Introduction
Animal-assisted therapies have a variety of applications in human medicine (Grandgeorge & Hausberger Reference Grandgeorge and Hausberger2011) and been used and described for over two decades (Stanley-Hermanns & Miller Reference Stanley-Hermanns and Miller2002). Equine-related treatments, in particular, have been investigated and although original reviews considered it controversial for use in psychotherapy due to most studies being methodologically flawed especially in their lack of randomised controlled trials between patients (Anestis et al. Reference Anestis Michael, Anestis, Zawilinski, Hopkins and Lilienfeld2014), hippotherapy has proven beneficial in the clinical progress of disabled patients (Koca & Ataseven Reference Koca and Ataseven2015; Wood & Fields Reference Wood and Fields2021). The improvements have been observed after horseback riding sessions on patients affected with various types of physical and/or mental disabilities. Recent, well-designed, controlled studies and meta-analyses demonstrating benefits include, amongst others, patients affected with: multiple sclerosis (Bronson et al. Reference Bronson, Brewerton, Ong, Palanca and Sullivan2010; Vermöhlen et al. Reference Vermöhlen, Schiller, Schickendantz, Drache, Hussack, Gerber-Grote and Pöhlau2018), cerebral palsy (Zadnikar & Kastrin Reference Zadnikar and Kastrin2011; Park et al. Reference Park, Rha, Shin, Kim and Jung2014; Martín-Valero et al. Reference Martín-Valero, Vega-Ballón and Perez-Cabezas2018; Matusiak-Wieczorek et al. Reference Matusiak-Wieczorek, Dziankowska-Zaborszczyk, Synder and Borowski2020), Down syndrome (Champagne & Dugas Reference Champagne and Dugas2010; Portaro et al. Reference Portaro, Cacciola, Naro, Cavallaro, Gemelli, Aliberti, De Luca, Calabrò and Milardi2020), postural instabilities (Silkwood-Sherer et al. Reference Silkwood-Sherer, Killian, Long and Martin2012), acute brain injury (Marquez et al. Reference Marquez, Weerasekara and Chambers2020), children with mental impairments and developmental delays (Kraft et al. Reference Kraft, Weisberg, Finch, Nickel, Griffin and Barnes2019), attention deficit and hyperactivity disorder (Oh et al. Reference Oh, Joung, Jang, Yoo, Song, Kim, Kim, Kim, Lee, Shin, Kwon, Kim and Jeong2018), autism spectrum disorders (Ajzenman et al. Reference Ajzenman, Standeven and Shurtleff2013), post-traumatic stress disorders (Johnson et al. Reference Johnson, Albright, Marzolf, Bibbo, Yaglom, Crowder, Carlisle, Willard, Russell, Grindler, Osterlind, Wassman and Harms2018; Shelef et al. Reference Shelef, Brafman, Rosing, Weizman, Stryjer and Barak2019) and schizophrenia (Jormfeldt & Carlsson Reference Jormfeldt and Carlsson2018). Ethical concerns have been voiced in recent years with the need for assessment and perhaps the use of animals for human therapies undergoing regulation (Loeb Reference Loeb2019; Fine & Andersen Reference Fine and Andersen2021). With increasing knowledge about animal welfare, behaviour and stress-related pathologies, multiple studies in horses have investigated the effects of working on stress parameters. These parameters include heart rate, and heart-rate variability, cortisol and behaviour (Munsters et al. Reference Munsters, Visser, van den Broek and Sloet van Oldruitenborgh-Oosterbaan2012, Kang & Yun Reference Kang and Yun2016; Uldahl et al. Reference Uldahl, Christensen and Clayton2021). Behavioural ethograms have been established to help assess signs of discomfort during ridden sessions (Henry et al. Reference Henry, Fureix, Rowberry, Bateson and Hausberger2017; Dyson & Pollard Reference Dyson and Pollard2020; Haddy et al. Reference Haddy, Burden, Prado-Ortiz, Zappi, Raw and Proops2021). Welfare investigations during, in particular, therapeutic riding have also been attempted and subsequent behavioural analyses have not been suggestive of horses experiencing significant levels of stress (Kaiser et al. Reference Kaiser, Heleski, Siegford and Smith2006; Mendonça et al. Reference Mendonça, Bienboire-Frosini, Menuge, Leclercq, Lafont-Lecuelle, Arroub and Pageat2019; Arrazola & Merkies Reference Arrazola and Merkies2020; Watson et al. Reference Watson, Davis, Splan and Porr2020), nevertheless better understanding of how this horse-human interaction