Salivary analysis to unveil the paradigma of stress of domestic horses reared in the wild

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Collectively, it is supposed that a horse kept in the wild has a lower level of stress than other housing system, and the aim of the present study was to investigate the level of stress in domestic horses reared in the wild and then moved to human controlled housing, through saliva analysis. Twelve clinically healthy Catria (Italian local breed) mares, usually reared in the wild, were moved into collective paddocks for a folkloric event. Saliva samples were obtained before and after the change of housing condition to evaluate stress biomarkers including salivary cortisol, salivary alpha-amylase, and butyrylcholinesterase. The mares were also scored using the Welfare Aggregation and Guidance (WAG) Tool to highlight the presence of abnormal behaviors. Despite the absence of differences in behavioral scores between wild and paddocks, salivary cortisol and butyrylcholinesterase were found to be overexpressed in the wild and statistically decreased when mares were moved to paddocks. The highest concentrations in stress biomarkers like salivary cortisol and butyrylcholinesterase in the wild was unexpected, but the need for managing hierarchical relationships, and the exposure to feral animals, predators, and weather changes, might explain these findings. The overall results of the present study may provide further knowledge toward stress response in domesticated horses living in the wild moved to human controlled housing system. Health sciences/Biomarkers Health sciences/Medical research Stress Horse Welfare Saliva Cortisol Alpha amylase Butyrylcholinesterase Figures Figure 1 Figure 2 Figure 3 Introduction In the last few years, an increasing interest has been pointed towards the use of saliva as a diagnostic fluid both in human and veterinary medicine. 1 – 3 It is well known that saliva composition can be affected by organic or psychological disorders and may reflect general metabolic changes. 4 In comparison with other biological samples (e.g. blood), saliva has the advantage of being easily collected by non-invasive and non-stressful procedures, which is extremely important when sampling animals. 5 Recently, some salivary markers including cortisol and salivary alpha-amylase (sAA) have demonstrated the potential to make diagnosis of chronic stress or to discriminate between stress, anxiety and depressive disorders in people. 4 , 6 Salivary cortisol has been used in most studies for the assessment of adrenocortical response to potentially stressful situations in horses 7 so that it is recognized as an indicator of acute stress. 8 , 9 The concentration of alpha-amylase in saliva increases abruptly following both acute and chronic stress, which makes it an important salivary biomarker of stress. 4 Van Veen et al. (2008), suggested that sAA could be a potential salivary stress marker in people sensitive to a negative social assessment. Together with cortisol and sAA, salivary butyrylcholinesterase (BChol) is considered a reliable indicator of stress in equine 1 , 7 , 8 , 10 and swine 11 . A recent study demonstrated as the administration of BChol can reduce stress-induced aggression in mice and is considered a candidate for treatment of anxiety in human. 12 Equids are prey animals, and they are inclined to live in group for safety. In order to maintain group membership and social arrangement, horses maintain their social relationship with the herd by scratching and rubbing, playing, resting, and following each other. 13 Their prey behavior influences also feed tendency, as for other species in nature; in fact, free roaming equines spend 16–20 hours a day 14 grazing large amounts of high fiber food 15 . It has been reported that horses continuously living on pasture with conspecifics display a more natural expression of the species’ behaviors such as feeding and social interactions 16 , 17 and less health impairments 18 , suggesting a better welfare state than horses kept in individual boxes 19 , 20 . Chronic stress induced by missing contact with other horses or by diseases causing pain (e.g. chronic lameness), have the potential of affecting the horse’s physical health. In this perspective, the evaluation of stress biomarkers can be relevant when assessing horse welfare, health or disease predisposition. According to some Authors, domestic horses are informative models to investigate the impact of stress on the HPA (hypothalamic–pituitary–adrenal) axis, as they commonly experience conditions impairing their welfare related to social, spatial, and feeding restrictions. 21 HPA axis response to acute stress has been studied by several authors but its interpretation in cases of prolonged stress is far from straight forward. 22 – 25 It is assumed that stabled horses are more prone to be stressed and to develop stereotypic and redirected behaviors that are generally used as an indicator of poor welfare associated with stabling. 26 Stress and abnormal behaviors are less likely to develop in animal that spent significantly longer time outside or that are kept in pasture. 