The effect of tongue-tie application on stress responses in resting horses

Tongue-ties (TT) are commonly applied to both Standardbred and Thoroughbred racehorses to increase control, by preventing them from getting their tongue over the bit, and as a conservative treatment for equine respiratory conditions, principally dorsal displacement of the soft palate. This study investigated responses to TT application in horses, at rest, using both behavioural (head-tossing, ear position, gaping and lip licking) and physiological (salivary cortisol concentrations, eye surface temperature and heart rate) indices. Twelve Standardbred horses (six of which were naïve to TT) were used in a randomised crossover design. The study comprised 3 phases; Phase 1 (Baseline), Phase 2 (Treatment), and Phase 3 (Recovery). At phase 2, tongue tie application (TTA) was performed using a rubber band that was looped around the tongue and secured to the mandible for 20 minutes. The control treatment (C) incorporated 30 secs of tongue manipulation, at the start of the 20 min, however no TT was applied. Behaviours (head-tossing, ear position, mouth gaping and lip-licking) and heart rate (HR) were recorded for the duration of the study and analysed in ten minute intervals. Salivary samples were taken at the end of each phase for subsequent cortisol assays and infrared thermography images were taken of each eye at 5-minute intervals. Statistical analyses were performed in SPSS using linear mixed effects models and repeated measures general linear models, to determine differences between treatments and within treatments, over time. Compared to control, there was more head-tossing/shaking (p<0.001), gaping (p<0.001) and backwards ear position (p<0.001) and less forward ear position (p<0.001) during TTA, in Phase 2. Horses with previous experience of TT showed more head-tossing (p=0.040) and gaping (p=0.030) than naïve horses. Lip-licking was more frequent after TTA treatment than control, during Phase 3 (p<0.001). Salivary cortisol concentrations increased after TTA (1846.1pg/mL ± 478.3pg/mL vs 1253.6pg/mL ± 491.6pg/mL, p=0.047). Mean HR, and mean right and left eye temperature did not differ significantly between treatments in any phase (all p> 0.05). The findings of this study suggest the application of a tongue-tie causes changes to both behavioural and physiological parameters suggestive of a stress-related response. Further research is needed that will enable racing and sport horse regulatory bodies to make informed decisions about the appropriate use of tongue-ties in horses.


28
The tongue-tie (TT is a form of tack modification that has been used in horses for over 100 years (Fleming, 1889).

29
The device is used to hold the tongue in a fixed position and may be made from a rubber band, leather strap, nylon 30 stocking or similar material, that is tied below the jaw or at either side of the horse's mouth to the bit, after being 31 looped around the tongue. Early reports suggest that TTs were used to prevent abnormal noise and airway 32 obstruction arising as a result of the horse "retracting the tongue so as to force back the soft palate to such an extent 33 that it interferes with the passage of air between the nasal passages and the larynx" (Fleming, 1898). Today, TT 34 use remains commonplace in both Standardbred and Thoroughbred racehorses, throughout the world (1-6). Their 35 primary putative purposes are firstly to conservatively address airway patency issues, principally due to dorsal 36 displacement of the soft palate (DDSP) and improve performance and secondly, to aid control of the horse by 37 preventing it from getting its tongue over the bit (3,6,7). Dorsal displacement of the soft palate, one of the most 38 common causes of dynamic airway obstruction during strenuous exercise is thought to affect approximately 20% 39 of racehorses (Pollock et al. 2009;Priest et al., 2012.). Tongue-ties are frequently used as the first line of 40 conservative treatment by trainers and may also be used in combination with surgical management (Franklin et 41 al., 2001;Barakzai and Dixon, 2005;Barakzai et al., 2009a). However, the efficacy and the exact mechanism by 42 which the TT may aid in prevention of DDSP remains controversial (Beard et al. 2001;Cornelisse et al. 2001; 43 Franklin et al. 2002;(5, 8).

44
Over recent years, concerns regarding potential welfare issues associated with TT use have been raised by animal 45 welfare organisations (Barakzai, 2009b). This has led to these devices being banned from a number of equestrian 46 disciplines under Federation Equestre Internationale regulations (9). Anecdotal reports suggest that routine TT 47 application may cause damage to the tongue (including lacerations, dysphagia, bruising, swelling, discolouration 48 and paralysis) (10). A recent South Australian survey identified that 26.3% of Standardbred racehorse trainers 49 reported complications relating to tongue-tie use, mostly associated with swelling of and superficial cuts to the 50 tongue, as well as changes in behaviour including head shyness (11). A study in the UK also reported that tongue-51 ties were not well tolerated in young Thoroughbred racehorses (12). This implies that the horses must habituate to 52 the aversiveness of the procedure (McGreevy and McLean, 2010). It is not clear how long horses take to habituate 53 to TTs and whether they ever do so completely. Whenever sustained pressure is used to modify horse behaviour, 54 the principles of ethical training as espoused by the International Society for Equitation Science (ISES 2011) are 55 compromised and negative reinforcement that relies on the release of pressure, cannot take place. The application 56 of various training devices in horses, including bit attachments and restrictive nosebands, have been reported to 57 result in pain and stress responses, thus compromising welfare (13-15).

