Effects of the method of restraint for shearing on behaviour and heart rate variability in alpacas

Journal Pre-proof Effects of the method of restraint for shearing on behaviour and heart rate variability in alpacas Susanne Waiblinger, Franziska Hajek, Bianca Lambacher, Thomas Wittek

PII:

S0168-1591(19)30187-X

DOI:

https://doi.org/10.1016/j.applanim.2019.104918

Reference:

APPLAN 104918

To appear in:

Applied Animal Behaviour Science

Received Date:

12 March 2019

Revised Date:

12 October 2019

Accepted Date:

17 November 2019

Please cite this article as: Waiblinger S, Hajek F, Lambacher B, Wittek T, Effects of the method of restraint for shearing on behaviour and heart rate variability in alpacas, Applied Animal Behaviour Science (2019), doi: https://doi.org/10.1016/j.applanim.2019.104918

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Effects of the method of restraint for shearing on behaviour and heart rate variability in alpacas

Susanne Waiblinger*1, Franziska Hajek1, Bianca Lambacher2, Thomas Wittek2

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Institute of Animal Welfare Science, Department for Farm Animals and Veterinary Public

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Health, University of Veterinary Medicine, Vienna (Vetmeduni Vienna) Veterinaerplatz 1, 1210 Vienna, Austria

*corresponding author:

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Tel: +43 1 25077 4905

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Fax: +43 1 25077 4990

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E-mail: address: [email protected]

Highlights

We compared three methods of restraint for Alpaca with and without shearing

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Alpaca were restrained standing or tied up while lying on the ground or a table

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Many animals screamed continuously during shearing in all methods of restraint

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Manual restraint while standing seems to be the least aversive method

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Abstract Alpacas are increasingly kept in Europe for different purposes including fibre production. Yearly shearing is necessary to harvest fibre and for welfare reasons. Different methods of restraint are used during shearing, which may affect the welfare of the animals differently. The aim of the study was to compare three common restraint methods: Standing, the animal is standing and held by hand; Ground, the animal is laid down and its legs are tied up and stretched out by ropes; Table, the animal is restrained on a special table, legs are tied up by

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ropes. We recorded behaviour continuously during restraint and, afterwards, at 10-min intervals from 30 to 120 min in alpacas sheared during restraint (ExpRS, 45 animals, 15 animals per treatment) or only restrained (ExpR, 15 animals, repeated measures). HRV and HR was measured in ExpR.

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Large individual variation was found, especially in ExpRS. One Standing animal in ExpRS

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could not be shorn due to strong defence reactions. During restraint, fewer Standing animals vocalised in ExpRS (p=0.07) and showed least flinching in both experiments (p<0.05), but

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showed leg movements (such as stepping, kicking, leg twitches, escape attempts) more often (p<0.001). After restraint, Standing animals showed or tended to show more lying, ruminating (ExpRS) or feeding (ExpR) and less agonistic (ExpRS) or affiliative interactions (ExpR) (p<0.05

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to p<0.1 for all), although both tie up treatments did not always differ. Accordingly, HRV (SDNN) was higher in Standing compared to Table. Differences between Ground and Table

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were rare, in contrast to Ground all Table animals vocalised during shearing; in ExpR Table animals showed more often flinching during restraint (both p<0.05) and tended to move legs

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and to vocalise more compared to Ground. In sum results indicate that Standing is the least aversive method with the shortest duration of the whole procedure and thus should be preferably used. However, shearing can induce strong reactions in some of the animals independent from treatment which may preclude shearing in those animals when standing. Habituation to handling procedures should be used to reduce stress and avoid the necessity for more invasive restraint. Effects of Ground and Table might depend on the exact body position which should be investigated further. 2

Keywords:

South American camelids, handling, restraint, shearing, stress, heart rate

variability

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Introduction

Alpacas (Vicugna pacos) were domesticated in South America around 6000 years ago and

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still play an important role for the Andean population not only for producing economically valuable fibre but as well for meat and dung for fuel (Wheeler 2012). Recently numbers of Alpacas have increased in Europe, where they are kept for fibre or breeding as well as for use

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in animal-assisted intervention or just for hobby (Gauly 2011 , Lambacher et al. 2015). Yearly shearing is a necessary management procedure not only to harvest fibre but also to reduce

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the risk of heat stress during summer (Navarre et al. 2001, Gerken 2010). Different methods of restraint are used during shearing in Europe (Wiede 2014): animals may be left standing

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and restrained manually, with or without a halter, or in a special chute for shearing; animals may be laid down on the ground (on a soft mat) and either restrained manually or tied up in a

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stretched-out position by ropes fixed at the front and hind legs; or animals may be restrained on a shearing table again by stretching them out with ropes at the legs. These restraint

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methods likely affect stress and welfare of the animals differently. The methods that involve tying up and stretching by ropes have been especially criticized as eliciting suffering (and

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potentially pain) and thus to conflict with animal welfare law in Germany (Wiede 2014). To our knowledge, no study has previously investigated reactions of Alpacas, kept by humans, to shearing or to different methods of restraint during shearing. There are only studies on reactions of wild South American camelids, guanacos (Lama guanicoe) and vicunas (Vicugna vicugna), to being caught and handled for shearing to harvest fibre. Behavioural and physiological stress reactions were affected by the duration of handling (Carmanchahi et al.