works, substantiated with a variety of behavioural and physiological parameters is required (Fine & Andersen Reference Fine and Andersen2021; Hovey et al. Reference Hovey, Davis, Chen, Godwin and Porr2021) and many studies lack a sound research design, including small sample sizes and control groups. Due to the bias in assessing behaviour as a single stress indicator, the need for other potential biological markers of stress in horses has been investigated, e.g. cardiovascular effects of catecholamine release (sympathetic-adrenal-medullary axis), especially heart rate and heart-rate variability, as well as the hypothalamo-pituitary-adrenal axis endocrine stress response (von Lewinski et al. Reference von Lewinski, Biau, Erber, Ille, Aurich, Faure, Möstl and Aurich2013; Ferlazzo et al. Reference Ferlazzo, Cravana, Fazio and Medica2020). Good correlation between circulating markers supports analysis of multiple parameters to increase accuracy (Ferlazzo et al. Reference Ferlazzo, Fazio, Cravana and Medica2018a,Reference Ferlazzo, Cravana, Fazio and Medicab,Reference Ferlazzo, Cravana, Fazio and Medicac). Previous findings have tended to suggest the interactions between horses and riders during therapeutic riding not to be deleterious for the horses when mentally able and mentally impaired riders are compared via behavioural analysis and heart parameters (Cravana et al. Reference Cravana, Fazio, Ferlazzo and Medica2021) and other biological parameters over time especially the hypothalamo-pituitary-adrenal axis (Fazio Reference Fazio, Medica, Cravana and Ferlazzo2013; Ferlazzo et al. Reference Ferlazzo, Fazio, Cravana and Medica2018a,Reference Ferlazzo, Cravana, Fazio and Medicab,Reference Ferlazzo, Cravana, Fazio and Medicac). In order to increase knowledge on horses’ stress status and focus on whether their welfare suffered in regard to use in therapeutic activities, our study aimed to investigate different behavioural and physiological stress indicators during hippotherapy sessions. The goal being to understand whether hippotherapy was indeed more stressful than a beginners’ riding session which functioned as a control group since horses’ physical activity was typically comparable in both sessions.
Materials and methods
Ethical statement
This experiment protocol was subjected to examination from the Ethics Committee for animals’ protection of VetAgro Sup - Campus Vétérinaire de Lyon, France. All procedures and protocol design were authorised under agreement number 1512-2 (meeting 18, 2015) and Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes (and feed legislation, if appropriate) were adhered to.
Pre-clinical study: Development of the specific ridden ethogram scale
A personalised ethogram with weighted scores, based on those seen in other studies into the behaviour of ridden horses was devised and is presented in Table 1. Briefly, parameters included each horse’s general attitude (Sénèque et al. Reference Sénèque, Lesimple, Morisset and Hausberger2019), the position of the neck, tail movements, position of the ears and mouth (Dyson & Pollard Reference Dyson and Pollard2021a,Reference Dyson and Pollardb; Torcivia & McDonnell Reference Torcivia and McDonnell2021). The grid and weighted scores were established specifically for the experiment according to ridden ethogram scores (Dyson et al. Reference Dyson, Berger, Ellis and Mullard2017) and taking into account the opinions of a physiology expert (PhD); to differentiate horses displaying stress behaviour from those more relaxed. Video analysis of a ridden session took place to enable validation of the ethogram by the physiology expert, ensuring that different individuals obtained scores that were diverse enough to warrant interpretation. Session scores ranged from 0 (no evidence of stress) to 20 (severely distressed) and took place in the equestrian centre; inside an indoor arena familiar to all participants (riders and horses). Riders had attained an intermediate level of experience, thus were able to contest low-level showjumping competitions (Strunk et al. Reference Strunk, Vernon, Blob, Bridges and Skewes2018). The scores that ended up being associated with each parameter are shown in Table 1, one weighted mark is used per parameter observed.
