Acute Physiological Stress Response of Horses to Different Potential Short-Term Stressors

Accepted Manuscript Acute physiological stress response of horses to different potential short-term stressors S. Ishizaka, J. Aurich, N. Ille, C. Aurich, C. Nagel PII:

S0737-0806(16)30560-3

DOI:

10.1016/j.jevs.2017.02.013

Reference:

YJEVS 2272

To appear in:

Journal of Equine Veterinary Science

Received Date: 24 September 2016 Revised Date:

23 February 2017

Accepted Date: 26 February 2017

Please cite this article as: Ishizaka S, Aurich J, Ille N, Aurich C, Nagel C, Acute physiological stress response of horses to different potential short-term stressors, Journal of Equine Veterinary Science (2017), doi: 10.1016/j.jevs.2017.02.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Acute physiological stress response of horses to different potential short-term

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stressors

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S. Ishizaka1, J. Aurich2, N. Ille1, C. Aurich1, C. Nagel1

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Horses, Vetmeduni Vienna, 1210 Vienna, Austria

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Vienna, 1210 Vienna, Austria

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Obstetrics and Reproduction, Department of Small Animals and Horses, Vetmeduni

Corresponding author´s address

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Prof. Dr. Jörg E. Aurich

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Obstetrics and Reproduction

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Department of Small Animals and Horses

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Vetmeduni Vienna

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1210 Vienna, Austria

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Phone +43 1 25077 6401

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Fax +43 25077 5490

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E-mail [email protected]

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Artificial Insemination and Embryo Transfer, Department of Small Animals and

ACCEPTED MANUSCRIPT Abstract

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In order to avoid stress in horses, it has to be known to what extent the animals

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perceive a challenge as stressful. In this study, salivary cortisol, heart rate and heart

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rate variability (HRV) parameters SDRR (standard deviation of the beat-to-beat

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interval) and RMSSD (root mean square of successive beat-to-beat differences) were

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determined in Shetland ponies (6 stallions, 5 mares) in response to a flashlight,

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exercise without a rider, road transport and a non-treatment control. Saliva was

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collected from 1 hour before to 24 hours after the tests and cardiac activity was

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recorded from 1 hour before to 2 hours after tests. Salivary cortisol concentration

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increased in response to transport (p<0.001) and remained unchanged in response

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to exercise, flashlight and no treatment (p<0.001 among tests). Heart rate increased

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during exercise (150±7 beats/min), followed by transport (99±12 beats/min) and

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remained unchanged in response to flashlight exposure and no treatment (over time

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p<0.001, among tests p<0.001). The SDRR decreased during exercise (p<0.01 over

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time) but not flashlight and control treatment (p<0.001 among tests). Changes in

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RMSSD were similar (p<0.001) except for a lack of changes in response to the

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flashlight. The SDRR differed between mares and stallions (p<0.01). In conclusion,

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horses were not stressed by exposure to the flashlight and exercise without a rider

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while road transport was perceived as stressful. The response did not differ markedly

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between stallions and mares.

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keywords: horse; stress; transport; exercise; flashlight

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Introduction

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Exposure of horses to potentially stressful challenges is seen increasingly critical. An

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acute stressor initiates a physiological response aimed at the regain of homeostasis.

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In animals, this response can be assessed by behavioural observations and analysis

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of physiological parameters, such as the release of cortisol [1-5] and catecholamines

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[6] or changes in heart rate and heart rate variability [1,2,5,7]. Short-term stress

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increases cortisol release and non-protein-bound cortisol rapidly diffuses from blood

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into saliva. Salivary cortisol concentration thus mirrors changes of free cortisol in

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blood of horses [8]. Acute stress may also elicit a shift of the autonomous nervous

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system towards sympathetic dominance with increased epinephrine release, an

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increase in heart rate and a decrease in heart rate variability (HRV). Heart rate

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variability, i.e. short-term fluctuations of the cardiac beat-to-beat interval, reflects the

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oscillatory antagonistic influence of the sympathetic and parasympathetic branch of

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the autonomous nervous system on the sinus node of the heart. A decrease in HRV

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due to high sympathetic or low parasympathetic activity is interpreted as part of the

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stress response [9]. The adrenocortical, cardiac and behavioural components of the

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stress response, however, may be regulated in part independently.

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Although the determination of physiological stress parameters is an established

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technique, there is nevertheless considerable disagreement with regard to

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interpretation of data and comparison among studies. While some authors consider

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already small changes in cortisol release, heart rate or HRV as indicative of

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disturbances in the animals´ homoestasis, others interpret changes not exceeding

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diurnal or circannual variations as less relevant [10,11].

