Transportation has been identified as a stressor for horses, and has been associated with several adverse outcomes including injury, respiratory and gastrointestinal disease [1
]. We have recently shown that 12 hours’ transportation is associated with ulceration of the gastric squamous mucosa in fasted horses, associated with increased pH of gastric content, and possibly with decreased gastrointestinal motility in horses fed 1 h and 6 h prior to transportation [7
]. Animal management during transportation may influence disease outcomes [8
], and international regulations on land transportation of live animals have been updated based on recent publications to safeguard the welfare of transported animals. However, there is still no agreement in mandatory requirements between countries and evidence in support of some recommendations is limited.
The adverse effects of transportation may be affected by confinement, isolation, direction of travel, and the size of the compartment in which the horse is transported [11
]. Several studies have been performed in order to determine the effects of direction of travel on a horses’ ability to maintain balance during transport of different duration (from 17 min to 3 h) [12
], but results have often been conflicting due to differences in trailer design, journey duration, and lack of simultaneous comparisons. Similarly, there is no agreement on the space allowance needed in transit [16
], and variable minimal space allowances are reported in current transport regulations of different countries [18
]. It has been reported that the most commonly observed body posture in horses during transportation involves standing with the front and hind limbs apart and the forelegs stretched forward [21
] a postural adaptation likely to help the horse to retain its balance. However, to assume this position, horses need sufficient space between their body and the vehicle partitions or other horses. Similarly, beneficial effects associated with lowering of the head below the height of the withers have been well characterised during journeys longer than 8 h [5
], and this posture also requires a space allowance greater than currently available in many transport vehicles.
This study documented the effects of 12 hours’ confinement in comparison with 12 h of transportation in single or wide bays, and in backward or forward positioning, on behavioural, physiological, laboratory and gastroscopy parameters. It was hypothesized that behaviours relating to stress and balance and physiological, laboratory and gastroscopy parameters would be increased in transported horses relative to those observed in confined horses, and that transportation in a rear-facing position and a wider bay size would attenuate such changes. We also hypothesized that behaviour would predict the severity of gastric ulceration (increased squamous and glandular ulcer scores), and that the frequency of behaviours related to stress would be correlated with increases in cortisol, muscle enzymes, rectal temperature and heart rate in transported horses.
The current study compared behaviours in horses transported for 12 h with those observed in horses confined for a similar time period. The findings supported the main hypothesis that behaviours relating to stress and balance occurred more frequently in transported horses than in confined horses, and horses transported in a rear-facing position and in a wider bay size showed less balance-related behaviour. In particular, horses travelling in a single bay showed a higher frequency of behavioural events (both related to stress and balance) and, after the journey, demonstrated increases in cortisol, neutrophils and WBC that were not observed in horses travelling in wider bays. These observations suggest that during 12 hours’ transportation, rear-facing position and wider bays may reduce the impact of transport on horse health and welfare. The second hypothesis was partially supported, as balance-related behaviours, particularly loss of balance, were associated with the severity of gastric ulceration and the increase in muscle enzymes after transportation, while stress behaviours (licking and total stress behaviours) correlated with decreased gastrointestinal sounds but not with cortisol. Increases in heart rate and rectal temperature observed after transportation were also positively associated with balance and stress behaviours, and with the development of clinical ESGD in the univariate regression model. These findings suggest that behaviour in transit and physiological measurements after transportation might identify horses at risk for development of transport-related disease and, for the first time, this study documented the effects of space during a long journey on behaviour, health and physiological parameters. Adequate space allowance and rear-facing positioning would appear to facilitate better balance, but effects of bay size and direction of travel on stress behaviours and long-term outcomes were less clear and require further study.
Transportation is considered stressful because horses are confined in a small space [33
]. However, in our study, horses showed a different behavioural repertoire during confinement and transportation. Transportation was associated with increased head movements (head tossing, turning and surveying), as well as with increased touching of the tie cord, relative to behaviours observed during confinement. The observed head movements were interpreted as indicative of increased arousal (i.e., anxiety/vigilance/alert response). Touching the tie cord during transportation has been previously correlated with cortisol [5
], and it may be interpreted as a redirected behaviour [27
]; the horse would like to do something else but instead starts touching the tie cord, which often is the only behaviour it is free to do. During transport, the horses repeatedly licked the surface in front of them. Licking in this manner may be considered as an oral stereotypy, because it was abnormal in its frequency and with no obvious purpose (there was no evidence that horses were seeking feed material or other substances such as salt). Both licking and touching the tie cord were behaviours observed infrequently during confinement and have been well characterised as indicators of stress [27
]. Unfortunately, chewing/licking, another behaviour associated with stress [35
], was excluded from the current study because available camera angles inconsistently precluded continuous observation of these behaviours during transportation, and we cannot confirm previous results which licking was positively correlated with the time horses spent with the head in a upper position [5
]. The high frequency of behaviours related to stress and the high frequency of total behaviours, demonstrate that transportation was much more stressful than confinement for our horses, confirming that transport stress is multifactorial [36
]. In our study, confined horses appeared to be able to keep their circadian pattern of sleeping, at least the standing sleep (low-wave sleep), intermittently. This was not possible for the horses during transportation. Overnight travel is recommended during summer to minimise the effects of high ambient temperatures, but there are currently no studies on the effects of sleep deprivation due to transport in horses. As sleep deprivation in other species has been associated with altered immune function [37
] and with impaired athletic performance [38
], the physiological implications of travelling overnight warrant further evaluation.