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2011, Taraborelli et al. 2017) or the exact way of catching, with higher levels if vehicles were involved as compared to only people driving the animals into the trap (Arzamendia et al. 2010). The aim of our study was to compare short-term behavioural and physiological reactions of Alpaca to three common restraint methods, held manually while Standing, or lying with legs tied up on the Ground or Table. We performed two experiments: in the first experiment the animals were shorn, while in the second experiment animals experienced only restraint. Physiological parameters, specifically salivary and faecal cortisol have been evaluated previously (Wittek et al. 2017). In this paper we present the behavioural effects of the restraint

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methods, with and without shearing, during and up to 2 hours after the procedure. We also present effects of restraint on heart rate variability, which was assessed in the second experiment to gain more insight into effects on the autonomic nervous system. We expected

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the fewest signs of stress in the Standing treatment, whether individuals were shorn or not. Because Alpaca often lay down on the ground as a natural defence, we expected Ground to

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elicit less stress than Table. Behavioural signs of stress in this study included more frequent vocalisations and stronger escape and defence reactions, and physiological signs of stress

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included decreases in heart rate variability. Two hours after restraint we expected higher stress to be reflected by less feeding and rumination, less lying, and a higher level of social

Methods

Animals and experimental design

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interactions.

Three methods used to restrain animals during shearing were compared: (i)

Standing, the animal is standing and held by hand;

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Ground, the animal is laid down on the ground and its legs are tied up and stretched out by ropes;

(iii)

Table, the animal is restrained on a special table, and again, its legs are tied up and stretched out by ropes.

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We investigated animals’ reactions to restraint in two experiments: either when being restrained and shorn (Restraint plus Shearing) or when being restrained only without shearing (Restraint alone). These two experiments were performed on two different commercial farms, thus with different animals. Consequently, comparisons were performed only within experiments. Experiment RS – Restraint plus Shearing In ExpRS animals were restrained with one of the methods while being sheared. ExpRS was conducted in May 2015 on three consecutive days on a commercial farm in Piesig, Germany,

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that keeps a total of ca. 160 alpacas. Animals were kept outdoor on pasture with shelter all year round. 45 healthy, female alpaca (age 1-11 years, mean±SD: 6.1±3.29 years) were randomly selected from all female animals except those in the final trimester of pregnancy and

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were randomly allocated to treatments (age in the different treatments, mean±SD, range: Standing, 5.5±2.76, 1.8 – 11.6; Ground, 5.8±3.2, 1.0 – 11.6; Table, 6.9±3.8, 1.2 – 11.6ver). All

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animals except two yearlings (one Ground, one Table) had been sheared by the usual procedure of the farm (i.e. restraint on ground, see 3.2.1 for more details) one or several times

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the years before. Fifteen animals were sheared per day, five animals per treatment in a block, resulting in a sample size of 15 animals per treatment, with order of treatments balanced over

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days (day 1: Ground, Table, Standing; day 2: Table, Standing, Ground; day 3: Standing, Ground, Table). Animals were bred on the farm (origin: own breeding) or purchased (own

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breeding / purchased: Standing 7 / 7; Ground 6 / 9, Table 7 / 8). Experiment R – Restraint alone

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In ExpR animals were only restrained. ExpR was conducted in June 2015 on a commercial farm in Tauplitz, Austria that keeps a total of 88 alpaca. Six castrated and nine uncastrated male alpaca from two herds (five uncastrated males from one herd, the others from the second herd) were selected randomly (age 1-8 years, mean±SD: 3,8±2,48 years). Animals from these two herds were kept separately throughout the experiment. All animals had been sheared by the usual procedure of the farm (restraint on ground) three weeks before ExpR started. A within-subject design was adopted, i.e. the 15 animals were subjected to each of the three 5

restraint methods consecutively with a one-week delay in between; order of treatment was balanced, i.e. 5 animals started with Ground, 5 with Table and 5 with Standing. The study was discussed and approved by the institutional ethics and animal welfare committee in accordance with Good Scientific Practice (GSP) guidelines and national legislation (ExpRS, Germany: Federal State Brandenburg, GZ 2347-11-2015; ExpR, Austria: BMWF GZ 68.205/0081-WF/V/3b/2015).

2.2

Procedure and measurements

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Experiment RS – Restraint plus shearing In ExpRS the 15 experimental animals that were selected for shearing on the respective day were separated from their herd and all moved into a separate fenced outdoor area of about

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50m². There the animals had no contact to the herd but were directly besides the shearing area, which was a roofed area in an open front housing. In the determined order individual

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animals were moved towards the area for shearing where they were caught by the farmer. After short sampling and data recording (see below; S-1) animals were restrained during the

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duration of shearing. When shearing was finished, the animals were immediately released from their fixation in Ground and Table conditions and held by the neck until 20 min after the start

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of the fixation in the same manner as the animals in the Standing condition. Then the animals were moved to another area for animals already sheared on this day and the next individual was caught. Animals stayed in the post-shearing pen for the whole day.