Table 1. Study ethogram showing associated weightings for each parameter
Experiment
Eight healthy horses comprising six geldings and two mares were utilised, with age ranging from 12 to 21 years. None of the horses were stressed or in pain. Pre-experiment clinical exams were normal with no horses displaying any discernable signs of lameness. Horses were housed together in their usual environment of an open barn (25 × 10 m; length × width) bedded with straw, with constant access to pasture (700 × 400 m). They were fed ad libitum dry hay and provided with permanent access to fresh water via a water trough. Horses had previously been ridden weekly by beginners and disabled people for a minimum of six months prior to the start of the experiment. This took place over a 15-day period in early spring, with mild and cloudy weather and an average temperature of 17°C.
Measurements of basal biological markers and clinical examinations were carried out at rest prior to the onset of the study to rule out any abnormalities, especially any hypothalamo-pituitary axis dysfunction or resting cardiac dysrhythmia. The impaired riders (IR) consisted of four women and four men, aged between 19 and 47 years and presented with various physical or mental disabilities. The beginner riders (BR) consisted of three women and five men aged between 22 and 28 years, who had never ridden a horse before this experiment.
The different parameters measured were chosen for their practicality and feasibility during the trial without interfering with the ridden examination for an excessively long period of time per horse. The parameters were assessed at different time-points before, during or immediately after the session. All parameters, including basal samples, were taken at the same times of the day to rule out any influence of circadian rhythm on secretions (Cordero et al. Reference Cordero, Brorsen and McFarlane2012; Diez de Castro et al. Reference Diez de Castro, Lopez, Cortes, Pineda, Garfia and Aguilera-Tejero2014) and all sampling for analyte concentrations were taken at five-day intervals to avoid any major seasonal interference with the results (Durham et al. Reference Durham, Clarke, Potier, Hammarstrand and Malone2021).
Session and sampling protocol
Session plans with sampling timings are shown in Table 2. Briefly, the horses were tacked up and taken into the indoor arena by helpers where heart rate and blood samples were obtained (basal samples). Riders mounted their respective horses for a total duration of 1 h, during which time a combination of dexterity pedagogical exercises were performed on their own (no side-walkers were present) under the supervision of the same instructor (GV: 1st degree BEES and Handi’Cheval training diplomate). The horses remained mainly at walk or stood still. A few strides of trot were added towards the end of the session for a maximum duration of 5 min. Horses were brought back to the midline in the arena 20 min after the start of the session, which gave riders sufficient time for a few tasks to be performed, thereby ensuring samples were representative of the ongoing ridden work, before a new blood sample was drawn (T1 samples). Similarly, immediately prior to the end of the session, all horses returned to the midline where a repeat heart rate recording and salivary samples were obtained (T2 samples). Samples were not taken all together in order to limit any disturbance caused to the ongoing session.
Table 2. Time-line, description of the ridden sessions and associated samples obtained
Heart rate
A smartphone (iPhone 6®, Apple Inc, Cupertino, CA, USA) was used to record an electrocardiogram (ECG) using a case with contact electrodes and an ECG application downloaded from the App Store (AliveCor® Veterinary Heart Monitor, AliveCor Inc, San Francisco, CA, USA). The device has been previously validated for its use in horses with comparable accuracy to a more classic base-apex derivation (Kraus et al. Reference Kraus, Rishniw, Divers, Reef and Gelzer2019). The device was positioned as per manufacturers’ recommendations at a 45° angle behind the left elbow (Figure 1). To improve contact, the skin and coat were dampened with surgical spirit prior to application of the electrodes. The heart rate was evaluated from the trace obtained over a minimum period of 2 min. Close examination of the recording was also performed, including regularity measurements, to detect the presence of arrhythmias.
