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Topic:Salivary Cortisol

Salivary cortisol is a biomarker used to assess stress and physiological responses in horses. It is a glucocorticoid hormone produced by the adrenal cortex and is released into the bloodstream in response to stressors, with measurable levels detectable in saliva. The non-invasive collection of saliva makes it a practical method for evaluating cortisol levels in equine studies. Salivary cortisol is utilized in research to explore stress-related conditions, welfare assessments, and the effects of training or environmental changes on horses. This page compiles peer-reviewed research studies and scholarly articles that investigate the measurement, variability, and implications of salivary cortisol in equine health and behavior.
Salivary alpha-amylase activity and concentration in horses with acute abdominal disease: Association with outcome.
Equine veterinary journal    February 7, 2019   Volume 51, Issue 5 569-574 doi: 10.1111/evj.13066
Contreras-Aguilar MD, Martínez-Subiela S, Cerón JJ, Martín-Cuervo M, Tecles F, Escribano D.Salivary biomarkers could be useful to objectively evaluate critical illness and prognosis for survival in horses with acute abdominal disease. Objective: To compare salivary alpha-amylase (sAA) activity and concentration in healthy horses and horses with acute abdominal disease, and evaluate the association between sAA activity and concentration with disease severity and outcome. Methods: A prospective cohort. Methods: sAA activity, measured using a colorimetric commercial kit, and concentration, measured using a Time-resolved immunofluorometric assay, in 25 healthy horses and in 33 horses wi...
Replication Pilot Trial of Therapeutic Horseback Riding and Cortisol Collection With Children on the Autism Spectrum.
Frontiers in veterinary science    January 14, 2019   Volume 5 312 doi: 10.3389/fvets.2018.00312
Pan Z, Granger DA, Guérin NA, Shoffner A, Gabriels RL.We aimed to determine whether results of our prior randomized control trial [RCT; NCT02301195, (1)] of Therapeutic Horseback Riding (THR) for children and adolescents with autism spectrum disorder (ASD) could be at a different riding center and if treatment effects also included differences in the expression of associations between problem behavior and the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Participants with ASD ( = 16) ages 6-16 years were randomized by nonverbal intelligence quotient to either a 10-week THR group ( = 8) or no horse interaction barn activity (BA) cont...
Adolescents’ Affective and Physiological Regulation Shape Negative Behavior During Challenging Equine Assisted Learning Activities.
Frontiers in veterinary science    December 4, 2018   Volume 5 300 doi: 10.3389/fvets.2018.00300
Pendry P, Carr AM, Vandagriff JL.This study examined associations between adolescents' ( = 59; = 11.63) diurnal and momentary activity of the Hypothalamic Pituitary Adrenal (HPA) axis as marked by salivary cortisol, and affective and behavioral responses to their first, mounted equine assisted learning (EAL) activity. The introduction to riding occurred during the fifth week of an 11-week EAL program for at-risk and typically developing adolescents. Before the 11-week program began, participants collected 6 salivary cortisol samples at prescribed times (wakeup, 4 p.m., bedtime) over 2 days, from which indices of diurnal cor...
Efficacy of equine-assisted psychotherapy in veterans with posttraumatic stress disorder.
Journal of integrative medicine    November 16, 2018   Volume 17, Issue 1 14-19 doi: 10.1016/j.joim.2018.11.001
Burton LE, Qeadan F, Burge MR.Posttraumatic stress disorder (PTSD) is a common and debilitating disorder among war veterans. Although complementary and alternative therapies are gaining acceptance in the treatment of PTSD, the efficacy of animal-based therapies in this disorder is unknown. The goal of equine-assisted psychotherapy (EAP) is to improve the social, emotional, and/or cognitive functions of individuals with PTSD. Objective: This study aims to explore the effects of EAP on PTSD symptoms. We hypothesized that veterans with PTSD who participate in a standardized EAP program for 1 h per week for 6 weeks would exp...
Bone Marrow Aspiration Does Not Induce a Measurable Pain Response Compared to Sham Procedure.
Frontiers in veterinary science    October 1, 2018   Volume 5 233 doi: 10.3389/fvets.2018.00233
Rowland AL, Navas de Solis C, Lepiz MA, Cummings KJ, Watts AE.Bone marrow is commonly collected from horses for regenerative medicine applications. Little information is available regarding pain experienced by the horse during bone marrow aspiration. The objective of this study was to characterize horse reaction and pain response during bone marrow aspiration (BMA) compared to a sham (SHAM) procedure. We hypothesized there would be significantly greater horse reaction or pain response measured by salivary cortisol, heart rate variability, and depth and duration of sedation between BMA and SHAM. Twelve university owned horses underwent a BMA and sham proc...