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ACCEPTED MANUSCRIPT Based on salivary cortisol concentration, heart rate and heart rate variability, we have

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analysed the response of horses to 3 different potential stressors: transportation by

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road, running exercise (movement without a rider) and exposure to a stationary

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flashlight. Transportation and exercise are complex stressors differing in the degree

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of physical activity requested from the animal while flashlight exposure is an

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exclusively visual stimulus. A test without any treatment was added as a control. We

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hypothesized that cortisol concentration and heart rate increase while HRV

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decreases in response to all three experimental stressors with the most pronounced

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response during and after road transport.

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Materials and methods

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Animals

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A total of 11 Shetland ponies were included into the study (6 stallions, 5 early-

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pregnant non-lactating mares, day 22.2±3.2 of pregnancy at the beginning of the

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study). They were separated by sex and kept in long-term established groups in

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paddocks with access to a stable. The ponies were fed hay twice daily, mineral

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supplements and water was freely available at all times. Age of the ponies was

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9.6±1.4 years with no significant difference between stallions (7.3±0.6 years) and

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mares (11.8±2.6 years).

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Experimental procedures

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Animals were exposed to 3 different test situations and a control treatment always

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one to two weeks apart. Tests included transportation by road (test T), running

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exercise (free enforced movement without a rider, test E) and exposure to a

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stationary flashlight (test F) and no treatment (control, test C). The order of tests was

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C, F, E and T in both stallions and mares. Twelve hours before determination of

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baseline values (60 and 30 min before beginning of tests) all horses of a group were

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brought from their paddock into an indoor group stable.

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All tests started at 0700 h. For test T, all stallions or mares of one group were loaded

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onto a commercial standard horse trailer (total area for the animals 1.7 x 3.0 meters)

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and transported over a distance of 50 km (transport time approximately 70 min). To

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obtain the same number of animals on the trailer, one cyclic mare kept in the same

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group was included into the mare transport but not included for data analysis. The

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horses were able to move freely on the trailer during transport. Saliva was collected

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at 60 min, 30 min and directly before loading, immediately after the end of transport

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and unloading and 2, 4, 6, 8, 10, 12, 18 and 24 hours thereafter. Cardiac beat-to-beat

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(RR) intervals were recorded continuously from one hour before to one hour after

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transport. For test E, all stallions or mares of one group were exercised in trot or

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canter in an indoor riding arena (size 20 x 40 meters). If necessary they were

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encouraged to move by voice or with a lunging whip but without touching the horses

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with the whip. Saliva was collected and RR intervals were recorded before, during

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and after the exercise phase as described for test T. For test F, all lights in the stable

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were turned off and the animals were exposed to a stroboscope flashlight (Stairville

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Strobe 1500 DMX PMaster Bundle, Thormann, Burgebrach, Germany) for three

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minutes. Saliva was collected and RR intervals were recorded before, during and

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after flashlight exposure as described for test T. As control (test C), ponies were

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given a one-hour rest period in the indoor group stable. Saliva was collected and RR

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intervals were determined before, during and after this test phase as described for

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test T.

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Cortisol analysis

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Saliva was collected with a cotton-based swab (Salivette cortisol, Sarstedt,

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Nümbrecht-Rommelsdorf, Germany) as described [1]. The Salivette was inserted at

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the angle of the lips into the mouth of the horse and placed gently onto the tongue for

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1 min until it was well soaked. After centrifugation for 10 min at 1000 g, 1 mL of saliva

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was aspirated, transferred into polypropylene tubes (Sarstedt) and frozen at -20 °C

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until analysis. Collection of saliva was tolerated by the horses of all groups without

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resistance. Cortisol concentration was determined with a commercial enzyme

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immunoassay validated for equine saliva in the authors` laboratory [12]. The intra-

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assay coefficient of variation determined from duplicates of a control plasma in each

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assay was 4.2% and the interassay coefficient of variation was 5.3%. The minimal

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detectable concentration defined as 2 standard deviations from zero binding was

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0.005 ng/mL.