Figure 1. Smartphone case used for ECG recording and the area for correct positioning (purple line).
Blood analyses: Plasma ACTH and serum total cortisol
Blood samples were obtained via jugular venipuncture using a Vacutainer® system, taken firstly in a tube without anticoagulant and then a tube with ethylene diamine tetra-acetic acid (EDTA) anticoagulant. Less than 4 h post-sampling, both dried and EDTA tubes were centrifuged (2,000g for 10 min and 2,000g for 5 min, respectively) and a minimum of 0.5 mL of serum or plasma were kept in Eppendorf tubes and frozen at –80°C until analysis. All adrenocorticotropic hormone (ACTH) and total cortisol samples were analysed in the same batch, via Chemiluminescence Immuno Assay (Immulite 2000, Siemens Medical Solutions Diagnostics. Erlangen, Germany) with a protocol previously validated for use in horses (Irvine et al. Reference Irvine, Burt, Hill, Shaw and Papasouliotis2016; Banse et al. Reference Banse, Schultz, McCue, Geor and McFarlane2017).
Salivary cortisol
Salivettes (Sarstedt, Nümbrecht-Rommelsdorf, Germany) were used to collect saliva using a haemostatic clamp. The salivette swab was placed in the mouth in the interdental space next to the bit when the horse was bitted, or as shown in Figure 2. It was left in place for a few minutes until soaked, depending on the intensity of the horse’s salivation. As these often took a long time to soak (more than 3 min), samples were not taken before the riding sessions (T0) to avoid any delay in the time-scale. Once collected, the salivette swabs were frozen less than 4 h after sampling and until analysis, to help decrease viscosity and remove salivary mucins (Garde & Hansen Reference Garde and Hansen2005). On the day of analysis, samples were thawed and at least 1 mL of saliva was obtained via centrifugation at 1,000g for 10 min. An ELISA (enzyme-linked immunosorbent assay) kit, previously validated for use on saliva in horses, was used to measure salivary cortisol (Sauer et al. Reference Sauer, Gerber, Frei, Bruckmaier and Groessl2020).
Figure 2. Insertion of a salivette swab in the horse’s mouth using a haemostatic clamp.
Stress score and behavioural assessment
A digital camera (E-M10 Mark III OM-D, Olympus, Tokyo, Japan) was placed in one corner of the arena, allowing visualisation of more than 70% of the riding area. The entire session was recorded for assessment at a later stage. Each horse’s behaviour was evaluated simultaneously during different time-points when the riders were performing the same exercises (Table 2). Horses’ demeanours were observed over a total length of 15 min taken over three different time-points for each horse: 5 min immediately following the start of the session, 5 min straight after the first sampling, and 5 min following the second sampling. Behaviour was scored as per the specific ethogram established in the pre-clinical study (Table 1), and according to the worse behaviour displayed during the observational time-frame. The final stress score consisted of the mean of the scores obtained following the three separate observations.
Statistical analysis
The statistical analysis was performed using R software (R Development Core Team 2008). Basal data were examined for normality with the distribution and using QQ plots. The Wilcoxon signed ranks test was used to compare values not normally distributed. Samples obtained at rest were compared to basal T0 samples before IR or BR sessions, when available. This was done to assess the stress levels of horses prior to riding and evaluate if sessions were then going to be comparable. If a significant difference was present, the variation between T1 or T2 values and T0 values were compared between IR and BR groups. If no difference was found, T1 or T2 values were directly compared between IR and BR groups. The investigation of a potential linear correlation between the biological stress markers and the stress score was performed graphically separately for each session, and then using Spearman’s correlation test. A P-value < 0.05 was considered to be of statistical significance.