27 The present study aimed at investigating changes in salivary cortisol, sAA, and BChol, considered as affordable biomarker of stress, in domesticated horses usually living in the wild and then exposed to a potential stressful condition like a local event. The study herein described, the methods applied and results obtained are reported in accordance with ARRIVE guidelines ( https://arriveguidelines.org ). Results According to the WAG tool, all the mares expressed the same behavioral score, resulting in grade A (good welfare) both in the wild and in paddock, irrespective of the different setting. The mouthpiece used for sampling was well tolerated by all horses (no signs of discomfort or stress were displayed during collection) and allowed an easy-going collection in all horses. The concentrations of sAA, BChol and cortisol in saliva samples collected from the same horses in the wild and in paddock are reported in Table 1 . The concentration of sAA were similar between pasture and paddock (p = 0.7742), with a wider range (from 19.70 to 86.40 U/L) in the wild compared to paddock housing (from 27.10 to 65.50 U/L) (Fig. 3 ). Butyrylcholinesterase was found to be statistically overexpressed during pasture stable and decreased in a statistically significant way when horses moved to paddock (p = 0.0068). The same trend was registered for cortisol, for which significant higher salivary concentration was found when horses were stabled in pasture (p = 0.0002). Table 1 Descriptive statistics for salivary biomarkers. Data are expressed in U/L, mU/mL, and ng/mL for salivary alpha-amylase (sAA), butyrylcholinesterase (BChol), and cortisol, respectively. Abbreviations: min: minimum concentration; max: maximum concentration. wild paddock mean ± sd min. max. range mean ± sd min. max. range sAA 46.25 ± 19.44 19.70 86.40 66.70 47.52 ± 13.11 27.10 65.50 38.40 BChol 12.44 ± 6.30 6.50 23.80 17.30 5.58 ± 2.39 3.70 11.80 8.10 Cortisol 33.59 ± 13.44 12.20 49.40 37.20 5.99 ± 5.70 0.40 16.80 16.40 Discussion In the planning phase of the study, we assumed that the modification of horse environment from wild setting to paddock restraining could have had a direct influence on both horse behavior and stress parameters. Unexpectedly, the mares did not change any behavioral aspect when observed in collective paddocks during the event compared to wild environment. A different result was obtained recently by Ruet and collaborators 20 that studied the reverse of the present study. Individual box housing is supposed to potentially compromise animal welfare: for this reason, a common practice is to offer horses a short-term period on pasture with conspecifics to alleviate the impact of long-term deprivation triggered by box shelter. When on pasture, horses increased the expression of natural behaviors such as locomotion, exploration and social behaviors which may have not been easily expressed when stabled in boxes; in addition, they did not display any stereotypies and aggressive behaviors towards humans. Interestingly, during the initial days on pasture horses were unresponsive to the environment in a significant manner than what was observed when stabled in individual boxes. After a proper period of adaptation, such behavior reverted to the level previously observed when horses were in boxes. Authors concluded that pasture can positively influence the welfare state of horses usually stabled individually, but also that several days of adaptation are needed, probably due to the novelty of the environmental and the needing to establish social conditions. 20 Conversely from Ruet and collaborators conclusions, in the present study the behavioral evaluation of horses did not change when passing form wild to paddock. A possible explanation to our finding could be that the mares were moved into collective paddocks and not in single boxes, maintaining their herd. Differently from behavioral observations, objective salivary parameters of horse stress namely BChol and Cortisol significantly changed during the study, while sAA remained unchanged. Regarding sAA, it is considered a potential salivary stress marker of acute and chronic stress in people especially in those sensitive to a negative social assessment. 28 , 29 In horses experimenting an acute stress, for example immediately after a sudden scare, sAA increase, to return at lower level after few minutes. 30 Since in our study horses were not exposed to an acute stress, the lack of change we observed could be due to the lack of reliability of sAA in evaluating non-acute stress in horses. Butyrylcholinesterase, a serine hydrolase biochemically related to the cholinergic enzyme acetylcholinesterase, is widely distributed in the nervous system, and it seems to be involved in neural function serving as a co-regulator of cholinergic transmission. The function of BChol in saliva is not fully understood but it has been associated with acute stress in humans, rats, and pigs. 31 In our study, Bchol salivary concentrations were significantly higher in saliva samples collected in the wild compared to samples collected on the same mares restrained in paddocks. A similar trend was observed in salivary cortisol showing the highest mean values when the mares stayed in the wild. Salivary cortisol represents the unbound biologically active fraction of total plasma cortisol, that is mainly bound to carrier proteins, thus reflecting the hypothalamic–pituitary–adrenal (HPA) axis activation. 32 , 33 The unexpected findings of the present study can be explained by an adaptive approach called “coping strategy” that defines the behavioral and physiological struggles of animal to face a new, potentially stressful situation. The coping strategy attracts the interest of different authors dealing with applied horse welfare and behavior. 34 When a change in environment occurs, as in the present study, animals need to cope with their environment using both behavioral and physiological stress responses. Different parameters are related to such coping strategy and may be monitored over time such as changes in form and frequency of behavioral response, changes in animals-based index (such as heart rate, body temperature, hormones, neutrophils and lymphocytes ratio, and other blood indicators). In this context, the implementation and study of novel biomarkers as sAA, salivary cortisol, and Bchol that can be easily measured and may serve as indicators over time, supporting in the understanding of individual adaptive capacity to stressful events. 35 Environment has a major impact on horse welfare and behavior (influencing for example the chance of feeding, protection from weather condition, and sleep-awake cycle) 36 and horse may reveal specific behavior patterns to adapt to challenging situations especially in man-made environment 37 . The results of the present study provide new knowledge in the multidimensional approach toward horse behavior understanding, hopefully providing valuable information for specific, possibly challenging situations. Animals living in man-made environments (as paddocks in the present study), are believed to be influenced by different kinds of stressor agents that causes adaptive responses. 