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Experiment R – Restraint alone

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In ExpR the 15 experimental animals were brought into two areas, 5 males in one area and the other 10 in another area, separated only by a fence so that interactions were possible. All animals were restrained in the respective method for 15 min, thereafter animals were held at the halter or neck for a further 5 min. Then animals were moved back to their respective area. A within-subjects design was adopted: the 15 animals were subjected to each of the three restraint methods consecutively with a one week delay in between. Order of treatment was balanced, i.e. 5 animals started with Ground, 5 with Table, and 5 with Standing. 6

2.2.1

Restraint methods

In both experiments the same three restraint methods were used, but details differed slightly. On both farms restraint on the ground was normally used for shearing and the same method as common on the farm was applied in the experiment. Neither of the farms normally used a table during shearing so a table was borrowed from other farms. Experiment RS – Restraint plus shearing For Standing all animals were held gently at the neck by hand. Some animals had to be held

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by more than one person for some time during shearing due to fierce defence / flight reactions. Animals in the Ground condition were laid down on a soft mattress in sterno-ventral position (see Photos in Wittek et al. 2017) with legs outstretched to the front and back, and sheared at the back, side, neck, head and proximal extremities. They were then turned onto each side to

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shear the extremities except the distal parts where ropes for fixation were attached. Lastly, they were again turned into the sterno-ventral position for shearing the distal parts of first the

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front and then the hind legs.

The table could be tilted. Thus, the animals in treatment Table were led besides the table and

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fixated at the body by a belt around the belly; the table was then tilted, and the legs were fixed by ropes so that they were stretched slightly to the front and back; when secured, the belt was

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unfastened. However, some animals showed strong defence reactions when trying to fasten the belt. For these animals the table was tilted before and animals were lifted onto the table.

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Animals were first sheared on one side, then turned to the other one. Experiment R – Restraint alone

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For Standing the animals were held at a halter if they were used to it, or by hand at the neck if not used to a halter. For Ground the animals were laid down on a soft mat directly on one side and were turned only once to the other side (in our experiment after 7.5 min) as was usual on that farm during shearing. For Table all animals were lifted onto the table and legs were fixed by ropes with legs stretched as in ExpRS.

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2.2.2

Behavioural Observations

Behaviour was observed both by continuous focal sampling and by instantaneous sampling from catching in the group until two hours after the start of restraint in several time frames. In the course of the study heart rate by auscultation, body temperature by rectal thermometer measurement, salivary cortisol and faecal cortisol metabolites were measured but are reported elsewhere (Wittek et al. 2017). Behavioural observations excluded those times where some form of handling was necessary due to those physiological measures, and additional breaks

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were included to avoid observer fatigue, resulting in the sampling schedule reported below. After shearing continuous observations were limited to 16 minutes in total due to overlapping observations of several animals and only one observer.

Behavioural reactions to catching were recorded on a qualitative score to identify potential

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individual differences in reactivity that could interfere with reactions to restraint. Continuous behaviour observations were conducted according to the following schedule with the start of

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restraint (i.e. the animal is in the final position) being minute 0: min 0 to 10 (excluding min 6 where physiological data were recorded, K0_10), 15 to 20, 23 to 26, 50 to 55 and 67 to 75.

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For further analysis, K0_10 was examined separately in both experiments, reflecting a period where all animals were restrained in the respective method. Minute 15 to 20 (K15_20) was

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analysed separately in ExpR reflecting the minutes directly after end of restraint in Ground and Table. In ExpRS, observations during min 0 to 10 (K0_10) were summed with the observations

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during min 15 to 20 to create the variable “K0_20”, thus comprising most of the period of shearing for all animals, but excluding minute 10-14. Observations after shearing were

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summed (K_after). In ExpRS, observation of minute 23 to 26 was lacking for several animals because duration of shearing took longer, thus these minutes were excluded for all animals. Table 1 lists and defines the behaviours recorded during continuous observations. All behaviours were events and thus frequencies were recorded. However, in case of vocalisations during shearing some animals showed continuous vocalisations, i.e. without interrupting it for the whole observation period, which was noted down as “continuously”. For observations during restraint/shearing all types of vocalisations were summed up (i.e. moaning 8

and screaming; growling was observed only by one animal in either of the two experiments). Treatment conditions resulted in non-uniform behavioural expressions of the other behaviours during restraint as outlined in Table 1 and were aggregated into categories accordingly or with respect to functional areas of behaviour (Table 1). Instantaneous behaviour recording was performed every 10 minutes from minute 30 to 120 after start of restraint. Lying (animal lying in sterno-ventral position, laterally, or lying down), standing, walking, and running were scored as mutually exclusive behaviours. Feeding,

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drinking, vigilance, ruminating, defecation, or urination were noted additionally.

Table 1 about here

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During restraint / shearing the vocal and tactile behaviour of handlers towards the animals were noted down as positive (calming talking, gentle touch, stroking) or negative (hectic

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movements, shouting, hits with the hand). However, as negative interactions were never observed, we did not analyse human behaviour further.

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Behaviour before and during restraint / shearing was recorded by the same trained observer in both experiments. Behaviour after shearing was observed by three other trained people in

2.2.3

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total, two in ExpRS and one other person in ExpR.

Duration of shearing and injuries

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The duration of shearing was measured by a stopwatch and defined as the duration of the

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clipper being first turned on until it was finally turned off. Injuries were noted down during shearing and further the animals were inspected for injuries after the end of shearing by two observers (the person recording behaviour during shearing plus another person taking some physiological measures).