Progressive habituation to separation alleviates the negative effects of weaning in the mother and foal.
Psychoneuroendocrinology    July 5, 2018   Volume 97 59-68 doi: 10.1016/j.psyneuen.2018.07.005
Lansade L, Foury A, Reigner F, Vidament M, Guettier E, Bouvet G, Soulet D, Parias C, Ruet A, Mach N, Lévy F, Moisan MP.Early and definitive separation between offspring and their mothers has negative consequences on behavioral and physiological responses. This study compared sudden and definitive weaning (Sudd group, N = 16) and weaning involving progressive habituation to separation using a fence line during the month preceding definitive separation (Prog group, N = 18). The impact of these two methods was assessed in both foals and their mothers through behavioral and biological parameters, including salivary cortisol, telomere length and blood transcriptomes. On the day of definitive separation, Pro...
Diagnostic accuracy of post-ACTH challenge salivary cortisol concentrations for identifying horses with equine glandular gastric disease.
Journal of animal science    May 16, 2018   Volume 96, Issue 6 2154-2161 doi: 10.1093/jas/sky074
Sauer FJ, Bruckmaier RM, Ramseyer A, Vidondo B, Scheidegger MD, Gerber V.The aims of this study were to better characterize the adrenal response to i.v. adrenocorticotropic hormone (ACTH) in horses with and without gastric disease and to validate and simplify the ACTH stimulation test by determining the diagnostic accuracy of six consecutive sampling time points after ACTH administration for equine glandular gastric disease (EGGD) and equine squamous gastric disease (ESGD). Twenty-six endurance and eventing horses without clinical disease [Sport Horse Population (SHP)] and an independent population of 62 horses [General Population (GP)] were grouped by gastroscopic...
Free and bound cortisol in plasma and saliva during ACTH challenge in dairy cows and horses.
Journal of animal science    January 30, 2018   Volume 96, Issue 1 76-84 doi: 10.1093/jas/skx008
Schwinn AC, Sauer FJ, Gerber V, Bruckmaier RM, Gross JJ.Cortisol levels reflect hypothalamic-pituitary-adrenocortical (HPA) axis activity. While most plasma cortisol is supposed to be bound to the soluble corticosteroid-binding globulin (CBG), only free cortisol (FC) actively regulates metabolic and immunological processes. We aimed to establish a multispecies suitable method to assess FC in cows and horses which in combination with total cortisol (TC) allows interpreting proportional changes of cortisol in saliva as well as in blood in response to a standardized HPA axis activation via ACTH. We further investigated if the ratios of cortisol fracti...
Effect of butorphanol, midazolam or ketamine on romifidine based sedation in horses during standing cheek tooth removal.
BMC veterinary research    December 6, 2017   Volume 13, Issue 1 381 doi: 10.1186/s12917-017-1299-6
Müller TM, Hopster K, Bienert-Zeit A, Rohn K, Kästner SBR.Standing surgery, especially dental procedures, are commonly performed in horses. This leads to an increasing demand for reliable sedation protocols. Therefore, it was the purpose of this study to investigate the influence of butorphanol, midazolam or ketamine on romifidine based sedation in horses during cheek tooth removal. Methods: Forty horses presented for tooth extraction were divided in four groups using matched pair randomization. Group R was sedated with romifidine (bolus 0.03 mg/kg, followed by a constant rate infusion (CRI) 0.05 mg/kg/h) and group RB with romifidine (same dose) an...
Increased adrenocortical response to adrenocorticotropic hormone (ACTH) in sport horses with equine glandular gastric disease (EGGD).
Veterinary journal (London, England : 1997)    September 11, 2017   Volume 228 7-12 doi: 10.1016/j.tvjl.2017.09.002
Scheidegger MD, Gerber V, Bruckmaier RM, van der Kolk JH, Burger D, Ramseyer A.This study tested the hypothesis that adrenocortical function would be altered in horses with equine gastric ulcer syndrome (EGUS). Twenty-six sport horses competing at national or international levels in eventing (n=15) or endurance (n=11) were subjected to a gastroscopic examination and an adrenocorticotropic hormone (ACTH) stimulation test. Salivary cortisol concentrations were measured before (baseline) and after (30, 60, 90, 120 and 150min) IV ACTH injection (1μg/kg bodyweight). Within EGUS, two distinct diseases, equine squamous gastric disease (ESGD) and equine glandular gastric diseas...
