used during implantation did not affect the responses to norepinephrine obtained post-mortem. Smaller mice of both strains had a significantly greater post-operative weight loss than larger animals and this was maintained for the experimental period. The results show that implantation of telemetry transmitters has a significant impact in mice that weigh < 25 g.

B3: Does Conditioning Influence the Increase of Heart Rate and Body Temperature as Provoked by Handling in the Mouse?
Klaas Kramer¹, Anne Mulder², Heleen van de Weerd³, Vera Baumans³, René Remie², and Bert Van Zutphen³. ¹Department of Safety and Environmental Affairs, Free University, Amsterdam, The Netherlands; ²Department of Laboratory Animal Science, Solvay Pharmaceuticals b.v., Weesp, The Netherlands; ³Department of Laboratory Animal Science, Utrecht University, Utrecht, The Netherlands. k.kramer@dienst.vu.nl.

Handling of mice or cleaning their cages results in an acute increase in heart rate (HR) and body temperature (BT). Radio-telemetry with an implantable transmitter provides an accurate and reliable way for obtaining these physiological parameters from freely moving animals in their home cage. In previous studies we found that the HR of freely moving mice, implanted with a transmitter, increased from 400–450 beats per minute (bpm), measured in their home cages at rest, to 750–800 bpm measured after handling and/or placing the animals in a novel cage. BT, also measured radio-telemetrically, increased by 1.5° C after handling. The purpose of the present study was to investigate whether conditioning of the mice with an acoustic stimulus, generated by a tone generator (10 kHz, 60 dB), might reduce the stress of a handling procedure. Male C57Bl/6N mice were used in three different treatment groups (Group 1–3; n = 6). The animals of Group 1 were not handled for two weeks, but in the third week these animals were handled according to the procedure as in Group 2. The animals of Group 2 were handled three times per day for six days per week during three weeks at arbitrary times. The animals of Group 3 were housed in another animal room and the handling procedure was executed as in Group 2, however after a warning with the acoustic stimulus. In the third week the animals of Group 3 were handled without the acoustic stimulus before the handling procedure. From the results, it can be concluded that entering the animals' room, even without performing handling procedures, increases HR and BT. After two weeks the animals have habituated to this type of disturbance. Repeated handling does not reduce the stress effects. After three weeks, HR and BT of the mice in Group 2 are still higher after handling. In contrast, if repeated handling is preceded by the acoustic stimulus (Group 3), the stress effect is reduced after two weeks. It is concluded that an acoustic stimulus can be effective in conditioning and thus in reducing the stress response in mice.

B3: Evaluation of a Behavior-based Post-operative Pain Scoring System in Rats
P.A. Flecknell and J.V. Roughan. Comparative Biology Centre, Medical School, University of Newcastle upon Tyne, Framington Place, Newcastle upon Tyne NE2 4HH,UK. p.a.flecknell@ncl.ac.uk.

We have demonstrated clear behavioral changes following laparotomy in rats, but this required prolonged observation under controlled conditions. To develop these observations into a pain scoring system, Fisher rats undergoing laparotomy as part of an unrelated project (n=57) were studied. Animals received either saline (0.2 ml/100 g s/c) or meloxicam (0.5, 1 or 2 mg/kg s/c) or carprofen (2.5, 5 or 10 mg/kg s/c) one hour prior to surgery. Following recovery from anesthesia, rats were placed in a cage for a 10-minute observation period. The frequency of back arching, fall/stagger, squirm and poor gait were used to compute a composite behavior score. Analysis of variance showed that the behavior score (4.6) was significantly greater (p < 0.01) in animals given saline than in those given 1 or 2 mg/kg of meloxicam (1.7, 1.8) or carprofen 2.5 (1.7), 5 (2.0) or 10 (1.7) mg/kg (values are geometric mean pain scores). These data indicate that this technique of quantifying post-operative pain can be used effectively and can be carried out in a sufficiently short period (10 minutes) to be practical and useful.

B3: Refinement of the Lethal Endpoint for Testing of Leptospiral Vaccines in the Hamster Model: Infrared Body Temperature Measurement and Other Strategies
Paul Townsend. Veterinary Laboratories Agency–Weybridge, New Haw, Addlestone, Surrey KT15 3NB, UK. p.townsend@vla.defra.gsi.gov.uk.

The batch potency testing of leptospiral vaccines for use in animals is carried out using animal models, primarily the hamster. The commonest vaccines tested at this establishment are the multivalent leptospiral vaccines against L. canicola and L. icterohaemorrhagica. Testing involves the challenge of hamsters vaccinated with the material under test (as well as unvaccinated positive control animals), using lethal doses of the organism. The aim of the study was to attempt to identify alternative, i.e. more humane, endpoints to replace the death of the animal as the endpoint of the test. Techniques employed include maintenance of animals on reverse lighting, clinical scoring of the animal, and measurement of body temperature using a non-invasive infrared temperature measuring device to identify a cut-off temperature predictive of death. The results of the studies will be presented.

B3: A CD-ROM on the Whole Cell Pertussis Vaccine Potency Test for Reducing Test Variability and for Implementing Humane Endpoints
Coenraad F.M. Hendriksen^1,2, Henk van Wilgenburg³, Klaus Cussler⁴, David Morton⁵, and Björn Steen². ¹Netherlands Centre on Alternatives to Animal Use (NCA), The Netherlands; ²National Institute of Public Health and the Environment, RIVM, PO Box 1, 3720 BA Bilthoven, The Netherlands; ³UvA, The Netherlands; ⁴PEI D; ⁵Univ. Birmingham, UK. c.f.m.hendriksen@las.vet.uu.nl.

The Mouse Protection Test (MPT) is legally required for the quality control of whole cell Pertussis vaccine. The MPT is based on an immunization-challenge procedure in mice. The test is characterized by a high variability, the use of large numbers of animals, and severe pain and suffering due to the lethal challenge procedure. Unfortunately, no in vitro methods are yet available as an alternative to the challenge procedure. Recently, we described the use of clinical signs to replace lethality as a test parameter. Although several regulatory bodies now allow for the use of humane endpoints, implementation of these endpoints in routine testing is still limited, due to their subjectivity. In a follow-up study, we showed that training of technicians considerably improves the correct scoring of animals. For training purposes, we have now developed an interactive CD-ROM that shows the successive clinical stages in the disease process, as well as the clinical signs that are predictive for lethal progress. Furthermore, in order to discuss ways to reduce test variability, we have recorded the various critical steps in the MPT, such as the making of vaccine dilutions, vaccine administration, challenge procedure, euthanasia, and so forth. Finally, the CD-ROM includes case studies for trouble-shooting as well as a wealth of information on test-related matters. The CD-ROM is produced by the Working Group HELP (Humane Endpoint for Lethal Parameters) and will be distributed free of charge by the World Health Organization (WHO). It is expected that the CD-ROM will contribute to both reduction and refinement of the use of animals.