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Heart rate and heart rate variability

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In the ponies, the cardiac RR interval was recorded with a mobile recording system

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(S810i, Polar, Kempele, Finland) set to RR interval as described elsewhere [1]. From

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the RR interval, heart rate and HRV variables, the standard deviation of RR interval

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(SDRR) and root mean square of successive RR differences (RMSSD) were

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calculated. Heart rate variability was analysed using Kubios HRV software

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(Biomedical Signal Analysis Group, Department of Applied Physics, University of

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Kuopio, Finland).To remove trend components, data were de-trended and an artefact

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correction was made as described elsewhere [1].

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For comparisons of heart rate and HRV over time and among tests, the one hour

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recording periods before and after the respective tests were divided into 1 min

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intervals. Baseline values were determined for 1 min intervals starting at 60 and 30

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min before the test. During tests E, T and C five additional 1 min recordings

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distributed evenly throughout the test period were evaluated (i.e. test E every 7.5

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min, tests T and C every 15 min). For test F, 5 subsequent 1 min intervals were

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evaluated, with the first three intervals during flashlight exposure and the other two

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intervals directly thereafter. In addition, for all tests, intervals starting at 60 and 120

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min after the end of the respective test were evaluated.

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Statistical analysis

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For statistical analysis the SPSS statistics package (version 22, IBM SPSS, Armonk,

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NY, USA) was used. Data were analysed by ANOVA using a general linear model

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(GLM) for repeated measures with test and sex as between subject factors and time

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as within subject factor. In addition, for cortisol the increase after the different tests (∆

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cortisol, peak values immediately after tests minus pre-test baseline) were compared

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by analysis of variance followed by Duncan´s test for pairwise comparisons. For

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heart rate, SDRR and RMSSD, changes from pre-test baseline values were

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calculated as the area under the curve (∆ AUC) for 5 recording intervals after the

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start of the individual test, with an increase resulting in positive and a decrease in

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negative AUC values. The ∆ AUC values were then compared by analysis of

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variance followed by Duncan´s test for pairwise comparisons. Furthermore, ∆ cortisol

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and ∆ AUC for heart rate, SDRR and RMSSD were compared between mares and

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stallions for each test by analysis of variance. For all statistical comparisons, a p-

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value below 0.05 was considered significant. Data are given as means±SEM.

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Results

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Cortisol concentration in saliva increased in response to road transport (test T) but

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not flashlight exposure, exercise and control treatment (tests F, E and C; time and

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interaction time x test p<0.001, test p=0.01; Figure 1). There was an overall

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difference in cortisol concentration between mares and stallions (p<0.05).

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Comparison between mares and stallions for individual tests revealed a small

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increase in cortisol concentration in mares and a decrease in stallions (∆ cortisol

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p<0.05 between mares and stallions) and cortisol release in response transport (test

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T) tended to be more pronounced in stallions than in mares (Figure 4a).

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Heart rate of horses increased most pronounced during test E, followed by test T

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(over time p<0,001), while heart rate did not change in response to tests F and C

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(among tests p<0.001, time x test p<0.001; Figure 2). For heart rate, no significant

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differences between male and female horses existed but time x sex interactions were

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significant (p<0.05). Heart rate calculated as area under the curve (∆ AUC) for test T

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was slightly, but significantly higher in mares than in stallions (p<0.05; Figure 4b).

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The SDRR decreased in test E and increased during test T (time p<0.05, test and

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interaction time x test p<0.001; Figure 3a). The RMSSD decreased in test E and

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remained largely unchanged during tests T, F and C (time p<0.05, test and

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interaction time x test p<0.01; Figure 3b). There was an overall difference in SDRR

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between mares and stallions with more pronounced changes in stallions than in

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mares (sex p<0.05) but no significant difference for RMSSD. Comparisons of ∆ AUC

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for each test indicated significant differences between stallions and mares neither for

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SDRR nor for RMSSD, Figure 4c and 4d).

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Discussion

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This study demonstrates differences in the response of horses to three challenges

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considered potentially stressful for the animals. Such challenges are usually

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investigated in individual studies, therefore, differences with regard to horse breed,

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history and experience of experimental animals, management and stabling often

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hinder comparisons among studies. Although the test situations in the present study

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differ in the way they affect the animal, the repetition of all three tests in the same

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horses allows direct comparisons among tests.

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Road transport is a complex, multi-factorial stressor, inducing an acute response with

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an increase in salivary cortisol concentration and heart rate, confirming previous

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studies on transport-associated stress in horses [2,4,13,14]. In horses, as in other

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species, salivary cortisol concentration follows a diurnal rhythm with highest values in

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the morning and a nadir in the late afternoon and evening [11,15,16]. To exclude any

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diurnal effect on cortisol concentration, all tests in the present study were performed

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at the same time of the day. The increase in salivary cortisol concentration during

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transport clearly exceeded the magnitude of diurnal changes detected in previous

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studies [11,16,17] and thus must be interpreted as physiologically relevant [10,11].