Effect of housing system on reproductive behaviour and on some endocrinological and seminal parameters of donkey stallions.
Reproduction in domestic animals = Zuchthygiene    August 14, 2017   Volume 53, Issue 1 40-47 doi: 10.1111/rda.13050
Rota A, Sgorbini M, Panzani D, Bonelli F, Baragli P, Ille N, Gatta D, Sighieri C, Casini L, Maggiorelli MM, Aurich C, Camillo F.Reproductive management of male donkeys employed for artificial breeding has been poorly studied. The aim of this study was to evaluate the effect of housing system, with the animals grouped together in a paddock or kept in individual boxes, on sexual behaviour, cortisol and testosterone concentration and seminal characteristics of adult male donkeys. The study included four Amiata donkey jacks (stallions) from which ejaculates, saliva and blood were collected during two distinct 3 weeks periods, one in the group and one in the box housing system. Time needed for semen collection was shorter ...
The Effects of Weaning Methods on Gut Microbiota Composition and Horse Physiology.
Frontiers in physiology    July 25, 2017   Volume 8 535 doi: 10.3389/fphys.2017.00535
Mach N, Foury A, Kittelmann S, Reigner F, Moroldo M, Ballester M, Esquerré D, Rivière J, Sallé G, Gérard P, Moisan MP, Lansade L.Weaning has been described as one of the most stressful events in the life of horses. Given the importance of the interaction between the gut-brain axis and gut microbiota under stress, we evaluated (i) the effect of two different weaning methods on the composition of gut microbiota across time and (ii) how the shifts of gut microbiota composition after weaning affect the host. A total of 34 foals were randomly subjected to a progressive (P) or an abrupt (A) weaning method. In the P method, mares were separated from foals at progressively increasing intervals every day, starting from five min ...
The effects of intrauterine infusion of peanut oil on endometrial health, salivary cortisol and interovulatory period in mares.
Theriogenology    July 13, 2017   Volume 102 116-125 doi: 10.1016/j.theriogenology.2017.07.009
Campbell MLH, Hampshire D, Hamstead LE, Rose BV, Smith KC, de Mestre AM.Intrauterine infusion of peanut oil at Day 10 post-ovulation has been reported to prolong dioestrus in mares. However, the effects of peanut oil treatment on the endometrium and whether the technique is painful have not been assessed. The objectives of this study were, (i) to determine the effect of intrauterine infusion of peanut oil on endometrial health, (ii) to determine whether use of intrauterine peanut oil is painful and (iii) to confirm that peanut oil causes prolonged dioestrus. Six mares aged 3-12 years old were used in a cross-over design with each mare administered both 1 ml of in...
An exploratory study of competition scores and salivary cortisol concentrations in Warmblood horses.
Domestic animal endocrinology    June 28, 2017   Volume 61 108-116 doi: 10.1016/j.domaniend.2017.06.007
Munk R, Jensen RB, Palme R, Munksgaard L, Christensen JW.The main objective of this explorative study was to describe the relationship between competition scores and salivary cortisol concentrations in young horses during dressage and showjumping competitions. The study also investigated whether the diurnal rhythm of salivary cortisol concentrations was affected by competition over consecutive days compared with the home environment. Saliva samples were collected from 126 dressage horses and showjumpers in their home environment and at 3 different events. The relationship between scores given by judges at the competition and cortisol concentrations ...
Use of fecal glucocorticoid and salivary cortisol concentrations as a measure of well-being of New York City carriage horses.
Journal of the American Veterinary Medical Association    January 25, 2017   Volume 250, Issue 3 316-321 doi: 10.2460/javma.250.3.316
Mercer-Bowyer S, Kersey DC, Bertone JJ.OBJECTIVE To use noninvasive approaches to assess stress in New York City (NYC) carriage horses during the course of their daily routine to determine whether use of these horses affected their well-being. DESIGN Prospective case control study. ANIMALS 13 (5 mares and 8 geldings) stabled working NYC carriage horses and 5 pastured (nonworking) NYC carriage horses (1 mare and 4 geldings). PROCEDURES Samples for determination of fecal glucocorticoid and salivary cortisol concentrations were collected on 3 successive days from 10, 8, and 9 working carriage horses during rest (time 1), preparation f...
