Research Paper
Challenges of thermal nociceptive threshold testing in the donkey

https://doi.org/10.1111/vaa.12182Get rights and content

Abstract

Objective

To evaluate a thermal nociceptive threshold (TNT) testing device in the donkey, and the influence of potential confounding factors on TNTs.

Animals

Two groups (Group 1 and Group 2) of eight castrated male donkeys aged 4–9 years, weighing 105–170 kg.

Methods

TNTs were measured by heating a thermal probe on skin until an end-point behaviour (threshold temperature) or a cut-out temperature (51 °C) was reached. The withers and the dorsal aspect of the distal limb were used as sites for TNT testing. The effects on TNT of different confounding factors: the limb tested; rate of heating; and ambient temperature were evaluated. Data were analyzed using general linear models, and Mann-Whitney tests, p < 0.05 was considered significant.

Results

End-point behaviours (skin twitch or donkey looking at test device) when the thermal probe heated the withers were observed in approximately half of tests. TNT was (mean ± SD) 46.8 ± 2.85 °C. Subsequently the limb was evaluated as the test site in Group 1 followed by Group 2 donkeys; end-point behaviour being a foot-lift. In Group 1, 72% of tests ended in an end-point behaviour but the response rate was lower in Group 2 (20%), although TNTs were similar [(47.6 ± 3.3) and (47.3 ± 3.0) °C respectively] for responding animals. Rate of heating, ambient temperature and laterality (right or left) did not affect thresholds, but mean TNT was significantly higher in the forelimb (48.5 ± 2.8 °C) than the hind limb (47.4 ± 2.8 °C) (p = 0.012).

Conclusions

When a thermal probe cut-out temperature of 51 °C was used in TNT testing in the donkey a high proportion of tests did not produce an identifiable end point behaviour. Higher cut-out temperatures damaged the skin. Under these conditions, thermal nociceptive threshold testing appears not be an appropriate analgesiometry technique in the donkey.

Clinical relevance

TNT testing under these conditions is not suitable form of analgesiometry for donkeys.

Introduction

The donkey has a reputation of stoicism with more subtle behavioural pain expression than horses. Current pain assessment techniques in animals depend heavily on behavioural cues, making assessment of pain difficult in donkeys. Nociceptive threshold testing (NTT) is a technique used to quantify nociceptive thresholds and analgesic efficacy, and has been used in various species of animals including horses. In equidae, three modalities of noxious stimuli have been applied; mechanical (Love et al. 2012) thermal (Robertson et al. 2005; Love et al. 2012) and electrical (Spadavecchia et al. 2003). Delivery of more than one type of noxious stimulus is recommended in order to fully evaluate the functional status of the nociceptive pathways (Nielsen et al. 2009).

In laboratory animals, thermal NTT is widely employed in classical experimental tests such as the tail flick test, tail withdrawal, paw withdrawal or hot plate test (Le Bars et al. 2001). Either the time taken (latency) for an animal to respond to an applied constant temperature, or the temperature at which an animal responds (if there is a ramped change in temperature) is measured. Thermal nociceptive threshold (TNT) testing has not been evaluated in the donkey previously, although the modality has been used in horses to evaluate the effectiveness of alpha-2 adrenoceptor agonists (Wegner et al. 2010), local anaesthetics (Robertson et al. 2005) and opioids (Love et al. 2012).

An ideal NTT stimulus should fulfil certain criteria. The stimulus should be easy to apply and repeatable, the behavioural response should be clear and easily identifiable and the stimulus should produce no lasting harm to the animal (Beecher 1957).

In this study we aimed to evaluate a thermal probe-based TNT device that had been developed for horses (Wegner et al. 2010; Love et al. 2012), in the donkey. The initial probe location to be evaluated was the withers (Study 1), and subsequently the limb (Studies 2 and 3). We investigated whether experimental factors such as whether right or left side of the body (laterality) (Studies 1 and 2), limb tested (Study 2), presence or absence of a companion (Study 2), level of distraction (Study 2), rate of heating (Study 2) and ambient temperature range experienced (Study 3) influenced the TNTs. Data collected in two groups of donkeys separated by 12 months were also compared. Certain experimental conditions (i.e. a companion present, limb tested, rate of thermal probe heating) were standardized as per Study 2, although location and level of prior training of the donkeys were not identical.

Section snippets

Ethical approval

This study received ethical approval from the University of Bristol (UB/10/019) and Ross University Institutional Animal Care and Use Committee.

Animals

Two groups of eight adult castrated male donkeys were studied. Group 1 donkeys were aged between four and eight years, and weighed between 105 and 160 kg. This group was studied in November and December 2010. Group 2 donkeys were aged between four and nine years and weighed between 152.5 and 170.5 kg. Group 2 donkeys were studied in November 2011.

Study 1: Evaluation of different cut-out temperatures and the influence of laterality and whether a companion was present or absent on TNTs measured over the withers

In three donkeys the thermal probe was heated to 53 °C, and in two of these donkeys, small raised scabs, similar in size and shape to the thermal probe, developed over the sites of application within 18 hours of testing. The lesions persisted for 14 days. Consequently, all subsequent TNT tests used a cut-out temperature of 51 °C and no skin lesions were observed.

Mean ± SD ambient temperature was 30.7 ± 2.1 °C, and mean baseline skin temperature was 34.9 ± 1.5 °C. Neither presence or absence of

Discussion

This study is the first to investigate TNT testing in the donkey and evaluation of the device followed methods used by researchers testing thermal probe based systems on horses (Robertson et al. 2005; Love et al. 2012). From Study 1 it was clear that a thermal stimulus applied to the skin over the withers of the donkey does not meet the ideal characteristics of a nociceptive stimulus for analgesiometry (Beecher 1957): a cut-out temperature of 53 °C led to the development of skin lesions

Conclusion

The difficulty in interpreting end-point behaviours when heating skin over the withers and the development of skin lesions when the skin was heated to 53 °C suggests that the withers is not an appropriate site for TNT testing in the donkey. Decreasing the cut-out temperature to 51 °C and heating skin over the dorsal aspect of the limb produced a more useful model, with a clear end-point behaviour of a foot-lift, and no skin lesions produced. However, when the limb site was tested in a second

Acknowledgements and conflicting interests

The authors would like to thank Drs Polly Taylor and Mike Dixon of Top Cat Metrology Ltd and Dr Tammi Krecek and the research assistants at RUSVM for their support.

This project was generously funded by Top Cat Metrology Ltd, RUSVM Research Committee, Langford Trust, Association of Veterinary Anaesthetists Educational Trust, Vetronics, Pfizer, Boehringer Ingelheim and Vetoquinol.

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