58
Appropriate assessment of stress in animals involves the integration of measurement of both behaviour and 59 physiology. Changes in behaviour can provide a useful and immediate means of assessing the response of an 60 animal to its environment, and shifts in demeanour, posture, gait and interactive behaviour may be associated with 61 presence of pain or stress (16). Head behaviours (such as head-shaking/-tossing and ear positioning) as well as 62 oral behaviours (gaping and lip-licking), have been used to estimate an animal's affective state (17). To date, the 63 physiological assessment of stress in horses has been based primarily on changes in endocrine function (18), as 64 well as parameters that reflect changes in autonomic functioning including heart rate and eye temperature (ET) 65 (18)(19)(20). Salivary cortisol concentration is established as a non-invasive indicator of stress because it reflects 66 activation of the hypothalamo-pituitary-adrenocortical axis (HPA) (21,22). Measurement of maximum ET using 67 infrared thermography has the potential to assess both acute and chronic stress in animals, reflecting changes to 68 peripheral blood flow during increased sympathetic output (22)(23)(24)(25). Heart rate variability (HRV) is a measure of 69 autonomic function and can be used to determine the balance between sympathetic and parasympathetic tone (20, 70 24, 26, 27).

71
The effect of tongue-tie application on stress responses in horses has not yet been investigated. The aim of this 72 study was to determine the effect of TT application on stress responses in resting horses. It was hypothesised that 73 the application of a TT would induce a stress response, resulting in increased concentration of salivary cortisol,

74
ET, HR and conflict/agitated behaviours. We also hypothesised that the stress response would be exacerbated in 75 horses that were naive to TT application.

80
This study used Standardbred horses (n=12), comprising mares (n=8) and geldings (n=4) aged 11.5 ± 3.0 years 81 (mean ± s.d) and weighing 487 ± 33.9 kg (mean ± s.d). Animals were subjected to a health check prior to the trial, 82 and deemed free from illness or injury. On experimental days, horses were brought in from the paddock, and 83 housed in day yards before being secured in stocks during the experiment. Horses wore a halter only, with no bit 84 and were loosely tied with a rope to the side bar of the stocks. All procedures were approved by the University of

88
The study was based on a randomised crossover design comprising two treatment groups: tongue-tie application 89 (TTA), and control (C). Horses were classified as either naive (n=6) or having previous experience of TTA during 90 their racing careers (n=6). Horses were pseudo-randomised and assigned to treatments so that 6 horses (3 91 experienced and 3 naïve to TT), would have TTA on day 1 followed by C on Day 2, and 6 horses (3 experienced 92 and 3 naive), received C on Day 1 followed by TTA on Day 2. For each horse, treatments were performed at the 93 same time on both days to take account of diurnal rhythm in physiological parameters. For each treatment, horses 94 were observed for a total of 80 minutes, which was divided into three phases (Phase 1: 30-minute baseline; Phase 95 2: 20-minute treatment; and Phase 3: 30-minute recovery). The application of a TT was performed by the same 96 operator, using a rubber band measuring 152mm x 15mm (Belgrave rubber band; size 106; 97 www.quillstationary.com.au). Following industry practice, the band was looped twice around the tongue, and then 98 secured to the lower jaw by looping the TT around the mandible (Figure 1). A new tongue-tie was used for each 99 horse. The duration of 20 minutes was based on the median duration of TT use reported during training and racing 100 in a previous study (Findley et al.,2015). During C, tongue manipulation was performed for 30 seconds at the start 101 of phase 2. This involved grasping of the tongue to mimic the initial process required for application of a TT, 102 however no TT was placed. On each experimental day, horses were brought into the barn in pairs, and individually 103 restrained within stocks for 10 minutes to acclimatise to the surroundings and to allow instrumentation of the 104 horses. During each treatment period, as one horse was treated, the other stood by, as a companion.