Results
Heart rate
No arrhythmias were detected at any point on the recording at rest or during the ridden sessions. Median resting HR at T0 was 35 bpm, not significantly different from the median resting HR of 36 bpm at T2. There was also no significant difference between the median resting HR (35 bpm) and the median value recorded at T0 for HR (37 bpm) and for BR (38 bpm). The median HR with BR at T2 was 38 bpm, no significant difference was found with IR at T2 (39 bpm) or with the resting value at T2. Results are summarised in the first column of Table 3.
Blood analyses: Plasma ACTH and serum total cortisol
Plasma ACTH results were within the laboratory seasonally adjusted reference intervals, confirming the absence of Pituitary Pars Intermedia Dysfunction (PPID): the median resting ACTH at T0 was 12.5 pg mL–1 and the median resting ACTH at T1 was 11.5 pg mL–1. Before the ridden sessions, median ACTH values were not significantly different from the resting ACTH and were 13.9 pg mL–1 for both IR and BR groups. For the total serum cortisol, the median for IR at T0 was 121.5 nmol L–1 which was significantly higher than the resting result 70.4 nmol mL–1 (P = 0.02) and the BR at T0 81.8 nmol L–1 (P = 0.05). Considering these and to avoid basal stress levels interference, differences between T1 and T0 for IR and BR were compared rather than values at T1. During the ridden sessions, for IR the median plasma ACTH at T1 was 10.3 pg mL–1, which did not differ significantly from BR at T1: 10.5 pg mL–1. Comparison of the difference between session and basal values from the same day can be seen in Figure 3. The median variation in serum cortisol with IR (T1–T0) was –30.9 nmol L–1, which was significantly lower than for BR at 4.7 nmol L–1 (P < 0.01). All medians and ranges are shown in Table 3.
Figure 3. Comparison of serum total cortisol variation for impaired (IR) and beginner riders (BR). ** P < 0.01. Lines connect the values obtained for the same horse, red line = no variation observed.
Table 3. Medians (range) of the physiological parameters obtained from all eight horses at T0 prior to the sessions, and ACTH and serum cortisol at T1 and heart rate, salivary cortisol and stress score at T2
IR = impaired riders; BR = beginner riders.
Salivary cortisol
The median resting salivary cortisol concentrations were not significantly different between T0 (1.2 nmol L–1) and T2 (1.3 nmol L–1). For the values obtained during the sessions, the median salivary cortisol at T2 with IR was 0.9 nmol L–1 and with BR it was significantly greater at 1.3 nmol L–1 (P = 0.04): distributions are shown in Figure 4, medians and ranges in Table 3.
Figure 4. Comparison of the salivary cortisol distributions during the sessions (T2) between the impaired (IR) and the beginner riders (BR). * P = 0.04. Lines connect the values obtained for the same horse.
Stress score
The final stress scores were also not significantly different between BR (score 3.83) and IR (score 3.00) (Table 3). Examination of plotted stress scores in relation to the different biological parameters (including the variation between T1 and T0 serum total cortisol) did not reveal any elliptical distribution which could have evoked a linear correlation (Figures 5[a] and [b]). This was confirmed by the Spearman’s correlation test between stress score and every other parameter which was not significant for any of the parameters analysed separately for both sessions.
Figure 5. (a) Stress scores plotted according to the related physiological parameter obtained during the IR sessions. No significant correlation was found.
Figure 5. (b) Stress scores plotted according to the related physiological parameter obtained during the BR sessions. No significant correlation was found.