38 On the other hand, it could also be hypothesized that although life in the wild allows horses to shows their natural behavior in freedom, at the same time this could represents a stress factor, linked to the need for managing hierarchical relationships within the herd, and the exposure to feral animals, predators, and weather changes, might explain these findings. We aimed to appraise the relationship between behavioral and physiological factors involved in the horse stress response and, surprisingly, salivary biomarkers revealed that stress level was higher in the wild. It should also be underlined that a large heterogeneity between the studies exists, because of the lack of standardized methods for measuring salivary cortisol, heterogeneity of studied populations and the bias of risk factors. In human being, for example, adolescents with severe symptoms of depression have not different concentration of salivary cortisol when compared to adolescents with mild symptoms of depression but had a significantly higher ratio of morning to evening levels of salivary cortisol. 39 This should be taken in account in future studies also in veterinary research. The findings of the present study are related to a typical Italian horse breed and the same evaluation considering different breeds/attitudes is warmly welcome, to provide more details to interpret horse behavior. Exploring coping strategies in horses and in any captive animal can be successful when a multidimensional approach including behavioral, neural, hormonal, and hematological measures is considered. Knowledge on stress and related coping styles can provide valuable information to prevent potentially challenging situations, in order to maximize animal welfare. Materials and methods Animals and behavioral assessments A total of 12 clinically healthy mares were included in the study with informed owners’ consent. The animals belonged to the Italian local breed Catria Horse known for its endurance, resistance, and well-balanced attitude (Fig. 1 ). Horses’ mean age was 6.5±6 years, and BCS ranged from 6 to 7 out of 9 points scale 40 . As inclusion criteria, all animals were domesticated and accustomed to handling before the beginning of the study, and they were maintained at pasture in the wild throughout the year. The study design consisted of behavioral evaluations and saliva collections on the same animals in the wild (usual environment) and in collective paddocks on occasion of a two-day folkloric event (Fiera del cavallo del Catria). Behavioral indicators were scored using the Welfare Aggregation and Guidance (WAG) Tool 41 evaluating the presence of abnormal behaviors like aggressiveness, unresponsiveness to the environment, hypervigilance, etc. All assessments were performed by an experienced observer. The first saliva sampling was performed with the free-ranging mares in their usual habitat (wild). The second sampling was collected from the same mares moved in paddocks on occasion of a local event. To avoid any interference on studied parameters, the procedures of the second time point were performed after 48h from relocation to allow the animals to familiarize with the new condition. Saliva samples were collected in the morning (10:00 a.m.-11:00 a.m.), using cotton swabs (Salivette® Sarstedt AG & Co., Nümbrecht, Germany) inserted into a customized hollow mouthpiece that horses chewed for 5 minutes (Fig. 2 ). After each collection, cotton swabs were removed from the mouthpiece by means of surgical clamps, cooled immediately and delivered to the laboratory within 2h. After behavioral evaluations and saliva collections, the mares were examined by an experienced clinician to verify the health conditions of each subject. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Camerino University (Registration number: 10/2023) and were in accordance with the standards recommended by the EU Directive 2010/63/EU for experiments on animals. Laboratory analysis Saliva samples were obtained by centrifugation at 3000g for 20 minutes (Universal 32, Hettich Zentrifugen, Tuttlingen, Germany) of Salivette® tubes (SARSTEDT AG & Co. KG, Nümbrecht, Germany) and 0.5 mL aliquots of saliva were stored at − 20 ◦C until analysis. At the day of the analysis saliva samples were thawed and centrifuged (10 min at 1000g) to eliminate debris residues. Surnatants were collected and cortisol, butyrylcholinesterase, and amylase levels were measured by using Horse Cholinesterase ELISA Kit, Horse Cholinesterase ELISA Kit, Horse Amylase ELISA kit (Bioassay Technology Laboratory), respectively, following the Manufacturer’s instructions. The absorbance was determined with Thermo Scientific Multiskan Sky amounted to 450 nm. Statistical analysis Statistical analysis was performed using GraphPad Prism version 8.2.1 for macOS, GraphPad Software, La Jolla California USA. sAA activity, salivary butyrylcholinesterase and cortisol concentrations were checked for normality using Shapiro-Wilk test, showing sAA and cortisol normally distributed while butyrylcholinestrase was not. Then, paired t test was applied to alpha-amylase and cortisol, and Wilcoxon test to butyrylcholinesterase to assess if concentrations of salivary biomarkers were statistically different between pasture and paddock. Values of p < 0.05 were considered significant. A post hoc analysis (G*Power 3.1.9.6 for Mac OS) was performed to assess the significance level and power obtained. Including a number of twelve horses gave a significance level of α = 5% (P < 0.05) and a power of 80%. Declarations Acknowledgements The Authors sincerely thank AACC - Associazione Allevatori Cavallo del Catria. This paper and the research behind it would not have been possible without their exceptional support and commitment. Authors’ contribution Conceptualization: MB; Data curation: MB, AM; Formal analysis: MB, AM, FLG, BP, AS, FL; Investigation: MB, AM, FLG, BP; Methodology: FLG, BP; Writing - original draft: MB, AM, FLG, BP, AS; Writing - review & editing: MB, AM, FLG, BP, FL Competing interests The authors declare no competing interests. Data availability statement Data generated during the study are available upon author’s request (please email to [email protected] ) References Contreras-Aguilar, M. D. et al. 