As expected, transportation was associated with increased balance behaviours (learning on rails, forward, backwards and lateral movements) in comparison with confinement. It is well recognised that maintaining balance in a truck is difficult, causing an increase in muscle enzymes [25
], and that factors like vehicle design, type of suspension, driver ability and type of road all have an effect on the horse’s ability to balance with more or less effort [11
]. The truck used in the current study was equipped with good suspension and a driver of many years of experience in live animal transportation, but the chosen route was a mix of minor and major roads, with straight and winding sections to mimic commercial transport routes. The variations of balance-related behaviour throughout the journey, in particular their decreased frequency during the third hour of transport, may therefore be related more on the type of road and route (a straight tract on a major road) than to other animal-related factors. Instead the high frequency of balance-related behaviour during the first and the last hour may be related to both route and arousal conditions. Horses can sense when they are close to their home range or stable and can get more restless during the last part of the trip. The horses tended to move more when aroused, a trend which was also evident during confinement, where the horses showed more turning the head, total stress and total behaviour during the first and the last hour and exhibited postures associated with increased arousal such as head elevation and pricking of the ears. The observed changes in haematological, blood biochemistry, cortisol concentrations and squamous ulcer scores observed in the current study were expected and are consistent with previous studies [7
], and may be explained by the higher arousal and difficulty of maintaining balance observed during transit in comparison with confinement.
The novelty of this study was the effects of space on the behaviour, health and welfare of the transported horses. Horses travelling in wider bays showed less balance-related movements, leaning less on the partitions and losing their balance less often, and less behaviour related to vigilance, possibly because their arousal was lower. Studies have demonstrated that horses experiencing loss of balance, scrambling, abrupt braking and cornering were more agitated and anxious during the journey, possibly due to fear of falling inside the trailer [46
]. The recommended loading density for horses loaded in groups is y = (54.837) × W0.325
, where y = density in kg/m2
and W = average animal weight in kilograms, about 1.2 m2
for a 500 kg horse [16
]. However, the latter study considered slaughter horses and injuries as the only welfare outcome evaluated. Minimal space allowance for an adult horse during a long journey is 1.75 m2
in Europe [19
], or 1.2 m2
in Australia [20
]. However, in many other countries minimal space allowance is not reported or a general and vague recommendation in line with the OIE regulation is provided (i.e., horses should have sufficient space to adopt a balanced position as appropriate to the climate and species transported) [47
]. The most recent European guidelines on transport of slaughter horses, derived from a Delphi survey method, suggest that horses should be transported with 10 and 20 cm of total space between animal and partitions [48
]. This is the first study to report animal-based evidence suggesting that horses travelling in a wide bay of 1.9 m2
are better able to balance, minimising the implications of transport on behaviour, health and welfare. Thus, findings may be useful for updating standards related to the minimum space allowances required for horses during long journeys.
Facing away from the direction of travel has been recommended in the literature [12
]. However, the majority of horse trailers and trucks are built to transport horses facing forward, and results from available studies are conflicting. Rear facing has been previously reported to be associated with fewer impacts against the slides and ends of the trailers, less frequent loss of balance, fewer total behaviour and less balance movement [12
]. Smith et al. [51
] found that horses travelling untethered preferred to travel facing backward, and Kusunose et al. [53
] confirmed this preference, demonstrating that yearlings learn quickly that facing backwards is advantageous during transport. However, these differences were not observed in other studies, and it was reported that the preference in the direction of travel is individual and may be related to the past experience [54
]. In the current study, rearward facing horses demonstrated fewer backwards, forwards and lateral movements and less total balance behaviour, as hypothesised. However, unexpectedly, rear-facing horses also demonstrated increased loss of balance in the current study. The latter results may be due to other factors, such as truck configuration, and therefore require clarification. Horses transported facing forwards demonstrated increased licking behaviour, increased interactions and total behaviour compared to rear facing and confined horses. This may be due to their difficulty maintaining balance which might increase anxiety and induce increased redirected behaviours and looking for a social calming effect. The fact that horses travelling facing the direction of travel are aroused is in line with the literature, where it was demonstrated that heart rate, HRV and salivary cortisol were higher in horses facing forward after a journey of short and middle duration [44
]. The current study demonstrated no significant effects attributable to direction of travel on clinical parameters (heart rate, respiratory rate, rectal temperature, GI auscultation scores), haematology or serum biochemistry, plasma cortisol or gastric squamous or glandular ulcer scores. This may therefore suggest that direction of travel is important for the ability of horses to keep their balance and may minimise anxiety, but it is does not substantially affect health.