Oxytocin treatment does not change cardiovascular parameters, hematology and plasma electrolytes in parturient horse mares.
Theriogenology    December 24, 2016   Volume 91 69-76 doi: 10.1016/j.theriogenology.2016.12.028
Nagel C, Trenk L, Wulf M, Ille N, Aurich J, Aurich C.In mares, foaling is associated with changes in hematology, plasma electrolytes, blood pressure and heart rate and it has been hypothesized that these are induced by oxytocin. To test this hypothesis, mares (n = 8-14/group) were treated with oxytocin (OT; 20 I.U.) or saline (CON) at 1 h (test A) and 12 h after foaling (test B) and during first postpartum diestrus (test C). Heart rate, heart rate variability (HRV), atrioventricular blocks, salivary cortisol concentration, blood pressure, plasma electrolytes and blood count were determined. Heart rate decreased from test A to C (P < 0.0...
Influence of transvaginal ultrasound-guided follicular punctures in the mare on heart rate, respiratory rate, facial expression changes, and salivary cortisol as pain scoring.
Theriogenology    June 6, 2016   Volume 86, Issue 7 1757-1763 doi: 10.1016/j.theriogenology.2016.05.040
Diego R, Douet C, Reigner F, Blard T, Cognié J, Deleuze S, Goudet G.Transvaginal ultrasound-guided follicular punctures are widely used in the mare for diagnosis, research, and commercial applications. The objective of our study was to determine their influence on pain, stress, and well-being in the mare, by evaluating heart rate, breath rate, facial expression changes, and salivary cortisol before, during, and after puncture. For this experiment, 21 pony mares were used. Transvaginal ultrasound-guided aspirations were performed on 11 mares. After injections for sedation, analgesia, and antispasmodia, the follicles from both ovaries were aspirated with a needl...
Validation of a novel saliva-based ELISA test for diagnosing tapeworm burden in horses.
Veterinary clinical pathology    May 24, 2016   Volume 45, Issue 2 335-346 doi: 10.1111/vcp.12364
Lightbody KL, Davis PJ, Austin CJ.Tapeworm infections pose a significant threat to equine health as they are associated with clinical cases of colic. Diagnosis of tapeworm burden using fecal egg counts (FECs) is unreliable, and, although a commercial serologic ELISA for anti-tapeworm antibodies is available, it requires a veterinarian to collect the blood sample. A reliable diagnostic test using an owner-accessible sample such as saliva could provide a cost-effective alternative for tapeworm testing in horses, and allow targeted deworming strategies. Objective: The purpose of the study was to statistically validate a saliva ta...
Repeatability of the ACTH stimulation test as reflected by salivary cortisol response in healthy horses.
Domestic animal endocrinology    May 12, 2016   Volume 57 43-47 doi: 10.1016/j.domaniend.2016.04.002
Scheidegger MD, Gerber V, Ramseyer A, Schüpbach-Regula G, Bruckmaier RM, van der Kolk JH.The aim of this study was to further characterize the ACTH stimulation test as reflected by salivary cortisol response and to measure the short- and long-term repeatability of it in healthy horses as a tool to assess the capacity of the adrenal cortex to secrete cortisol. Nineteen healthy horses were subjected to 3 ACTH stimulation tests. Intervals were 2 wk and 5 mo between the first and second and the second and third tests, respectively. A dose of 1-μg/kg BW synthetic ACTH was injected intravenously. Saliva samples were collected at baseline and at 30, 60, 90, 120, 150, and 180 min afte...
Influence of Horse and Rider on Stress during Horse-riding Lesson Program.
Asian-Australasian journal of animal sciences    March 22, 2016   Volume 29, Issue 6 895-900 doi: 10.5713/ajas.15.1068
Kang OD, Yun YM.The present study aims to confirm the influence of a horse-riding lesson program (HRLP) on the stress level of horses and riders by respectively analyzing their salivary cortisol concentration. Twenty-four healthy horses and 23 riders participated in this study. The horses were randomly classified into two groups for the horse riding lesson program: Class 1 (for the beginner lesson) and Class 2 (for the intermediate lesson). The Class 1 group consisted of 12 horses and 12 riders, while the Class 2 group consisted of 12 horses and 11 riders. Salivettes cotton wool swabs were used for saliva col...