2.2.4

Heart rate and heart rate variability

Heart rate and heart rate variability (HRV) was measured in ExpR. Non-invasive heart rate measurement devices from Polar® were attached to the chest of alpacas directly after the first 9

physiological measures were taken, i.e. directly before animals were restrained. After fitting the belts and starting the receivers, the animals were restrained as described in 3.2.1. Heart rate monitors remained in place until 60 minutes after start of restraint and measured interbeat-intervals (IBIs). IBI data were corrected by using the automated function of Polar Precision Performance SW with default adjustment (correction medium) under visual control in 1 min sections. Only 1 min sections with an error rate below 5% were used for further analysis with the program Kubios HRV version 2.1, calculating frequency domain heart rate variability parameters (SDNN, RMSSD, SDNN/RMSSD) and mean heart rate. The acceptable 1 min

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sections of the first ten minutes of restraint and of the last 10 min of measurement (minute 50 to 60 after start of restraint), respectively, were summarized by calculating mean values of HRV parameters and were used for further analyses. Due to poor signal or extensive device

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malfunction during the first two days, only the HRV data of the last test day could be used for analysis. Processing of HRV data was performed by one person not involved in data

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acquisition otherwise and blind to the treatments and aims of the study.

Statistical analysis

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All statistical analyses were calculated using IBM® SPSS® Statistics Version 22. Statistical tests with p≤0.05 are referred to as significant, while those with 0.5
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In both experiments, behavioural reactions to catching did not differ (p>0.1) between treatment

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groups and thus were not considered further in analysis. In ExpRS, on day 1, the last animal in the Standing condition (purchased) could not be sheared due to extreme flight and defence reactions; the trial was stopped after 3 min, reducing sample size for all observations to 14 in this treatment and 44 in total. Information on the animals’ origin was lacking for one animal. In ExpRS scan sampling after shearing was not recorded for some animals for some time points in the period 30 to 60 minutes. Therefore, the proportion of scans of a specific behaviour over

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the whole 1.5 hours of observation was calculated, while in ExpR the absolute number of scans in the first and second hour after restraint was used for analysis. Due to the discontinuous distribution during shearing in ExpRS (most animals showing either continuous vocalisation or no vocalisation at all), vocalisations were coded dichotomously (vocalisation yes/no). Further, behaviours that were low in frequency or observed only in a few animals were also dichotomised (ExpRS: lying, ruminating; ExpR: affiliative behaviour). For ExpRS, these data were analysed by Chi² test or Fisher Exact test, respectively. In case of p≤0.1 standardized residuals were used to identify cells where the observed frequency showed

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significant deviations from the expected frequency (residuals ≥ Ɩ1Ɩ). For ExpR with repeated measures design, CochranQ and McNemar test were used to analyse dichotomized variables. All other behavioural data were also analysed by non-parametric statistics for between-

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subjects (ExpRS) or within-subjects (ExpR) data due to non-normality and variance heterogeneity. Before using non-parameteric statistics generalized linear models using

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Poisson-distribution were calculated with treatment and origin of animals as fixed effects for ExpRS. However, models could not be fitted since data were over-dispersed even after

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deleting outliers. Thus, all three treatments were compared with Kruskall-Wallis tests (ExpRS) or Friedman tests (ExpR) first; in case of a trend (p≤0.1) pairwise comparison was performed

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with Mann-Whitney U tests (ExpRS) or Wilcoxon tests (ExpR). A possible influence of origin in ExpRS was tested for by comparing animals of different origin over all three restraint methods.

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The few differences found (vocalisation, head movement) always depended on only one outlier and thus are not reported further as it was not the aim of the project and animal origin was

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balanced over treatments.

Occurrences of behaviours are visualized in boxplots for all data except vocalisations during shearing in ExpRS. That is, boxplots are shown also for variables that were dichotomized for statistical analysis to ease overview. In boxplots, the box comprises the second and third quartile, the bold line corresponds to the median, the whiskers indicate the lowest and highest values except outliers (1.5 – 3 times the interquartile range, shown by circles) and extreme outliers (>3 interquartile range, indicated by stars). 11

HRV data in ExpR could be used only on the last test day (see 3.2.3.) and sample size was further reduced to only four, three and five for Standing, Ground and Table, respectively. Restraint methods were compared with regard to heart rate and the HRV parameters SDNN, RMSSD and RMSSD/SDNN by use of ANOVA and Bonferroni post-hoc tests. Additionally, we calculated Pearson correlations between the parameters across all animals.

3.1

Results Restraint plus Shearing (ExpRS)

3.1.1 3.1.1.1

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Behaviour during shearing Vocalisations

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Around half of the animals (24 animals) vocalised continuously throughout shearing, mostly

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screaming (14), without a difference between methods (p>0.1; Number of animals vocalising continuously for Standing/Ground/Table: 7/9/8; Screaming continuously 4/6/4). However,

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restraint methods tended to differ with respect to the number of animals showing any vocalisation in the first 10 min of shearing (Fisher exact, p=0.085, Fig 1a). According to the

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standardized residuals, more Standing animals showed no vocalisation at all (number of animals vocalising no/yes: 6/8, standardized residuals: 1.3/-0.8), but only one Table animal did not vocalise (1/14, -1.4/0.8), and Ground animals fell in between (4/11, 0,1/-0.1). When adding

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the last five minutes of the observation, i.e. for K0_20, the difference between methods

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remained the same (Fisher exact, p=0.088; vocalisation no/yes: Standing 4/10, 1.2/-0.5; Ground 3/12, 0.4/-0.2; Table 0/15, -1.5/0.7; Fig 1b).