Discussion
This is one of the first studies to assess both physiological (ACTH, serum and salivary cortisol, heart rate) and behavioural (stress ethogram) parameters at different time-points for horses being ridden by beginners and comparing them to those obtained from horses ridden by disabled people. A significant difference was observed for the serum total cortisol and salivary free cortisol, which were both found to be lower during the hippotherapy session. The serum cortisol was taken 20 min prior to taking the salivary cortisol, a peak in saliva following a serum peak usually by 15 to 20 min (Hurcombe et al. Reference Hurcombe2011). These findings are consistent with a possible reduced systemic stress response during the hippotherapy sessions in comparison with being ridden by beginners, although our sample size means the results should be interpreted with caution.
The main limitation of our study was the small number of horses involved; this is difficult to overcome using a study protocol in the horses’ usual environment since most riding lessons for beginners or hippotherapy sessions tend to take place in an indoor or outdoor arena with limited space. Thus limiting the number of horses involved for obvious safety reasons. In our experiment, the equestrian centre used a maximum of eight horses during all their usual sessions involving beginners or for hippotherapy sessions. The organisation of multicentric experiments or repeated analyses over several weeks could have helped improve the power of our tests if the data could have been assessed and been normally distributed, although significant differences were already found for two of the five parameters examined here. Also, repeating analyses over time can lead to other bias: for example, normal basal ACTH distribution can vary on a weekly basis throughout the year (Durham et al. Reference Durham, Clarke, Potier, Hammarstrand and Malone2021), therefore all measurements in our study were performed within 15 days to limit any such seasonal effect. It is also reasonable to assume that by examining a combination of markers related to stress, the likelihood of finding an abnormality would be greater, especially using physiological parameters which are highly sensitive (Padalino & Raidal Reference Padalino and Raidal2020). There is also a limitation concerning the use of our ridden ethogram, although based on different published ethograms (Dyson et al. Reference Dyson, Berger, Ellis and Mullard2017; Dyson & Pollard Reference Dyson and Pollard2020, Reference Dyson and Pollard2021a,Reference Dyson and Pollardb), the weights that were applied are perfectible and could be improved using horses ridden in different contexts. Adaptation of the grid could be improved by assessing the repeatability, testing for intra- and inter-observer variability, especially with observers familiar and unfamiliar with the grid. Such analyses could lead to the procurement of likelihood of stress from the occurrence of behavioural markers; but the lack of a gold standard method to evaluate the stress status in horses to validate such parameters is still lacking. The nature of the impaired riders’ physical or mental disabilities was not disclosed to us at the time of the experiment to respect the privacy of the riders but this could be investigated more specifically since the influence on the horse could be different. Thus, the use of biological parameters is essential in addition to behavioural analysis, due to the possibility of acquired shorter duration of stress responses (Marsbøll & Christensen Reference Marsbøll and Christensen2014).
The sympathetic nervous system and the hypothalamic-pituitary axis are the main actors of biological responses to stressors, where their activation results in adrenal secretions of catecholamines and corticosteroids, respectively (Ayala et al. Reference Ayala, Martos, Silvan, Gutierrez-Panizo, Clavel and Illera2012). These messengers modulate important responses, such as heightened alertness, glucose mobilisation/use, vasomotor tone, cardiac output to improve perfusion during times of physiological and pathological stress (Hurcombe Reference Hurcombe2011). Catecholamines are the first category of biological markers involved in the stress response. Despite their good correlation with systemic stress and sympathetic nervous system stimulation (Wagner Reference Wagner2010), their very short half-life (less than 30 s for adrenaline and noradrenaline) makes them impractical for handling and being measured precisely under field conditions (Snow et al. Reference Snow, Harris, MacDonald, Forster and Marlin1992), which is why these analyses did not feature in our study. Tachycardia remains one of the easiest physiological consequences of an increase in catecholamines to observe and was measured precisely via the ECG. Heart rate was found to remain below the expected increase associated with physical stress described in the literature, which lies between 30–50% from the baseline values (Wagner Reference Wagner2010). The ECG also underwent close examination for regularity and complex morphology as the occurrence of premature complexes and runs of tachycardia have been described in relation to stress in horses (Sandersen et al. Reference Sandersen, Detilleux, Delguste, Pierard, van Loon and Amory2005). Heart-rate variability accurately represents the state of stress over an entire session, and this could be used in the future to improve the overall stress assessment (Garcìa-Gòmez et al. Reference Garcìa-Gòmez, Guerrero-Barona, Garcìa-Peña, Rodrìguez-Jimenèz and Moreno-Manso2020).
