Contact heat thermal stimulation is suitable for standardized assessment of thermal cutaneous nociception in horses. However, the success of clear end point detection can be variable and depends on the body region stimulated, ambient temperatures and the test conditions.
Ambient temperature appeared to have significant influence on thermal threshold testing. In the present study thermal threshold temperatures at the nostril and withers were significantly lower at warm ambient temperature compared with cold temperatures. There is little information in the literature concerning the influence of ambient temperatures or skin temperatures and on determined thermal thresholds in horses. Tail flick latencies (TFL) in rats and mice were significantly increased and tail skin temperature was decreased when the tail was immersed in cold water (0°C, 5°C or 10°C) before testing [15, 20]. The current study indicated that skin temperature was strongly correlated with ambient temperature. Ambient temperature below 10°C resulted in significantly lower skin temperature but higher reaction temperatures compared to ambient temperatures above 20°C. It might be necessary to heat the probe up to a higher temperature to get heat transferred and reach the nociceptive threshold when the skin tissue is at a lower temperature. There was no increase in skin temperature over the three consecutive measurements, showing that the 20 minute interval was adequate for the skin to return to its normal temperature without active cooling. Cold ambient temperature and therefore low skin temperatures (13.9 to 21.9°C) resulted in prolonged heating time and reduced power of the thermal threshold testing device. So cut-out temperature couldn’t be reached in several measurements at the coronary band and the heating cycle was interrupted at 48 – 50°C. The data of the inaccurate measurements were excluded from analysis and therefore 47 thermal thresholds instead of 60 TT were considered. For further studies being performed at cold ambient temperatures more powerful probes and batteries need to be used.
The randomized delay in starting thermal stimulation in the current study allowed the duration of one heating cycle to be randomly varied. This prevented the horses from becoming conditioned to any audible clue such as a click when heating was started. It also ensured the assessor did not become accustomed to the normal time lapse from start of stimulation to response. Some studies suggest that horses can get conditioned to the noxious stimulus. They reacted as soon as they saw the lamp supplying radiant heat  or as they felt the stimulus before it became painful . In the present study thermal thresholds did not change when thermal stimulation was repeated at 20 minute intervals, suggesting that horses did not become accustomed to the noxious stimulus. In a further study which is in preparation for publication (Poller C, Hopster K, Kästner SBR) TT did not change over 10 repeated measurements in 30 minute to 2 hours intervals when horses were treated with a placebo (saline solution). Similar results were reported in another study where thermal thresholds remained constant over 24 hours .
The quick heat transfer to nociceptors is an important factor contributing to successful clear end-point detection and preferably low thresholds to prevent skin burns. Heavily pigmented epidermal tissues and hair covering impeded the relative transparency to near infrared light  and probably the thermal transfer in deeper layers. It was possible that hairy skin, despite clipping, isolated the deeper layers of epidermis from the heat source  leading to the conclusion that this could have contributed to the late or failed response to the thermal stimulus at the coronary band. Slower heating rates could improve the heat conduction to the nociceptors by warming up the skin for longer excursion but also increased the likelihood of skin burning. Unfortunately, it was not possible to perform the current study in a fully randomised design concerning experiments at warm and cold ambient temperatures for seasonal and logistic reasons as climatized rooms were not available. Therefore, possible conditioning of the horses to the thermal stimulus in the second set of tests (cold temperatures) could be considered. In case of conditioning, lower thermal thresholds at cold ambient temperatures compared to warm ambient temperatures would have been expected, but results showed the opposite with higher TT in November.
All horses were trained wearing the equipment to ensure that their reaction to thermal stimulation was not influenced by discomfort or stress. The horses developed individual behaviour when they were restrained in stocks: some of them became bored and distracted whilst others were very nervous or anxious. Nociceptive thermal thresholds were not constant or reliable when thermal stimulation was applied to horses tied in stocks, resulting in high thresholds reaching cut-out temperature. Different authors suggested that nociceptive threshold testing in horses should be performed within the animal’s normal environment and when unrestrained to avoid distraction of the horse . To the author’s knowledge so far there is only one more published study with thermal threshold testing in unrestrained horses .
When visible end-points at the different body sites (nostril, withers, coronary band) were compared, the reaction to the stimulus at the withers was the most clear and easy to identify, as the transformation of the noxious stimulus into a visible reaction is mainly mediated via a reflex pathway (skin flick response via the spinothalamic tract) . Coordinated head shaking or rubbing the nose against an object requires conscious perception (trigeminal nerve at the nostrils) with more individual variation . In this study, measurements at the coronary band had the lowest reliability in producing constant reactions to thermal stimulation. It has been suggested that the depth and density of Aδ and C-fibres and the distribution of nociceptors may be variable between species and body parts . The epidermis in horses has been reported to be nearly twice as thick as in cats or rodents . In addition to the interspecies differences in epidermal thickness there were also variable data between body parts within species . It is likely that nociceptors and nerve fibres in horses are located in deeper layers. This would lead to higher threshold temperatures recorded at the skin surface, as seen in the present study (49.9 ± 4.0°C at the withers), compared to cats or rodents (approximately 45°C) [13, 25] assuming that temperature equilibration between skin surface and nociceptors needed longer time in thick skin. Thermal threshold might also be affected by blood flow . Measurements of blood flow demonstrated significant differences between species and body parts . Failed reactions to a thermal stimulus on the distal limb in sheep at ambient temperatures below 8°C were considered as result of vasoconstriction in the skin and ischaemia of the small nerve fibres . It is likely that thermal stimulation at the coronary band of horses was probably affected by skin thickness, increased hair density and reduced blood flow, and, particularly at low ambient temperature, by vasoconstriction. A more proximal part of the limb as stimulation site, like mid cannon bone instead of the coronary band, might be less affected by temperature changes and blood flow allowing more successful end-point detection.
The uneven gender distribution and the lack of knowledge of the state of the estrous cycle in the 3 mares is a limitation of the study and might have influenced the results, because in other species, including humans, gender can influence nociceptive sensitivity. Women were more sensitive to cold, heat and ischaemic pain than men  or in female rats nociceptive sensitivity was decreased after ovariectomy . However, the influence of the estrous cycle on nociception and pain was controversial in these studies [26, 27]. To the author’s knowledge there are no published studies in horses comparing thermal nociceptive thresholds between mares and geldings or the influence of the estrous cycle. As the season might influence the occurrence of the oestrous in mares, it cannot be ruled out that the differences in thermal thresholds between warm and cold temperatures were also influenced by hormonal changes and are not only associated with temperature differences.