Changes in Salivary Cortisol Concentration in Horses during Different Types of Exercise.
Asian-Australasian journal of animal sciences    March 4, 2016   Volume 29, Issue 5 747-752 doi: 10.5713/ajas.16.0009
Kang OD, Lee WS.This study aimed to estimate the change of stress level in horses based on cortisol concentration levels in their saliva. A total of 61 horses were divided into the following three groups: i) tourist riding experience (TR, n = 23); ii) resting group (RR, n = 14); and iii) horse-riding education (ER, n = 24). The saliva samples of TR and ER groups were taken using plain cotton Salivettes four times a day: at 07:00 (basal), 11:00 (Exercise 1, after 1-hour exercise in the morning), 14:00 (Exercise 2, after 1-hour exercise in the afternoon), and 16:00 (Exercise 3, after 1-hour exercise in the afte...
The PGF2α agonists luprostiol and d-cloprostenol reliably induce luteolysis in luteal phase mares without evoking clinical side effects or a stress response.
Animal reproduction science    March 2, 2016   Volume 168 92-99 doi: 10.1016/j.anireprosci.2016.02.031
Kuhl J, Nagel C, Ille N, Aurich JE, Aurich C.In the present study we have evaluated a possible stress reaction in response to two different PGF2α analogs-luprostiol and D-cloprostenol--and their effects on estrous cycle characteristics. In a cross-over-design eight mares received in alternating order either luprostiol (Treatment LUP; 3.75 mg im), D-cloprostenol (Treatment CLO; 22.5μg im) or saline (Treatment CON; NaCl 0.9% 0.5ml im) on day 8 after ovulation. Injection of either LUP or CLO, but not of CON resulted in a significant decline of progesterone concentration in plasma to baseline concentrations within two days (time: p<0.00...
Sialolithiasis in horses: A retrospective study of 25 cases (2002-2013).
The Canadian veterinary journal = La revue veterinaire canadienne    December 15, 2015   Volume 56, Issue 12 1239-1244 
Carlson N, Eastman T, Winfield L.This study evaluated outcomes of surgical treatment of equine sialolithiasis, highlighting cases involving the proximal parotid salivary duct. Sialoliths in the proximal parotid duct were difficult to identify radiographically and more frequently associated with draining tracts and sialadenitis compared with sialoliths in the distal parotid duct. Ultrasonography confirmed the diagnosis of sialolithiasis in all cases in which there was no radiographic evidence of a sialolith. All cases of proximal parotid duct sialolithiasis required transcutaneous removal. A longer duration of illness was obse...
Stress Response of Veterinary Students to Gynaecological Examination of Horse Mares – Effects of Simulator-Based and Animal-Based Training.
Reproduction in domestic animals = Zuchthygiene    August 26, 2015   Volume 50, Issue 5 866-871 doi: 10.1111/rda.12600
Nagel C, Ille N, Erber R, Aurich C, Aurich J.Invasive procedures in animals are challenging for veterinary students who may perceive a gynaecological examination of mares as stressful. Simulator-based training may reduce stress. In this study, students received equine gynaecology training 4 times either on horses (group H; n = 14) or a teaching simulator (group SIM; n = 13). One day and 14 days thereafter, their diagnostic skills were tested on horses (skills tests 1 and 2). During the skills tests, the students' stress response was analysed by heart rate, heart rate variability (HRV) parameters SDRR (standard deviation of beat-to-b...
Presence and distribution of leptin and its receptor in the minor salivary glands of the donkey.
Acta histochemica    March 20, 2015   Volume 117, Issue 3 305-308 doi: 10.1016/j.acthis.2015.03.001
Dall'Aglio C, Bazzucchi C, Mercati F, Ceccarelli P.Leptin is a hormone widely diffused in the mammalian body in which it plays functions that go far beyond control of appetite and energy metabolism. The finding that it is present in the major salivary glands of various animal species is of interest for the functional implications that it may imply. Since there are no data on the presence of leptin and its receptor in the minor salivary glands, the aim of this study was to demonstrate their presence and distribution in such glands of donkeys. This latter was chosen as species of reference because the monogastric herbivore shows intense salivati...
Increased cortisol release and transport stress do not influence semen quality and testosterone release in pony stallions.