Figure 1 about here

3.1.1.2

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Other behaviours

Table 2 shows the number of animals showing a specific behaviour during the first 10 minutes or the whole period K0_20 min. No threatening was observed during shearing, and very few animals kicked or laid down in the first 10 min. Standing animals showed more LegMovements behaviour than Ground and Table both during the first 10 min (KW-test: Chi²=7.899, p=0.02; Fig 2i) and for K0_20 (KW: Chi²=13.122, p=0.001; Fig 2j), but less flinching (KW: Chi²=9.125, p=0.01), which they also tended to show less during the first 10 min (KW: Chi²=5.561, p=0.06; Fig 2a). However, treatments did not differ in the sum of all these defence behaviours (Defence) in the first 10 min (KW: Chi²=2.125, p=0.35) and only tended to do so for K0_20

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(KW: Chi2=4.983, p=0.08; Fig 2j).

Table 2 about here

Behaviour after shearing

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3.1.2

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Figure 2 about here

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After shearing, i.e. in the period 30 min to 120 min after start of restraint and shearing, animals of the three restraint methods differed with respect to ruminating (Fisher exact , p=0.04) and tended to differ in lying (Fisher exact, p=0.068) with fewer Ground animals ruminating and lying

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and more Standing animals lying than expected (according to standardized residuals): Both ruminating and lying were observed only in one animal once after Ground (ruminating yes/no:

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1/14; standardized residuals -1.6 / 1.1; lying: 1/14; -1,5/0.9). After Standing six animals displayed rumination (yes/no 6/8, st.res. 0.9/-0.6) and 6 animals displayed lying (6/8, st.res.

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1.1/-0.7). After Table, six animals ruminated (6/9, st.res. 0.7/-0.5) and five displayed lying (5/10, st.res. 0.4/-0.3, for proportions of scans see Fig 3). Animals did not differ with respect to walking, standing, feeding or vigilance (all p>0.1) Regarding continuous observations treatments differed in agonistic interactions (KW: Chi²=6.544, p=0.038): animals that had been shorn on the Table showed more agonistic behaviour than Standing animals and tended to show more than Ground animals (Fig 4). No

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difference was detected in affiliative social interactions (Chi²=1.324, p=0.516), vocalisations (Chi²=2.801, p=0.247) or comfort behaviour (Chi²=0.509, p=0.775).

Figure 3 and Figure 4 about here

3.1.3

Shearing duration and injuries

Shearing duration tended to differ between methods (Chi2=5.338, p=0.07), with the longest

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duration in Table (mean ± sd: 20.9 ± 3.52 min, min - max: 16 - 29 min), differing from Standing (17.5 ± 2.6 min, 10 – 21 min, U= -2.182, p=0.03), but not from Ground (19.0 ± 2.43 min, 15 – 24 min, U=-1.493, p=0.14); Standing and Ground did not differ (U= -0.982, p=0.326).

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Shearing induced injuries in 13 animals, all injuries were only superficial, not bleeding and one to three centimetre long. In Standing six of the 14 shorn animals got cuts (of those two were

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cut twice, i.e. 8 cuts in total), in Ground three animals were cut once and in Table four animals were cut once. However, methods did not differ regarding the number of injuries (Fisher exact

3.2.1 3.2.1.1

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Restraint alone (ExpR)

Behaviour during restraint Vocalisations

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3.2

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test, p =0,418).

In contrast to ExpRS, no animal screamed continuously during restraint in ExpR. No difference

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was found between methods of restraint with respect to any type of vocalisations or the sum of moaning and screaming with the Friedman test for the first 10 min (screaming: Chi²=4.083, p=0.13; moaning: Chi²=2.176, p=0.34; sum of vocalisations: Chi²=2.513, p=0.29; Fig 5). Only two animals screamed during standing, five animals each screamed when restrained on the ground or on the table. About half of the animals did not vocalise at all independent from the treatment (Standing, 7 animals; Ground, 8 animals; Table, 8 animals).

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In the five minutes after restraint (K15_20) methods differed with respect to vocalisations (Friedman-test: p=0.033, Chi²=6.821) with animals having been restrained on the Table vocalising more than Ground (p=0.02, z=-2.354, Fig 5).

Figure 5 about here

3.2.1.2

Other behaviours

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During the first 10 minutes, only two animals spit once each, one animal when restrained on the ground and one animal on the table. Further, animals restrained during standing did not flinch and only one showed MoveHead, while more of those behaviours were observed during

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restraint on the ground or on the table (Fig. 6 a,c; Friedman Test: Flinch: Chi²=19.633, p<0.001; MoveHead: Chi²=15.316, p<0.001). Animals showed more LegMovements when Standing

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than when on the ground or, as a trend, on the table (Fig. 6e). In their overall Defence behaviour animals only tended to differ (Chi²=5.547, p=0.06; Fig. 6g). In the five minutes after

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restraint (K15_20) animals differed in flinching (Chi²=6.000, p=0.050) with three Table animals

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flinching (Fig 6 b).

3.2.2

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Figure 6 about here

Behaviour after restraint

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Treatments differed with respect to feeding (Chi²=6.792, p=0.034) and tended to differ in walking (Chi²=4.696, p=0.096). When animals had been restraint Standing they fed most often compared to after restraint on the Ground and on the Table, where feeding was lowest (Fig 7). Vigilance followed the opposite pattern of lowest occurrence after Standing and highest after Table, but no significant difference was confirmed. Animals laid down rarely (three animals after Standing, two after Ground and one after Table) and ruminated rarely (Fig 7).

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The number of animals showing affiliative behaviour tended to differ between methods after restraint, with two animals after Standing, seven after Ground and three after Table (Cochrans Q=5.250, p=0.07), in pairwise comparison Standing tended to differ from Ground (p=0.06). There were no other differences in continuously observed behaviours.