111
Behavioural data were recorded using two digital cameras (GoPro® HERO3), each mounted on a tripod placed at 112 either side of the stock at 90 O to the horse's head, at a distance of 1.5m. Recording commenced at the start of Phase 113 1 and ceased after completion of Phase 3. The behaviours recorded from the video record included oral (gaping, 114 lip-licking) and head-related behaviours (head-tossing, ear positioning), that have previously categorised as either 115 positive/relaxed or negative/agitated (Table 1) (17). Behaviours were measured as a duration (% of time), apart 116 from lip-licking which was measured as a frequency (n). Video clips of ten minute duration (last ten minutes of 117 phase 1 all of phase 2 and the first and last 10 minutes of phase 3) were used for behavioural analysis using 118 behaviour analysis software (Mangold International GMbH, version interact 8) ( Table 2). Analysis could not be 119 blinded because it was not possible to obscure the observer's view of the tongue. All behavioural analysis was 120 conducted by the same observer to minimise inter-observer variation. Table 1: Head-related and oral behaviours recorded in horses during and after tongue-tie application or tongue 123 manipulation. Behaviours were categorised as either positive/relaxed or negative/agitated (17).

Behaviour Category Description Measurement
Head-tossing

131
Saliva samples were collected using Salivette® (Sarstedt, Sweden) swabs, at the end of each phase. The swab was 132 placed in the horse's oral cavity (between the cheek and the teeth) using surgical forceps and moved around gently 133 for 30 seconds. Swabs were stored on ice before being transported to the laboratory, where the tubes were 134 centrifuged (1000g for 10 minutes) and samples frozen at -20°C until later analysis. The saliva was analysed for 135 cortisol concentration, using a commercially available ABOR Enzyme-linked immunosorbent assay (ELISA)

136
(Ann Arbor, Michigan 48108-3284 United States). Sensitivity and limit of detection were determined to be 17.3 137 pg/mL and 45.4pg/mL, respectively. All samples were run in duplicate and results expressed as pg/mL. The intra-138 assay coefficient of variation determined from duplicates of a control saliva sample in each assay plate (n=3) was 139 16.7% and the inter-assay coefficient of variation was 5.9%.

140
Eye temperature

141
An infrared camera (ThermaCam S60, FLIR Systems AB, Danderyd, Sweden) was used to collect thermographic 142 images of the eye at 5-minute intervals for the duration of the study period, as per previous studies (Yarnell et al.,

185
No other factors had a significant effect on head-tossing.

263
Mean HR did not differ significantly between TTA and C at any time point (Figure 9). However, there was a 264 trend for horses in the TTA group to have a higher mean heart rate compared with C at T2 (43bpm ± 2.4bpm vs 265 37bpm ± 2.5bpm; p=0.079), followed by a reduction in HR in this group of horses after TT removal (

284
Head-tossing behaviour in horses has been shown to reflect agitated or conflict behaviour caused by irritation or 285 pain to the mouth (33). In the current study, horses exposed to TTA spent significantly more time head-tossing 286 during Phase 2 compared to C, suggesting that the presence of a TT may be irritating and/or results in discomfort 287 or pain, inducing a negative affective state in these animals.

299
Ear position

300
Backwards positioning of the ears is reported to reflect negative affective states, including fear, discomfort, 301 submission, avoidance, pain or aggression in horses (35-37). In our study, horses spent more time with their ears 302 backwards during TTA and immediately after removal compared to C. These findings align with those from a 303 study of horses ridden with their necks in a hyper-flexed position, in that horses spent more time with ears 304 backwards when hyperflexed than when they were not hyperflexed, possibly suggesting an association between 305 hyperflexion of the neck and a negative experience (38). During Phase 2, horses with TTA spent more time with 306 their ears backwards at T2 than at T1. This may suggest that over the course of the 20 minutes of the current 307 treatment, the horses' discomfort, submission, avoidance or discomfort increased. This is consistent with previous 308 research showing that the intensity, duration and frequency of a stressor generally correlates with the intensity of 309 the stress responses that manifest (39). During Post 1, the time spent with ears in a backwards position was also 310 higher with TTA than C. This may suggest that horses subjected to TTA may still be recovering from the effects

311
of treatment into what we anticipated to be a wash-out period.

312
When horses have their ears facing in forwards are often regarded being relaxed or in a positive affective state 313 (40). Another study reported ears facing forward as a feature of horses displaying interest or pleasure towards 314 experienced handlers in contrast to inexperienced handlers (41). During TTA in the current study, horses spent 315 less time with their ears forward at T1 and T2. This may suggest that during TTA, horses are less relaxed than 316 during C. As with head-tossing, time spent with ears forwards was associated with previous experience. Horses 317 with prior experience of TTA showed decreased time with ears in a forward position compared to naïve horses.