In parallel with the sympathetic-induced responses, inputs also stimulate the hypothalamus which triggers the release of corticotropin-releasing hormone into the pituitary portal system. This causes the rapid release of ACTH by the pituitary gland due to their close proximity. ACTH then binds to adrenal receptors to stimulate (mainly) cortisol release (Hurcombe Reference Hurcombe2011). All ACTH measurements in our study, especially initial resting samples, were relatively low. They were comprised within seasonally adjusted reference intervals; the experiments being performed in May close to when the yearly ACTH distribution curve shows a natural trough (Durham et al. Reference Durham, Clarke, Potier, Hammarstrand and Malone2021). Elevations above reference ranges are indicative of a systemic stress response, but they may also be influenced by Pituitary Pars Intermedia Dysfunction, a commonly observed endocrine disorder in older horses (Beech et al. Reference Beech, Boston and Lindborg2011). Horses were reasonably old in our study (aged between 12 and 21 years) but all resting results were thankfully not consistent with pituitary dysfunction. The aforementioned pathological ACTH elevation is not accompanied by an increased cortisol elevation, which could be due to a compensatory increase in urinary excretion (Morgan et al. Reference Morgan, Keen, Homer, Nixon, McKinnon-Garvin, Moses-Williams, Davis and Walker2018).
Cortisol, whether in its total or free form found in serum or the free component found in saliva, is a well-used and established stress indicator in horses (Peeters et al. Reference Peeters, Sulon, Beckers, Ledoux and Vandenheede2011; Sauer et al. Reference Sauer, Hermann, Ramseyer, Burger, Riemer and Gerber2019; Ferlazzo et al. Reference Ferlazzo, Cravana, Fazio and Medica2020). The relationship between serum total cortisol, serum free cortisol and salivary cortisol is also intricate and well correlated with stressful stimuli (Alexander & Irvine Reference Alexander and Irvine1998; Peeters et al. Reference Peeters, Sulon, Beckers, Ledoux and Vandenheede2011; Bohák et al. Reference Bohák, Szabó, Beckers, Melo de Sousa, Kutasi, Nagy and Szenci2013). Reports of the use of salivary cortisol are becoming especially common, essentially because of the ease of sampling and the fact that it is non-invasive (von Lewinski et al. Reference von Lewinski, Biau, Erber, Ille, Aurich, Faure, Möstl and Aurich2013; Kang & Yun Reference Kang and Yun2016), and reliable (Garde & Hansen Reference Garde and Hansen2005; Sauer et al. Reference Sauer, Gerber, Frei, Bruckmaier and Groessl2020). Interestingly, during our study sessions, the difference in serum total cortisol and salivary cortisol were the only markers found to be significantly lower during hippotherapy sessions. Baseline cortisol on the day of hippotherapy was significantly greater than on the other days (resting and beginners session day). This prompted us to perform the analysis using the difference between serum cortisol during the session and baseline cortisol from that day. The assumption being that this difference would be a more true reflection of the influence of the session on serum cortisol rather than an initial stress state due to factors external to the sessions. A number of tractors were in use on the yard on the morning of the IR session, but horses were used to occasionally encountering these so there is no certainty about the origin of the baseline cortisol elevation. Although the absence of statistically significant differences among the other parameters does not necessarily mean equivalence, the observations of the distributions were much more homogenous and therefore deemed comparable. In addition, the similar difference observed in the salivary cortisol results support the logic of our analysis. This appears to be in accordance with previous findings in the literature, whereby serum cortisol was equally found to be significantly lower during therapy sessions (Hovey et al. Reference Hovey, Davis, Chen, Godwin and Porr2021) or not affected by contact with people presenting post-traumatic stress disorders (Merkies et al. Reference Merkies, McKechnie and Zakrajsek2018). Other hormonal parameters that could be used to assess stress-coping mechanisms in horses include beta-endorphine and thyroid hormones, which have been studied in healthy horses under different conditions and could add some value to further studies (Ferlazzo et al. Reference Ferlazzo, Fazio, Cravana and Medica2018a,Reference Ferlazzo, Cravana, Fazio and Medicab,Reference Ferlazzo, Cravana, Fazio and Medicac), or oxytocin which has been studied in horses used for post-traumatic stress disorder equine-assisted therapies and activities (Malinowski et al. Reference Malinowski, Yee, Tevlin, Birks, Durando, Pournajafi-Nazarloo, Cavaiola and McKeever2018).