Figure 7 about here

Heart rate variability

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3.2.3

In the first 10 min of restraint HRV differed between treatments in SDNN (F=7,233, p=0.013, Fig 8). HRV (SDNN) was higher in Standing (median, min-max: 77, 66-88, N=4) compared to

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Table (34, 31-55, N=5; p=0.013); however, Ground did not differ from either group (70, 28-80, N=3; pS_G=0.536, pG_T=0.251,). No difference was found in HR, RMSSD or SDNN/RMSSD and

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in the period 35 to 45 min after the end of restraint, although the pattern of RMSSD in the first 10 min was similar to SDNN with highest values in Standing, lowest in Table and intermediate

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in Ground. HR tended to correlate with SDNN/RMSSD (r=0.52, p=0.09, N=12) but neither with SDNN nor with RMSSD. RMSSD was highly positively correlated with SDNN (r=0.74, p=0.006,

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N=12) and negatively correlated with SDNN/RMSSD (r= -58, p= 0.05, N=12), which did not show an association with SDNN.

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Figure 8 about here

4

Discussion

The results of both experiments - restraint alone in ExpR and restraint plus shearing in ExpRS are in line with our hypothesis that holding animals manually while standing (Standing) elicits the fewest stress response compared to tying them up with ropes when lying on the ground (Ground) or on a table (Table). This is reflected in less flinching (ExpRS and Exp R), higher heart rate variability (SDNN, measured only in ExpR) and a trend for lower number of animals 16

vocalizing (ExpRS) during restraint as well as less agonistic interaction, more ruminating and, by trend, more lying (ExpRS) or more feeding and, by trend, less affiliative interactions (ExpR) after restraint, although in some behaviours differences were found only in one of the two tying up treatments - Ground or Table. Further, against expectations from practice, shearing duration was shortest in Standing, being similar to Ground and shorter than Table, although the time taken to fix ropes and lay the animal down was not included. In addition, restraint methods did not differ with regard to injuries. However, escape attempts were observed more often in Standing animals and one animal in the Standing treatment had to be excluded from

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the experiment due to strong defence reactions that made shearing impossible. Differences between Ground and Table were very rare but supported our hypothesis that Table elicits even more stress reactions than Ground.

Behaviour during restraint with and without shearing

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A high percentage of animals were screaming continuously during shearing in ExpRS independent from the restraint method. Increased vocalisations, especially loud screaming,

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were found as indicators of stress during handling and shearing in wild South American camelids (SAC), guanacos and vicunas caught for fibre-harvesting (Arzamendia et al. 2010,

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Taraborelli et al. 2017), with female vicunas vocalising more in a capture-shearing event than males (Marcoppido et al. 2018). Thus, shearing can induce a high level of stress, which may be attributed to the intense handling which included tactile stimuli and clipper noise – in several

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animals, duration of screaming fully coincided with the clipper turning off. Also in sheep, wool-

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removal contributed most to the stress reactions during shearing as compared to the other shearing-associated factors, up-ending and isolation (Hargreaves and Hutson 1990). Thus, it is recommended to reduce potential stress due to clipper noise and handling by habituation or even positive conditioning (e.g. (Lund et al. 2012, Pratsch et al. 2018) and a general good animal-human relationship (Waiblinger et al. 2006, Hemsworth and Coleman 2011, Waiblinger 2019). Ideally alpaca would accept shearing without any restraint necessary. Positive reinforcement training has been successfully used in different animal species to train them to 17

voluntarily participate in veterinary or husbandry procedures (e.g. Grandin et al. 1995, Savastano et al. 2003, Schapiro et al. 2018). Such techniques may offer possibilities for even eliminating the need for restraint during shearing in alpaca and thus avoid stress and largely minimize the risk of injury. Although this may be difficult to achieve in a commercial setting with relatively large herd sizes and professional shearers being unfamiliar to the animals, strategies to reduce stress and avoid restraint by use of positive reinforcement should be developed and investigated further. When comparing restraint methods, being tied up on the ground or a table during shearing

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resulted in a trend for more animals vocalizing than when standing, indicating lower stress levels in Standing animals. Results of other behaviours during restraint are less clear. In wild South American camelids, guanacos and vicunas, a higher frequency of sudden movements

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such as kicking, struggling or attempts to stand up during handling and shearing were related to other indicators of heightened stress, e.g. behaviour in the pre-handling corral (Arzamendia

re

et al. 2010, Taraborelli et al. 2017, Marcoppido et al. 2018). While Standing animals showed less flinching than animals tied up in both of our experiments, and less head movements in

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ExpR, they moved their legs more. Fewer LegMovements in Ground and Table animals may be a sign of tonic immobility which is seen as a sign of intense fear with physical restraint

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(Fureix and Meagher 2015) and was suggested to be elicited in sheep restrained for shearing (Hargreaves and Hutson 1990). Besides, tied up animals may have given up in trying to escape

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due to restricted movement of their legs. This forced immobility is likely to cause more stress as compared to Standing animals (Grandin 1997), probably due to experiencing even lower

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level of control / escape possibilities. This interpretation is supported by the trend for a higher level of vocalisations, post-restraint results and physiological data (see 5.2. and 5.3.). LegMovements in Standing animals occurred disproportionately more in the last 5 min of observation which is likely due to the fact that legs are shorn at the end of shearing, eliciting respective defence movements. Thus, farmers may refrain from shearing the legs to reduce defence reactions and risk for injuries.