Theriogenology    February 19, 2015   Volume 84, Issue 1 70-75 doi: 10.1016/j.theriogenology.2015.02.015
Deichsel K, Pasing S, Erber R, Ille N, Palme R, Aurich J, Aurich C.The use of breeding stallions for equestrian competitions requires that fertility is not negatively affected by competition or transport to the competition site. In this study, effects of cortisol release induced by road transport (600 km), adrenocorticotropic hormone (ACTH) administration (3 × 0.5 mg synthetic ACTH) and placebo treatment on semen quality and testosterone release were investigated in Shetland stallions (N = 13) using a crossover design. Saliva for cortisol and blood for testosterone analysis were collected for 10 weeks after treatments. Semen was collected daily for 5 days di...
Effects of season, age, sex, and housing on salivary cortisol concentrations in horses.
Domestic animal endocrinology    January 21, 2015   Volume 52 11-16 doi: 10.1016/j.domaniend.2015.01.003
Aurich J, Wulf M, Ille N, Erber R, von Lewinski M, Palme R, Aurich C.Analysis of salivary cortisol is increasingly used to assess stress responses in horses. Because spontaneous or experimentally induced increases in cortisol concentrations are often relatively small for stress studies, proper controls are needed. This requires an understanding of the factors affecting salivary cortisol over longer times. In this study, we have analyzed salivary cortisol concentration for 6 mo in horses (n = 94) differing in age, sex, reproductive state, and housing. Salivary cortisol followed a diurnal rhythm with the highest concentrations in the morning and a decrease throug...
Heart rate and salivary cortisol concentrations in foals at birth.
Veterinary journal (London, England : 1997)    November 27, 2014   Volume 203, Issue 2 250-252 doi: 10.1016/j.tvjl.2014.11.013
Nagel C, Erber R, Ille N, Wulf M, Aurich J, Möstl E, Aurich C.Heart rate (HR), HR variability (HRV) and salivary cortisol concentrations were determined in foals (n = 13) during the perinatal phase and until 5 months of age. In the fetus, HR decreased from 77 ± 3 beats/min at 120 min before birth to 60 ± 1 beats/min at 5 min before birth (P <0.01). Within 30 min of birth, HR increased to 160 ± 9 beats/min (P <0.01). Salivary cortisol concentrations immediately after birth were 11.9 ± 3.6 ng/mL and within 2 h increased to a maximum of 52.5 ± 12.3 ng/mL (P <0.01). In conclusion, increases in HR and sa...
Influence of transrectal and transabdominal ultrasound examination on salivary cortisol, heart rate, and heart rate variability in mares.
Theriogenology    November 13, 2014   Volume 83, Issue 4 749-756 doi: 10.1016/j.theriogenology.2014.11.010
Schönbom H, Kassens A, Hopster-Iversen C, Klewitz J, Piechotta M, Martinsson G, Kißler A, Burger D, Sieme H.Pregnancy diagnostics in equine reproduction are routinely performed using transrectal ultrasonography, although it is also possible to visualize the fetus by transabdominal ultrasound examinations from the 90th day of gestation onward. We hypothesized that ultrasound examinations may stress the mare and that the gestational stage status and lactation may influence the mare's stress reaction. To investigate the stress reaction, 25 thoroughbred mares of different age, pregnancy and lactational status underwent a transrectal examination. In pregnant mares, an additional transabdominal examinatio...
Effect of different head and neck positions on behaviour, heart rate variability and cortisol levels in lunged Royal Dutch Sport horses.
Veterinary journal (London, England : 1997)    July 16, 2014   Volume 202, Issue 1 26-32 doi: 10.1016/j.tvjl.2014.07.005
Smiet E, Van Dierendonck MC, Sleutjens J, Menheere PP, van Breda E, de Boer D, Back W, Wijnberg ID, van der Kolk JH.Different head-and-neck positions (HNPs) are discussed in relation to potential welfare issues. To evaluate the effect on welfare, seven Royal Dutch Sport horses were studied in five predetermined HNPs: (1) unrestrained (HNP1); (2) neck raised, bridge of nose around the vertical (HNP2); (3) neck lowered and considerably flexed, bridge of nose pointing towards the chest (HNP4); (4) neck raised and extended, bridge of nose in front of the vertical (HNP5), and (5) neck lowered and flexed, bridge of nose pointing towards the carpus (HNP7). A standardised exercise test (SET) of 34 min consisted o...