The stress ethogram we used did not show a statistically significant difference, nor a correlation with any of the biological parameters measured. However, the overall scores observed during both riding sessions were low (3.83 and 3.00 for the IR and BR, respectively) on a scale that potentially could extent up to 20. This indicates an overall agreement with the low level of stress suggested by the other biological parameters. Developing and validating a stress ethogram in horses is problematic since most of the distressed behaviours expressed can have a subjective interpretation from the observer. Furthermore, it is possible that the focus of horses in accomplishing the task asked by the rider prevents them from expressing otherwise common pain behaviours that could be found at rest (Torcivia & McDonnell Reference Torcivia and McDonnell2021). More specifically, it has been shown that horses tend to not express certain discomfort behaviours when reins restrict their movements (Smiet et al. Reference Smiet, Van Dierendonck, Sleutjens, Menheere, van Breda, de Boer, Back, Wijnberg and van der Kolk2014). Examples of ethograms developed for ridden work include scales specifically targeting orthopaedic pain (Dyson & Pollard Reference Dyson and Pollard2020, 2021a,b); these show similarities to our scale although our objective was to investigate discomfort on a more generalised level. The ideal ethogram to assess the latter lies somewhere among all the aforementioned scales. The different weights affected to each parameter need to be established in a more robust mathematical manner, which was beyond the scope of this study.
Animal welfare implications and conclusion
Our study has shown that within the context of hippotherapy sessions, horses do not present with increased biological stress markers when compared to being ridden by beginners. Furthermore, a statistically significant decrease in cortisol was observed, potentially indicating that being involved in hippotherapy sessions is less stressful for the horses than being ridden by beginners. Although these results indicate that hippotherapy may be ethically justified as it benefits humans without harming the horses, the present study was small, and the results should be interpreted with caution.
Acknowledgements
The Lions Club Nîmes Maison Carrée (France) represented by Doctor Jean Delate, and Zoetis supported financially the project and helped to buy the ECG phone case, the Salivette swabs and cortisol ELISA kit for salivary analyses. The biochemistry department of VetAgro Sup Campus Vétérinaire de Lyon (France) kindly performed the blood analyses free of charge. The authors would like to thank Marie-Laure Delignette-Muller for helping with the initial statistical analysis, Marie-Christine Carlier from Université Lyon 1 (France) for her advice on processing the samples, and also Edwige Rousseliere and Benoît Rannou from the VetAgro Sup – Campus Vétérinaire de Lyon (France) biochemistry laboratory for their help with the biochemistry analyses. A warm thank you to Sara Marcelin, Gérard Violland and the equestrian centre Les Cavaliers du Bordelan (France) for welcoming us and leading the riding sessions. We are also thankful to Hugo Leonardi, Jonathan Rodriguez, Laura Chaumeil, Liane Dupon, Maleck Vasseur, Maxime Collin, Morgane Cornier and Thomas Chavalle for trying horseback riding for the first time.
Competing interests
None.
Open access