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Equine Vet J 15 , 371–372 (1983). Kubasiewicz, L. M. et al. The Welfare Aggregation and Guidance (WAG) Tool: A New Method to Summarize Global Welfare Assessment Data for Equids. Animals 2020, Vol. 10, Page 546 10 , 546 (2020). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Apr, 2024 Reviews received at journal 14 Apr, 2024 Reviews received at journal 11 Apr, 2024 Reviewers agreed at journal 07 Apr, 2024 Reviewers agreed at journal 06 Apr, 2024 Reviewers invited by journal 06 Apr, 2024 Editor assigned by journal 05 Apr, 2024 Editor invited by journal 02 Apr, 2024 Submission checks completed at journal 02 Apr, 2024 First submitted to journal 21 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3974986","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":286791565,"identity":"c41b93fb-4efd-4647-9031-dd6537de6c2b","order_by":0,"name":"Marilena Bazzano","email":"","orcid":"","institution":"University of Camerino, Matelica (MC)","correspondingAuthor":false,"prefix":"","firstName":"Marilena","middleName":"","lastName":"Bazzano","suffix":""},{"id":286791567,"identity":"000108b9-9755-4d94-b964-454b07409514","order_by":1,"name":"Andrea Marchegiani","email":"data:image/png;base64,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","orcid":"","institution":"University of Camerino, Matelica (MC)","correspondingAuthor":true,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Marchegiani","suffix":""},{"id":286791569,"identity":"6318bd4b-9631-4d78-a5b6-9a81f536f2ee","order_by":2,"name":"Francesca La Gualana","email":"","orcid":"","institution":"Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"La","lastName":"Gualana","suffix":""},{"id":286791571,"identity":"77a8ef60-ca59-4a4a-997f-f5197f6620a4","order_by":3,"name":"Begi Petriti","email":"","orcid":"","institution":"Sapienza University of Rome","correspondingAuthor":false,"prefix":"","firstName":"Begi","middleName":"","lastName":"Petriti","suffix":""},{"id":286791573,"identity":"6678b52f-a5f8-43a8-a33d-4df6fef594be","order_by":4,"name":"Andrea Spaterna","email":"","orcid":"","institution":"University of Camerino, Matelica (MC)","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Spaterna","suffix":""},{"id":286791575,"identity":"8dde70bf-65b2-4a4a-aaaa-715a6166c7c8","order_by":5,"name":"Fulvio Laus","email":"","orcid":"","institution":"University of Camerino, Matelica (MC)","correspondingAuthor":false,"prefix":"","firstName":"Fulvio","middleName":"","lastName":"Laus","suffix":""}],"badges":[],"createdAt":"2024-02-21 08:45:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3974986/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3974986/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54178246,"identity":"cb44f2ba-ef7f-4102-bc86-66f5e96c2b41","added_by":"auto","created_at":"2024-04-05 16:12:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7616501,"visible":true,"origin":"","legend":"\u003cp\u003eCatria horses.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3974986/v1/0db7e6725ea79cae4379e1d4.png"},{"id":54178261,"identity":"d0e0c695-01b5-4dad-be3f-f1631e1993b5","added_by":"auto","created_at":"2024-04-05 16:12:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":18487374,"visible":true,"origin":"","legend":"\u003cp\u003eMouthpiece during saliva collection.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3974986/v1/7c82c263b8c44618be7e390a.png"},{"id":54178248,"identity":"b1e5af08-cd78-4d3a-81ad-2cf1de336be9","added_by":"auto","created_at":"2024-04-05 16:12:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66927,"visible":true,"origin":"","legend":"\u003cp\u003eSalivary alpha-amylase (sAA) activity, butyrylcholinesterase, and cortisol from the same horses stabled in pasture and paddock. Asterisk indicates statistically significant differences (** p\u0026lt;0.01, *** p\u0026lt;0.001) between groups.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3974986/v1/d7f924ea1931f8c0dd3089f9.png"},{"id":54179658,"identity":"c50f7dbe-68ab-4808-9666-116336bdd121","added_by":"auto","created_at":"2024-04-05 16:21:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4499545,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3974986/v1/ba3a4bb0-01b2-492b-a329-502d10042504.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Salivary analysis to unveil the paradigma of stress of domestic horses reared in the wild","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn the last few years, an increasing interest has been pointed towards the use of saliva as a diagnostic fluid both in human and veterinary medicine.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e It is well known that saliva composition can be affected by organic or psychological disorders and may reflect general metabolic changes.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e In comparison with other biological samples (e.g. blood), saliva has the advantage of being easily collected by non-invasive and non-stressful procedures, which is extremely important when sampling animals.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Recently, some salivary markers including cortisol and salivary alpha-amylase (sAA) have demonstrated the potential to make diagnosis of chronic stress or to discriminate between stress, anxiety and depressive disorders in people.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Salivary cortisol has been used in most studies for the assessment of adrenocortical response to potentially stressful situations in horses \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e so that it is recognized as an indicator of acute stress.