18

Ground and Table showed few differences, but these were in line with our hypothesis of even higher stress when animals are restrained on a table: all Table animals vocalised during shearing in ExpRS; in ExpR Table animals showed flinching during restraint more often and tended to have higher frequency in LegMovements during restraint and more vocalisations in the five min after restraint compared to Ground. Different factors may have contributed to this result: Firstly, animals had to be lifted up to the table, while in Ground animals were made to lie down without being lifted off the ground. It is important to note that observations started only after animals were tied up; nevertheless, being lifted up and lying above the ground may be

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more stressful than being forced to lie on the ground in a manner resembling natural defence behaviour in South American camelids (Pollard and Littlejohn 1995). Secondly, the surface of the table was harder (wooden) than the ground (mat) potentially making lying on the table more

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uncomfortable. Lastly, all animals in ExpR and most in ExpRS had pre-experienced shearing on the ground as this was the method used by the farmer. Lying on the table thus may have

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added novelty as an additional stressor (Grandin 1997).

Behaviour after restraint with and without shearing

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Behaviour after restraint further supports the hypothesis of lower stress levels in Standing animals, which were seen more often ruminating, feeding and, by trend, lying, and less often involved in agonistic and, by trend, affiliative interactions. All these behavioural changes are

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known as indicators of stress in different animal species and also, except ruminating, in Alpaca

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and other SAC (Pollard and Littlejohn 1995, Arzamendia et al. 2010, Taraborelli et al. 2017, Marcoppido et al. 2018). We did find a trend for a higher frequency of affiliative behaviours in ExpR after Ground as compared to Standing. Affiliative social behaviour is rarely seen in studies on new world camelids’ social behaviour (Svendsen and Bosch 1993, Gerken et al. 1998). In other animal species affiliative behaviour or body contact was found to reduce stress and tension and to increase after stressful situations (Waiblinger et al. 2002, Napolitano et al. 2009, Rault 2012). 19

In captured wild vicunas, allogrooming occurred to a much higher extent after handling and shearing than before (Marcoppido et al. 2018, see also Arzamendia et al. 2010 for grooming behaviour including allo- and autogrooming). Our results are thus in line with the notion that affiliative interactions also help stress coping in alpaca. While the pattern of differences between Standing and the two tie-up treatments is consistent during restraint, it is partly inconsistent after restraint. Behavioural differences between treatments after restraint are caused by carry-over effects of stress during restraint, but different types of stressors likely elicit different emotions and specific behavioural responses

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(Veissier and Boissy 2007). Handling and social separation elicit fear, and higher stress levels may enhance affiliative behaviour to ease calming down (see above). In contrast, the exact way of restraint may differ in the level of physical discomfort elicited. In ExpRS animals were

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positioned most of the time in sternal recumbency with legs stretched to the front and back, while in ExpR they were positioned only on the sides. Strain on joints may have been higher

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in sternal recumbency, where outstretched joints had to bear the animal’s body weight, and thus may have caused discomfort or even pain lasting after restraint. We did not expect such

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differences beforehand and thus did not document angles in joints nor examine animals after restraint with adspection (locomotion score) nor palpation. This aspect clearly merits further

4.3

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research.

Heart rate variability

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The results of HRV during restraint, although being based on a very low sample size, are in

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line with behavioural data on lower level of stress during Standing: SDNN was higher in Standing compared with Table, and Ground was intermediate. SDNN represents the overall variability, it is influenced by both the sympathetic and the parasympathetic nervous system activity, and was shown to decrease with increasing stress in various animal species and humans (for review: von Borell et al. 2007, Shaffer and Ginsberg 2017). Dairy cows had lower SDNN when standing than when lying (Hagen et al. 2005), thus supporting the notion of psychological stress being responsible for lower SDNN in Table. Our behavioural data also 20

are in line with the results of other physiological measurements (saliva cortisol, heart rate, respiratory rate) of the same experiment published in Wittek et al. (2017), where Standing animals also had the least signs of physiological stress reactions, and large individual differences were found as well.

5

Conclusion

Being restrained manually in a standing position is less aversive for Alpaca than being tied up on the ground or a table without increasing the risk for injuries. Additionally, the standing

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position was associated with the shortest procedure duration. However, shearing elicited strong behavioural reactions in some of the animals regardless of treatment, resulting in a failed shearing attempt in one Standing animal. Habituation or positive conditioning to handling

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including touching all body regions and to clipper noise should be used for all animals to reduce

6

Acknowledgements

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stress and avoid the necessity for more invasive restraint due to extreme defence reactions.

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We are very grateful to the alpaca farmers (Eva Maria and Thomas Pötsch, Alpakaland Österreich, Tauplitz, Austria and Kerstin and Frank Niemann, Alpacas of Density, Sonnewalde

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(Piesig), Germany); allowing to conduct the experiment on their farm and devoting their time to it when shearing the animals or helping to handle them. We would like to thank Jason Yee

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for reviewing the English language and helpful comments on an earlier version of the paper, Larissa Kawasch for help in behavioural observation, and Teresa Salaberger and Sebastian

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Becker for help in performing the experiments. We acknowledge funding by the Austrian Buiatric Association (Austria) and the Alpaca Association e.V. (Germany).

21

7

References

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South American camelids. Animal 4(9): 1451-1459.