Correlations were observed between several behavioural parameters and physiological parameters measured in the current study. Changes in heart rate and in rectal temperature (i.e., the difference between values obtained prior to departure and on return from travel) were correlated with total stress behaviours, total balance behaviours and the total behaviour score. This may be due to the strong connection between behaviour, sympathetic system and thermoregulation [55
]. Similarly, the correlations between frequency of stress behaviours, interaction and licking with decreased GI motility may suggest that these behaviours are associated with both increased sympathetic tone and, therefore, with decreased GI motility. It has been reported that transportation causes decreased gut sounds [5
] and increased risk of colic, in particular colon impaction [56
]; however, more studies on this relationship are needed. Importantly the severity of ESGD was associated with several balance behaviours, in particular loss of balance. Gastroscopic observations reported previously for these horses suggested that ulceration was likely due to contact between the squamous mucosa and alkaline gastric fluid, and it is likely that the increased loss of balance observed in the current study facilitated such contact. The severity of ESGD after transportation was also correlated with stress-related behaviour, head surveying, tossing and licking. Horses with gastric squamous ulceration tended to be more reactive to a novel test, spend more time away from the novel object, pawed more and tended to show oral stereotypy [57
]. However, in the same paper, authors failed to demonstrate a difference in pain-related behaviour, heart rate, cortisol and other clinical parameters between horses with and without stomach ulcers. Similarly, we were not able to find any associations between glandular ulcers and behaviour, haematology or situations of travel. This might reflect the mild and clinically insignificant glandular lesions observed in the current study, or could be related to the difficult and still unclear aetiology of glandular ulcers [58
]. We also failed to find associations between behaviour and haematology or biochemistry changes, or with transport conditions tested (width of bay or direction of travel). This was surprising, as it was hypothesised that increased stress behaviours would be associated with increased cortisol concentrations and increased movement with increased muscles enzymes. Our findings suggest that behavioural changes and non-invasive measures of autonomic balance such as heart rate or heart rate variability and GI motility might be more sensitive indicators of horses’ response to transportation than haematology, biochemistry or endocrine parameters. Consequently, our study suggests that monitoring behaviour and physiological responses in transit and after transportation are likely to identify horses that are at risk for transport-associated disease.
Our results need to be interpreted with caution, because the study was limited by several factors. First of all, the number of horses travelling in wide bays was lower than those travelling in single bays, due to restrictions in the configuration of the truck. The truck configuration also prevented a balanced study design relating to the number of horses travelling backwards and forwards. The preferences and travel history of each horse were unknown, and we were unable to control for this, for example by use of a repeated measures or cross-over study. The truck also had different compartments, and the first compartment, located over the trailer’s connection to the prime mover, and where two horses were located in rear facing wide bays, was considered the least stable due to its height and the rotational forces during turning. Consequently, the configuration of the truck may have confounded our results, and the frequency of total balance behaviour reported by horses in wide bays and rear facing may be overestimated. Conversely, horses used in the confinement study and consequently fasted 12 h before the journey were all located in the two rear-most compartments. Random allocation into different compartments and bay sizes would have been a more desirable strategy, but this would have prevented other study outcomes. An equal number of horses used in confinement travelled forwards as backwards, and behaviours in this group were not significantly different from those observed in other horses, suggesting that feeding management prior to departure did not influence observed behaviours. Only a confinement/transportation of 12 h were tested, so our results may not be repeated in journeys of shorter duration. Finally, some behaviours, such as the frequency of yawning, chewing and lip smacking were not always visible during transportation and had to be excluded from the analysis. Notwithstanding those limitations, this study has reported behavioural changes associated with space allowance and direction of travel, and is the first study reporting the effects of a wider bay on behaviour and health of horses in comparison with single bay or confinement alone. As such, it has increased our knowledge of transport stress and how to mitigate it.