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003eThe concentration of alpha-amylase in saliva increases abruptly following both acute and chronic stress, which makes it an important salivary biomarker of stress.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e Van Veen et al. (2008), suggested that sAA could be a potential salivary stress marker in people sensitive to a negative social assessment. Together with cortisol and sAA, salivary butyrylcholinesterase (BChol) is considered a reliable indicator of stress in equine \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e and swine\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. A recent study demonstrated as the administration of BChol can reduce stress-induced aggression in mice and is considered a candidate for treatment of anxiety in human.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEquids are prey animals, and they are inclined to live in group for safety. In order to maintain group membership and social arrangement, horses maintain their social relationship with the herd by scratching and rubbing, playing, resting, and following each other. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003eTheir prey behavior influences also feed tendency, as for other species in nature; in fact, free roaming equines spend 16\u0026ndash;20 hours a day \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e grazing large amounts of high fiber food\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. It has been reported that horses continuously living on pasture with conspecifics display a more natural expression of the species\u0026rsquo; behaviors such as feeding and social interactions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and less health impairments \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, suggesting a better welfare state than horses kept in individual boxes \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChronic stress induced by missing contact with other horses or by diseases causing pain (e.g. chronic lameness), have the potential of affecting the horse\u0026rsquo;s physical health. In this perspective, the evaluation of stress biomarkers can be relevant when assessing horse welfare, health or disease predisposition. According to some Authors, domestic horses are informative models to investigate the impact of stress on the HPA (hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal) axis, as they commonly experience conditions impairing their welfare related to social, spatial, and feeding restrictions.\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e HPA axis response to acute stress has been studied by several authors but its interpretation in cases of prolonged stress is far from straight forward. \u003csup\u003e\u003cspan additionalcitationids=\"CR23 CR24\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e It is assumed that stabled horses are more prone to be stressed and to develop stereotypic and redirected behaviors that are generally used as an indicator of poor welfare associated with stabling.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e Stress and abnormal behaviors are less likely to develop in animal that spent significantly longer time outside or that are kept in pasture.\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe present study aimed at investigating changes in salivary cortisol, sAA, and BChol, considered as affordable biomarker of stress, in domesticated horses usually living in the wild and then exposed to a potential stressful condition like a local event. The study herein described, the methods applied and results obtained are reported in accordance with ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAccording to the WAG tool, all the mares expressed the same behavioral score, resulting in grade A (good welfare) both in the wild and in paddock, irrespective of the different setting. The mouthpiece used for sampling was well tolerated by all horses (no signs of discomfort or stress were displayed during collection) and allowed an easy-going collection in all horses.\u003c/p\u003e \u003cp\u003eThe concentrations of sAA, BChol and cortisol in saliva samples collected from the same horses in the wild and in paddock are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The concentration of sAA were similar between pasture and paddock (p\u0026thinsp;=\u0026thinsp;0.7742), with a wider range (from 19.70 to 86.40 U/L) in the wild compared to paddock housing (from 27.10 to 65.50 U/L) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Butyrylcholinesterase was found to be statistically overexpressed during pasture stable and decreased in a statistically significant way when horses moved to paddock (p\u0026thinsp;=\u0026thinsp;0.0068). The same trend was registered for cortisol, for which significant higher salivary concentration was found when horses were stabled in pasture (p\u0026thinsp;=\u0026thinsp;0.0002).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDescriptive statistics for salivary biomarkers. Data are expressed in U/L, mU/mL, and ng/mL for salivary alpha-amylase (sAA), butyrylcholinesterase (BChol), and cortisol, respectively. Abbreviations: min: minimum concentration; max: maximum concentration.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003ewild\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c9\" namest=\"c6\"\u003e \u003cp\u003epaddock\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emean \u0026plusmn; sd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emin.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emax.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003erange\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003emean \u0026plusmn; sd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emin.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emax.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003erange\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003esAA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.25 \u0026plusmn; 19.