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An increase in heart rate of horses during group transport on a vehicle as in the

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present experiment has been described previously [2,14,18]. Both basal heart rate

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and peak heart rate during transport were slightly higher in the present study than in

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previous experiments with comparable transport duration [2,14]. This is, however,

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most probably caused by a higher physiological heart rate in Shetland ponies

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compared to full-size horses [19] and does not represent a different cardiac response

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to transport in Shetlands.

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The slight increase in the HRV variables SDRR and RMSSD during transport is in

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partial disagreement with an acute stress response which is expected to cause a

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sympathetic dominance and thus a decrease in HRV [9]. An initial increase in HRV

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has been found in some [2] but not all transport studies [14] from our group. During

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transport, factors such as decelerations and accelerations of the vehicle will lead to

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an increased overall HRV and thus may mask stress-induced HRV decreases.

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Ideally, HRV recordings in horses are made with the animal quietly standing [20].

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During transport balancing movements and changes in muscle tonus of the animals

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cannot be avoided.

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Exercise without a rider elicited a threefold increase in heart rate, a decrease in

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SDRR and RMSSD but no change in salivary cortisol concentration. The increase in

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heart is in agreement with the situation in ridden horses [1,5,7]. Although a decrease

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in HRV often indicates an increase in sympathetic tone, marked increases in heart

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rate are associated with a certain reduction in HRV also without a stress response. In

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the present study, the increase in heart rate was clearly more pronounced than the

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decrease in HRV, suggesting the absence of a major stress response during

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exercise. The lack of any increase in salivary cortisol concentration indicates that the

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adrenocortical and sympathoadrenal components of the animals` response to

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exercise are regulated independently. The increase in heart rate is thus largely

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caused by physical exercise. Findings in ponies exercised without a rider in the

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ACCEPTED MANUSCRIPT present study are in contrast to the situation in ridden horses during training or

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equestrian competitions [3,5,21,22] but also horses lunged without a rider [16]. In

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ridden and lunged horses, not only heart rate but also salivary cortisol concentration

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increased and decreases in HRV were more pronounced than during the exercise

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test of the present study. This suggests that exercising with a rider or being lunged

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by a person also induces an emotional response in horses while free exercise in a

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familiar environment does not. Because no saliva samples were taken during the

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time the horse were running, a small and transient cortisol release in the initial

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phases of running cannot be totally excluded. However, in several studies where

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horses were ridden [5,21,22] or lunged [16] salivary cortisol concentration was

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always elevated until approximately one hour after the end of riding or lunging. The

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horses running without a rider or lunge thus clearly differ with regard to their cortisol

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response even if there may have been a transient cortisol peak early during the

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running phase.

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Exposure to a stroboscope flashlight in a darkened stable did neither induce a

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detectable increase in salivary cortisol concentration or heart rate nor a decrease in

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HRV. Thus, this non-moving, visual stimulus was not perceived as a stressor by the

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horses. To the best of our knowledge, the response of horses to a stroboscope

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flashlight has not been studied previously. In different species such as guinea pigs

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[23], rats [24] and starlings [25] exposure to a flashlight induced increases in cortisol

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release and heart rate that were interpreted as a stress response. The lack of such a

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response might be due to differences in vision between horses and other species.

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The eye of the horse is especially effective for dim-light vision but is less able to

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provide information about visual detail in a stationary scene than vision in humans

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[26]. The lack of a response to flashlight exposure in the horse, i.e. a flight animal

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ACCEPTED MANUSCRIPT with a highly developed visual system, is surprising. In partial agreement with our

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findings, flashlight exposure evoked a less pronounced stress response than more

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complex stressors like the approach of a human in starlings [25]. Horses respond to

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novel objects such as an umbrella or plastic tarp [27,28] with an increase in heart

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rate and decrease in HRV. Although with the sampling interval in our study a

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delayed, transient cortisol release cannot totally be excluded, the lack of any

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accompanying changes in heart rate makes such a cortisol response extremely

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unlikely. We could recently demonstrate an increase in salivary cortisol concentration

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and transient increase in heart rate of foals directly after hot iron branding [15].

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Branding is a complex acute stressor but as flashlight exposure of extremely short

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duration.

