**4. Discussion**

This report documents the development and evaluation of remote controlled systems for TT and MT testing in large animals. Remote control by IR of both thermal and mechanical systems proved reliable. The baseline data were consistent, and increased thresholds were detected after analgesic treatment. Although NT testing does not mimic the complexities of true clinical pain, enabling normal behaviour should bring the response to a drug closer to clinical reality than in a restrained animal and therefore provide information that more closely resembles how a drug will behave under clinical conditions. The adaptations required both for power to activate the stimulus and for the control circuits to be placed on the animal were successful. Thermal stimulation depended only on small electrical components producing heat, and the main requirement was sufficient miniaturization for the electronics and sufficient battery power to be carried on the back of an animal as small as the cat. The mechanical system, however, required generation of air pressure to drive the actuator pin, necessitating attachment of a gas reservoir and metered pressure control being fitted to the animal. Wired or handheld MT testing systems driving a pneumatic actuator can make use of manual compression of a syringe [21,22,26]. Both air reservoir (the syringe) and pressure control (e.g., following visual pressure indicators) are under operator control and not fixed to the animal. The system adopted for the remote control system incorporated solenoid metering valves and an air reservoir of at least 60 mL. This proved too large for dogs and cats, but suitable for larger animals.

Most of the criteria for NT testing outlined by Beecher [5] were met. The aspects concerning repeatability of the stimulus, minimal tissue damage, immediate cessation of the stimulus when the animal responds and automatic cut-o ff if there is no response are no di fferent from wired or handheld devices [8,22]. Features to prevent tissue damage and to ensure stimulus repeatability have been addressed elsewhere [17]; this report concentrates on aspects that allow normal behaviour and a clear escape response at threshold.

All the species tolerated wearing the equipment well. The larger, herbivore animals often required no acclimation to this at all. Young, playful dogs and cats required more "dummy" sessions, but reliable data could be collected in all cases with time and handling appropriate to the species. Complete free roaming was clearly not possible as the IR system would be out of range and the animal must be within sight of the tester for its response to be seen. However, testing was entirely feasible within a room or large kennel for small animals and in a stall without the use of stocks for large animals. Absence of wires and cables was particularly valuable in cats and young, playful dogs.

In contrast to handheld units, remote NT testing limits the stimulus site to body parts where the probe or actuator can be reliably fixed to the animal's body. Mechanical testing in particular is limited as the actuator must react against something to generate the force. A band around a limb provides the necessary configuration, but application to other parts of the body is more di fficult. Although fixing the thermal probe on the thorax was both straightforward and suitable for cats and dogs, in the larger animals it was sometimes di fficult to detect the end point, in part as the thorax was obscured by the bulkier equipment but also because the response itself was less clear. Independent investigations examined the response to NT at di fferent sites, and both concluded that the thorax was less reliable and NTs were higher [27,28]. Large animals in stalls are more likely to be bothered by flies, resulting in confusing skin twitches over the body. Testing the limb is generally preferable, except when opioid analgesia is employed when an alternative site maybe better. Opioids often produce locomotor stimulation in otherwise pain-free healthy horses and obscure the leg lift or stamp response. In cold weather, a further disadvantage of the limb site for thermal testing was the low skin temperature of the limbs requiring more power to heat to threshold temperature. It is of note that skin temperature and TT in the cats were higher with the remote system than the wired. This was considered likely to be due to the larger bulk of the remote band providing more insulation than the narrow band without circuit boards used for the wired system. The original wired probes also included a substantial aluminium support that conducted heat away, probably contributing to lower indicated skin temperature. However, in spite of the higher temperatures recorded with the remote system, the excursions (TT – skin temperature) were similar. This has led to use of the excursions in many reports [29], at least for statistical analysis, as it removes one source of variation.