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e66.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47.52 \u0026plusmn; 13.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e65.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e38.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBChol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.44 \u0026plusmn; 6.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.58 \u0026plusmn; 2.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e8.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCortisol\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e33.59 \u0026plusmn; 13.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e49.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e37.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.99 \u0026plusmn; 5.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e16.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e16.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the planning phase of the study, we assumed that the modification of horse environment from wild setting to paddock restraining could have had a direct influence on both horse behavior and stress parameters. Unexpectedly, the mares did not change any behavioral aspect when observed in collective paddocks during the event compared to wild environment.\u003c/p\u003e \u003cp\u003eA different result was obtained recently by Ruet and collaborators \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e that studied the reverse of the present study. Individual box housing is supposed to potentially compromise animal welfare: for this reason, a common practice is to offer horses a short-term period on pasture with conspecifics to alleviate the impact of long-term deprivation triggered by box shelter. When on pasture, horses increased the expression of natural behaviors such as locomotion, exploration and social behaviors which may have not been easily expressed when stabled in boxes; in addition, they did not display any stereotypies and aggressive behaviors towards humans. Interestingly, during the initial days on pasture horses were unresponsive to the environment in a significant manner than what was observed when stabled in individual boxes. After a proper period of adaptation, such behavior reverted to the level previously observed when horses were in boxes. Authors concluded that pasture can positively influence the welfare state of horses usually stabled individually, but also that several days of adaptation are needed, probably due to the novelty of the environmental and the needing to establish social conditions.\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eConversely from Ruet and collaborators conclusions, in the present study the behavioral evaluation of horses did not change when passing form wild to paddock. A possible explanation to our finding could be that the mares were moved into collective paddocks and not in single boxes, maintaining their herd.\u003c/p\u003e \u003cp\u003eDifferently from behavioral observations, objective salivary parameters of horse stress namely BChol and Cortisol significantly changed during the study, while sAA remained unchanged. Regarding sAA, it is considered a potential salivary stress marker of acute and chronic stress in people especially in those sensitive to a negative social assessment.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e In horses experimenting an acute stress, for example immediately after a sudden scare, sAA increase, to return at lower level after few minutes.\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Since in our study horses were not exposed to an acute stress, the lack of change we observed could be due to the lack of reliability of sAA in evaluating non-acute stress in horses. Butyrylcholinesterase, a serine hydrolase biochemically related to the cholinergic enzyme acetylcholinesterase, is widely distributed in the nervous system, and it seems to be involved in neural function serving as a co-regulator of cholinergic transmission. The function of BChol in saliva is not fully understood but it has been associated with acute stress in humans, rats, and pigs.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In our study, Bchol salivary concentrations were significantly higher in saliva samples collected in the wild compared to samples collected on the same mares restrained in paddocks. A similar trend was observed in salivary cortisol showing the highest mean values when the mares stayed in the wild. Salivary cortisol represents the unbound biologically active fraction of total plasma cortisol, that is mainly bound to carrier proteins, thus reflecting the hypothalamic\u0026ndash;pituitary\u0026ndash;adrenal (HPA) axis activation.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe unexpected findings of the present study can be explained by an adaptive approach called \u0026ldquo;coping strategy\u0026rdquo; that defines the behavioral and physiological struggles of animal to face a new, potentially stressful situation. The coping strategy attracts the interest of different authors dealing with applied horse welfare and behavior. \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e When a change in environment occurs, as in the present study, animals need to cope with their environment using both behavioral and physiological stress responses. Different parameters are related to such coping strategy and may be monitored over time such as changes in form and frequency of behavioral response, changes in animals-based index (such as heart rate, body temperature, hormones, neutrophils and lymphocytes ratio, and other blood indicators). In this context, the implementation and study of novel biomarkers as sAA, salivary cortisol, and Bchol that can be easily measured and may serve as indicators over time, supporting in the understanding of individual adaptive capacity to stressful events. \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eEnvironment has a major impact on horse welfare and behavior (influencing for example the chance of feeding, protection from weather condition, and sleep-awake cycle)\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e and horse may reveal specific behavior patterns to adapt to challenging situations especially in man-made environment \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe results of the present study provide new knowledge in the multidimensional approach toward horse behavior understanding, hopefully providing valuable information for specific, possibly challenging situations. Animals living in man-made environments (as paddocks in the present study), are believed to be influenced by different kinds of stressor agents that causes adaptive responses.