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Cortisol concentration, heart rate and HRV remained unchanged during and after

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control treatment, demonstrating that changes seen during and after the different test

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situations were due to these experimental challenges and not fluctuations around a

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baseline throughout the day.

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The response to different challenges did not differ to a major extent between mares

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and stallions. Cortisol concentration after flashlight exposure was slightly but

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significantly higher in mares than in stallions. The overall change, however, was

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much less than during transport and neither the cortisol response to any test except

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flashlight exposure nor baseline cortisol concentration in saliva differed between

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male and female horses. This is in agreement with a recent study, analysing salivary

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cortisol concentration in horses over several months, where no sex differences in

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basal cortisol concentration existed between female and male, 1 to 3 year-old horses

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[11]. In contrast, basal cortisol concentration was higher in men than in women [29]

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and, in adult dogs, was higher in males than females [30]. Similar to the findings of

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the present study in horses, the cortisol response to acute, experimental stressors

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was comparable between men and women or slightly more pronounced in women

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[29].

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Small, test-specific differences between sexes were detected in the present study

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with regard to heart rate and HRV. During transport, the increase in heart rate was

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more pronounced in mares than in stallions. In contrast, higher heart rates in

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response to novel challenges have been demonstrated in male compared to female

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young horses [31] while increases in heart rate were not affected by sex in yearling

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horses exposed to unfamiliar humans and a handling test [32]. In the same study,

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however, behavioral differences between male and female yearling horses existed

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and males initially showed less exploratory behavior towards an unfamiliar human

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than females [32]. Age and, by inference, experience may affect an animal´s stress

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response more than its sex. In the present study, mares and stallions were not

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transported on the same days and therefore minor differences between groups may

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be due to small differences in ambient temperature, humidity of road traffic between

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days.

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In conclusion, road transport evoked an adrenocortical and cardiac stress response

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in horses. Exposure to a flashlight did not induce changes in cortisol release and

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heart rate while exercise without a rider caused changes in heart rate and heart rate

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variability compatible with physical activity but no prolonged stress response. No

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major differences in the response of male and female horses to the different

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experimental challenges existed.

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Acknowledgement

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The study was supported by a research fellowship from the OeAD, Vienna, Austria,

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to Saori Ishizaka.

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Conflict of interest statement

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None of the authors of this article has a financial or personal relationship with other

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people or organizations that could inappropriately influence or bias the content of the

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article.

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Figure captions

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Figure 1: Cortisol concentration in saliva of horses (n=11) before and after control

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treatment (test C), flashlight exposure (test F), running exercise (test E) and road

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transport (test T). Significant differences over time (p<0.001) and among tests

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(p=0.01) and significant interaction time x test (p<0.001). Different superscript letters

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in legend indicate significant differences for increase in cortisol directly after tests (∆

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cortisol), values are means ± SEM.

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Figure 2: Heart rate of horses (n=11) before, during and after control treatment (test

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C), flashlight exposure (test F, see also insert), running exercise (test E) and road

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transport (test T); horizontal lines indicate duration of tests C, E, T and F (insert),

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significant differences over time (p<0.001) and among tests (p<0.001) and significant

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interaction time x test (p<0.001); different superscript letters in legend indicate

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significant differences for changes in heart rate (∆ AUC), values are means ± SEM.

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Figure 3: (a) Standard deviation of the RR interval (SDRR) and (b) root mean square

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of successive RR deviations (RMSSD) in horses (n=11) before, during and after

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control treatment (test C), flashlight exposure (test F, see also inserts), running

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exercise (test E) and road transport (test T); horizontal lines indicate duration of tests

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C, E, T and F (inserts); for SDRR significant differences over time (p<0.05) and

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among tests (p<0.001) and significant interaction time x test (p<0.001), for RMSSD

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significant differences over time (p<0.05) and among tests (p<0.01) and significant

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interaction time x test (p<0.01), different superscript letters in legend indicate

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significant differences for changes in SDRR and RMSSD (∆ AUC), respectively,

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values are means ± SEM.

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Figure 4: (a) Increase in cortisol (∆ cortisol), and changes in (b) heart rate, (c) SDRR

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and (d) RMSSD calculated as area under the curve (∆ AUC) in mares (n=5) and

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stallions (n=6) in response to control treatment (test C), flashlight exposure (test F),

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running exercise (test E) and road transport (test T), *significant differences between

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mares and stallions, p<0.05), values are means ± SEM.

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The response to transport, exercise and flashlight was determined in ponies.

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