318
Holding the ears back is recognised as a sign of pain (42) so this finding could is suggest that they have learned to 319 associate the sensation of having their tongues tied with a negative affective state. It may also suggest that the 320 experienced horses have learned that, rather than being swiftly transient, the restraint persists for some time.

322
Gaping or opening of the mouth has been categorised as an agitated behaviour, where increases in this behaviour 323 may reflect a negative affective state in horses (17)

334
The frequency of lip-licking behaviour during TTA showed a significant decline from Baseline. This probably 335 reflects the fact that horses with tongue-ties are unable to physically perform lip-licking when their tongues are 336 restrained. However, after TTA, horses showed significantly more lip-licking behaviour than at baseline which 337 may represent post-inhibitory rebound behaviour. Post-inhibitory rebound behaviour is the term given to an 338 increase in the expression of a behaviour following a period of restriction or deprivation (44,45), that may indicate 339 that the deprivation (of the opportunity to perform certain normal behaviours) has deleterious effects on welfare 340 (44,46). In a similar study that focussed on restrictive nosebands, a post-inhibitory rebound behaviour was 341 observed for yawning, swallowing and lip-licking behaviour (23). That said, the authors of that study noted that 342 post-inhibitory rebound behaviours were observed for all groups with varying noseband tightness, thus the horse's 343 response may have been related to the novelty of having had two bits in the mouth rather that noseband tightness 344 alone. Importantly, in the current study, no significant difference in lip-licking behaviour emerged between horses 345 experienced to TTA and the naive horses, suggesting the post-inhibitory rebound response observed was likely 346 due to the application of tongue-tie rather than any novelty effect. No significant difference in lip-licking was 347 observed in horses during C treatment between Phases 1, 2 and 3, suggesting that the brief tongue manipulation 348 performed had no effect on the horses' motivation to lick their lips.

357
A positive correlation has been shown between salivary and plasma cortisol in horses (51)

384
Heart rate

385
Changes in heart rate have previously been used as a non-invasive measure stress in horses (20,24,26,27). Heart 386 rate is regulated by both the sympathetic and parasympathetic branches of the autonomic nervous system (ANS).

387
Increased heart rate, due to an increase in sympathetic activity, has been associated with a number of husbandry 388 practices including branding procedures, restraint, transport, and social isolation (50, 56) In this study, no 389 significant difference was observed in HR between TTA and C groups over time. However, there was a trend for 390 heart rate to increase atT2 with TTA and to decrease following TT removal. The lack of significant findings may 391 relate to the low number of horses used in this study and the high variation in HR between individuals. In addition, 392 fluctuations in HR are labile and subject to both external and psychological influences (57) and may have been 393 affected by external environmental factors (extreme weather conditions, construction sounds and traffic noise) that 394 occurred on some days but not others. Other studies have used heart rate variability (HRV) as a non-invasive 395 measure of stress in horses (58, 59)). However, this was not deemed appropriate in this study due to the high 396 prevalence of 2 nd degree AV block that was observed. 2 nd degree AV block has been shown to significantly 397 influence HRV variables when based on RR intervals (60).

398
Limitations and future research

399
Although behaviour offers an immediate means of assessing an animal's response to potential stressors, the 400 interpretation of behaviours is often subjective between assessors. Therefore, future studies may benefit from 401 assessing the inter-and intra-observer reliability of the behaviours measured in this study, and thus reducing 402 limitations due to observer bias. This is particularly important in prey species (including the horse), as outward 403 behavioural signs of fear and distresses may be masked as a means of survival (25). The cortisol assay produced 404 large values for both inter and intra coefficient of variation, consequently limiting the reliability of results. This 405 may have been due to error associated with pipetting or contamination from feed material within the samples.

406
Heart rate measurements showed large variation between individuals in this study and may have been influenced 407 by extraneous factors in some horses. This study was also limited by the small sample size. A larger sample size 408 would reduce the variation among individuals and thus increase reliability of results.

410
This present study provides novel evidence on the effects of tongue-tie application on stress responses in resting 411 horses, suggesting the application of a TT results in increased agitated/conflict behaviour, decreased desirable 412 relaxed behaviours, and increased salivary cortisol concentration. Further investigation into the appropriate use of 413 TTs in horses should establish whether the costs to the horses, when wearing TT, are offset by the benefits to the 414 horses and other stakeholders. This will enable racing and sport horse regulatory bodies to make informed 415 decisions regarding the continued use of tongue-tie in horses.