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e On the other hand, it could also be hypothesized that although life in the wild allows horses to shows their natural behavior in freedom, at the same time this could represents a stress factor, linked to the need for managing hierarchical relationships within the herd, and the exposure to feral animals, predators, and weather changes, might explain these findings.\u003c/p\u003e \u003cp\u003eWe aimed to appraise the relationship between behavioral and physiological factors involved in the horse stress response and, surprisingly, salivary biomarkers revealed that stress level was higher in the wild.\u003c/p\u003e \u003cp\u003eIt should also be underlined that a large heterogeneity between the studies exists, because of the lack of standardized methods for measuring salivary cortisol, heterogeneity of studied populations and the bias of risk factors. In human being, for example, adolescents with severe symptoms of depression have not different concentration of salivary cortisol when compared to adolescents with mild symptoms of depression but had a significantly higher ratio of morning to evening levels of salivary cortisol.\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e This should be taken in account in future studies also in veterinary research. The findings of the present study are related to a typical Italian horse breed and the same evaluation considering different breeds/attitudes is warmly welcome, to provide more details to interpret horse behavior. Exploring coping strategies in horses and in any captive animal can be successful when a multidimensional approach including behavioral, neural, hormonal, and hematological measures is considered. Knowledge on stress and related coping styles can provide valuable information to prevent potentially challenging situations, in order to maximize animal welfare.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eAnimals and behavioral assessments\u003c/h2\u003e\n\u003cp\u003eA total of 12 clinically healthy mares were included in the study with informed owners\u0026rsquo; consent. The animals belonged to the Italian local breed Catria Horse known for its endurance, resistance, and well-balanced attitude (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Horses\u0026rsquo; mean age was 6.5\u0026plusmn;6 years, and BCS ranged from 6 to 7 out of 9 points scale\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. As inclusion criteria, all animals were domesticated and accustomed to handling before the beginning of the study, and they were maintained at pasture in the wild throughout the year. The study design consisted of behavioral evaluations and saliva collections on the same animals in the wild (usual environment) and in collective paddocks on occasion of a two-day folkloric event (Fiera del cavallo del Catria).\u003c/p\u003e\n\u003cp\u003eBehavioral indicators were scored using the Welfare Aggregation and Guidance (WAG) Tool \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e evaluating the presence of abnormal behaviors like aggressiveness, unresponsiveness to the environment, hypervigilance, etc. All assessments were performed by an experienced observer.\u003c/p\u003e\n\u003cp\u003eThe first saliva sampling was performed with the free-ranging mares in their usual habitat (wild). The second sampling was collected from the same mares moved in paddocks on occasion of a local event. To avoid any interference on studied parameters, the procedures of the second time point were performed after 48h from relocation to allow the animals to familiarize with the new condition.\u003c/p\u003e\n\u003cp\u003eSaliva samples were collected in the morning (10:00 a.m.-11:00 a.m.), using cotton swabs (Salivette\u0026reg; Sarstedt AG \u0026amp; Co., N\u0026uuml;mbrecht, Germany) inserted into a customized hollow mouthpiece that horses chewed for 5 minutes (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). After each collection, cotton swabs were removed from the mouthpiece by means of surgical clamps, cooled immediately and delivered to the laboratory within 2h. After behavioral evaluations and saliva collections, the mares were examined by an experienced clinician to verify the health conditions of each subject.\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Institutional Animal Care and Use Committee of Camerino University (Registration number: 10/2023) and were in accordance with the standards recommended by the EU Directive 2010/63/EU for experiments on animals.