The precise NTs recorded are dependent on many factors including the site, the style of the stimulus probe, the environment and the breed or strain of the species under study. This makes results from studies in di fferent laboratories di fficult to compare. Remote testing may reduce the variation by allowing normal behaviour but it still remains extremely important that all the conditions are detailed, and strict consistency within one study is essential. A further central source of disparity within a study is individual variation. There is usually a range of skin temperature and both TT and MT even within a group of similar animals [30], and reducing all other sources of variation is necessary to produce useful data. It is important to describe the site, heating rate, probe style and the environment in order to understand the data fully.

It is well recognized that distraction alters the perception of pain [31] and, however caused, may a ffect recorded NT. Furthermore, the importance of enabling a natural escape response to indicate the NT has been emphasized [32]. Any distraction or, particularly in prey species, anything threatening or frightening will illicit abnormal behaviour, particularly freezing, and may prevent a natural escape response [33]. The potential for the tester to be unseen by the animal subject is of particular benefit in prey species unaccustomed to contact with humans. This was illustrated by the sheep who did not respond when the tester was in the same room. Our own unpublished observations with rabbits

support this: there was no response to thermal stimulation unless the tester was hidden, when TTs similar to dogs and cats were recorded. The e ffect of close proximity of the tester was illustrated even in calm horses who were quite familiar with humans. The MTs in the HHAN horses were significantly lower when tested with the wired compared with the remote controlled system. This is presumably due to an element of anticipation and apprehension with close proximity, when there are visual and perhaps audible clues that the stimulus is coming. This e ffect is further illustrated in sows [22] where hand-held MT testing was compared with a limb-mounted actuator, albeit still connected by a light pressure line. Baseline MTs were 13–17 N with the handheld device and 18–23 N with the limb fixed actuator, incorporating the same probe tip.

This investigation did not address reproducibility of the NT in the same animals over days, weeks or more. Remote control would in theory probably foster stable baseline thresholds. Learning to anticipate an aversive experience is less likely if the animal's behaviour is una ffected and no cues reinforce anticipation. Dogs undergoing MT testing with a handheld device at several sites at 10–15 s intervals had lower MTs when tested 10–14 days later [34]. It was concluded that the dogs had learned to respond to the same stimulus more quickly. This e ffect was probably exacerbated by the necessary restraint, the high frequency of the tests and the use of a large probe tip (1 cm diameter), which requires a larger force to produce the same pressure as the smaller probe tips used here. Pain is experienced when the stimulus intensity is su fficient to stimulate nociceptive nerve endings by pressure; the high forces required to generate su fficient pressure with a large probe surface area may squash the dog and be unpleasant even before they produce pain. Remote control does not address all these potential confounders; care with the frequency of repeated stimuli and the forces applied are equally important as lack of restraint and freedom of movement.

In spite of this experience with a handheld device [35], a number of studies using remote controlled NT testing have demonstrated good reproducibility. In dogs, TTs were shown not to change over daily testing for 3 days [36] and in cats over several months [6]. In horses, neither TT nor MT changed significantly over several weeks [27,37–39]. Pastern MT remained stable or even increased with familiarity over a few weeks, even when attached to the operator by a long light pressure line [26]. The ability of remote control to avoid visual cues and allow normal behavior presumably contributes to this.

Neither TT or MT remote control testing were evaluated in cattle, nor MT testing in sheep. In principle, these should both be suitable species for remote controlled systems, being large enough to carry the bigger control boxes on the back. Sheep tolerated the back-mounted remote thermal control system willingly. Actuators applied to limbs have been used in cattle standing in stocks [12,13], and it seems likely that the remote systems would have similar advantages over wired in the same way as in horses. Thermal threshold has been measured successfully in both neonatal and young foals [40].

Camels reacted to MT testing in a similar manner to cattle and horses, and the remote system functioned well in relatively recalcitrant animals once all the equipment was in place [41]. Thermal testing in camels was challenging: the high limb thresholds are presumably a result of adaptation to withstand desert and sandstorm temperatures of over 50 ◦C.

The IR remote controlled systems have now been used for formal investigations into pain and analgesics in cats [42,43], dogs [29], sheep [25] and horses [37–39,44].