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eLaboratory analysis\u003c/h2\u003e\n\u003cp\u003eSaliva samples were obtained by centrifugation at 3000g for 20 minutes (Universal 32, Hettich Zentrifugen, Tuttlingen, Germany) of Salivette\u0026reg; tubes (SARSTEDT AG \u0026amp; Co. KG, N\u0026uuml;mbrecht, Germany) and 0.5 mL aliquots of saliva were stored at \u0026minus;\u0026thinsp;20 ◦C until analysis. At the day of the analysis saliva samples were thawed and centrifuged (10 min at 1000g) to eliminate debris residues. Surnatants were collected and cortisol, butyrylcholinesterase, and amylase levels were measured by using Horse Cholinesterase ELISA Kit, Horse Cholinesterase ELISA Kit, Horse Amylase ELISA kit (Bioassay Technology Laboratory), respectively, following the Manufacturer\u0026rsquo;s instructions. The absorbance was determined with Thermo Scientific Multiskan Sky amounted to 450 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism version 8.2.1 for macOS, GraphPad Software, La Jolla California USA. sAA activity, salivary butyrylcholinesterase and cortisol concentrations were checked for normality using Shapiro-Wilk test, showing sAA and cortisol normally distributed while butyrylcholinestrase was not. Then, paired t test was applied to alpha-amylase and cortisol, and Wilcoxon test to butyrylcholinesterase to assess if concentrations of salivary biomarkers were statistically different between pasture and paddock. Values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered significant.\u003c/p\u003e\n\u003cp\u003eA post hoc analysis (G*Power 3.1.9.6 for Mac OS) was performed to assess the significance level and power obtained. Including a number of twelve horses gave a significance level of \u0026alpha;\u0026thinsp;=\u0026thinsp;5% (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a power of 80%.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Authors sincerely thank AACC - Associazione Allevatori Cavallo del Catria. This paper and the research behind it would not have been possible without their exceptional support and commitment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: MB;\u003c/p\u003e\n\u003cp\u003eData curation: MB, AM;\u003c/p\u003e\n\u003cp\u003eFormal analysis: MB, AM, FLG, BP, AS, FL;\u003c/p\u003e\n\u003cp\u003eInvestigation: MB, AM, FLG, BP;\u003c/p\u003e\n\u003cp\u003eMethodology: FLG, BP;\u003c/p\u003e\n\u003cp\u003eWriting - original draft: MB, AM, FLG, BP, AS;\u003c/p\u003e\n\u003cp\u003eWriting - review \u0026amp; editing: MB, AM, FLG, BP, FL\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData generated during the study are available upon author\u0026rsquo;s request (please email to [email protected])\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eContreras-Aguilar, M. 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Relationship between condition score, physical measurements and body fat percentage in mares. \u003cem\u003eEquine Vet J\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 371\u0026ndash;372 (1983).\u003c/li\u003e\n\u003cli\u003eKubasiewicz, L. M. \u003cem\u003eet al.\u003c/em\u003e The Welfare Aggregation and Guidance (WAG) Tool: A New Method to Summarize Global Welfare Assessment Data for Equids. \u003cem\u003eAnimals 2020, Vol. 10, Page 546\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 546 (2020).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Stress, Horse, Welfare, Saliva, Cortisol, Alpha amylase, Butyrylcholinesterase","lastPublishedDoi":"10.21203/rs.3.rs-3974986/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3974986/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHorse welfare is the product of multiple factors, including behavioral and physiological adjustments to cope with stressful situation regarding environment and housing condition. Collectively, it is supposed that a horse kept in the wild has a lower level of stress than other housing system, and the aim of the present study was to investigate the level of stress in domestic horses reared in the wild and then moved to human controlled housing, through saliva analysis. Twelve clinically healthy Catria (Italian local breed) mares, usually reared in the wild, were moved into collective paddocks for a folkloric event. Saliva samples were obtained before and after the change of housing condition to evaluate stress biomarkers including salivary cortisol, salivary alpha-amylase, and butyrylcholinesterase. The mares were also scored using the Welfare Aggregation and Guidance (WAG) Tool to highlight the presence of abnormal behaviors. Despite the absence of differences in behavioral scores between wild and paddocks, salivary cortisol and butyrylcholinesterase were found to be overexpressed in the wild and statistically decreased when mares were moved to paddocks. The highest concentrations in stress biomarkers like salivary cortisol and butyrylcholinesterase in the wild was unexpected, but the need for managing hierarchical relationships, and the exposure to feral animals, predators, and weather changes, might explain these findings. The overall results of the present study may provide further knowledge toward stress response in domesticated horses living in the wild moved to human controlled housing system.\u003c/p\u003e","manuscriptTitle":"Salivary analysis to unveil the paradigma of stress of domestic horses reared in the wild","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-05 16:12:52","doi":"10.21203/rs.3.rs-3974986/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-18T07:18:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-14T14:44:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-11T14:33:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"ee92a8b3-0a9d-434c-9390-25d0b69054c3","date":"2024-04-07T07:29:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a690747d-c71a-4f4b-a776-c92f0f871470_SNPRID","date":"2024-04-07T03:40:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-06T23:21:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-05T15:26:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-02T10:10:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-02T09:42:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-02-21T08:43:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0df7d97a-5fde-4b67-8e10-283ce712f0bd","owner":[],"postedDate":"April 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":30188786,"name":"Health sciences/Biomarkers"},{"id":30188787,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2024-05-14T09:47:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-05 16:12:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3974986","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3974986","identity":"rs-3974986","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}