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Gerken, M. 2010. Relationships between integumental characteristics and thermoregulation in

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Grandin, T. 1997. Assessment of Stress During Handling and Transport. J. Anim. Sci. 75: 249–

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Hagen, K., Langbein, J., Schmied, C., Lexer, D. and Waiblinger, S. 2005. Heart rate variability

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Lund, K. E., Maloney, S. K., Milton, J. T. B. and Blache, D. 2012. Gradual Training of Alpacas to the Confinement of Metabolism Pens Reduces Stress When Normal Excretion Behavior Is Accommodated. Ilar Journal 53(1): E22-E30.

Marcoppido, G., Arzamendia, Y. and Vilá, B. 2018. Physiological and behavioral indices of short-term stress in wild vicuñas (Vicugna vicugna) in Jujuy Province, Argentina. Journal of Applied Animal Welfare Science 21(3): 244-255. Napolitano, F., Knierim, U., Grasso, F. and De Rosa, G. 2009. Positive indicators of cattle welfare and their applicability to on-farm protocols. Italian Journal of Animal Science 8 (SUPPL.

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Pollard, J. C. and Littlejohn, R. P. 1995. Effects of social isolation and restraint on heart rate and behaviour of alpacas. Appl. Anim. Behav. Sci 45: 165-174.

Pratsch, L., Mohr, N., Palme, R., Rost, J., Troxler, J. and Arhant, C. 2018. Carrier training cats

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Rault, J. L. 2012. Friends with benefits: Social support and its relevance for farm animal welfare. Applied Animal Behaviour Science 136(1): 1-14.

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Savastano, G., Hanson, A. and McCann, C. 2003. The development of an operant conditioning training program for new world primates at the Bronx Zoo. J Appl Anim Welf Sci 6(3): 247-261. Shaffer, F. and Ginsberg, J. P. 2017. An Overview of Heart Rate Variability Metrics and Norms.

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Svendsen, G. E. and Bosch, P. C. 1993. On the behavior of vikunjas (Vicugna vicugna Molina, 1782). Differences due to sex, season and proximity to neighbors. Z. Säugetierkunde 58: 337343.

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Taraborelli, P., Torres, M. E. M., Gregorio, P. F., Moreno, P., Rago, V., Panebianco, A., Schroeder, N. M., Ovejero, R. and Carmanchahi, P. 2017. Different responses of free-ranging wild guanacos (Lama guanicoe) to shearing operations: implications for better management practices in wildlife exploitation. Animal Welfare 26(1): 49-58. Veissier, I. and Boissy, A. 2007. Stress and welfare: Two complementary concepts that are intrinsically related to the animal's point of view. Physiology & Behavior 92(3): 429-433. von Borell, E., Langbein, J., Desprs, G., Hansen, S., Leterrier, C., Marchant-Forde, J., Marchant-Forde, R., Minero, M., Mohr, E., Prunier, A., Valance, D. and Veissier, I. 2007. Heart rate variability as a measure of autonomic regulation of cardiac activity for assessing stress

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B. 2006. Assessing the human-animal relationship in farmed species: a critical review. Appl.Anim.Behav.Sci 101: 185-242.

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Wheeler, J. 2012. South American camelids – past, present and future. Journal of Camelid

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Wittek, T., Salaberger, T., Palme, R., Becker, S., Hajek, F., Lambacher, B. and Waiblinger, S.

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2017. Clinical parameters and adrenocortical activity to assess stress responses of alpacas using different methods of restraint either alone or with shearing. Veterinary Record

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10.1136/vr.104232: 7.

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Fig 1: ExpRS: Number of animals that vocalised (dark grey) or did not (light grey) in a) the first ten minutes of shearing or b) over the whole period K0_20 while being restrained Standing

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(n=14), Ground (n=15) and Table (n=15).

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Fig 2: ExpRS: Boxplots of the frequency of Flinching (a,b), MoveHead (c,d), Spitting (e,f), LegMovements (g,h) and Defence (sum of the aforementioned behaviours, i,j) in the first 10 min (K0_10; left graphs) or over the whole 20 min (K0_20, right graphs). P-values refer to

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results of Mann-Whitney U tests; values in italics if PKW>0.05<0.1. Note different scales.

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Fig. 3: Exp RS: Proportion of scans animals were lying (a), walking (b), standing (c), feeding (d), ruminating (e) or vigilant (f) in the period 30 – 120 min after start of restraint and shearing. Lying and ruminating were dichotomized for statistical analysis and thus the statistical

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differences not shown, vigilance was not analysed due to low occurrence.

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Fig. 4: Exp RS: Frequencies of behaviours observed continuously around 1 h after start of restraint and shearing (observation times were 50-55min and 67-75 min after start of restraint). Number of occurrences of moaning (a), affiliative interactions (b), comfort behaviour (c) and agonistic interactions (d) per 13 min. P-values refer to results of Mann-Whitney U tests; values

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in italics if PKW>0.05<0.1. Note different scales.

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Fig 5: ExpR: Boxplots of the frequency of Moaning (a,b), Screaming (c,d), and sum of vocalisations (e,f), in the first 10 min (K0_10; left graphs) or the 5 min after restraint when all animals were standing (K15_20, right graphs). Numbers of extreme values or outliers refer to

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animals. P-values refer to result of Wilcoxon tests. Note different scales.

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Fig 6: ExpR: Boxplots of the frequency of Flinching (a,b), MoveHead (c,d), LegMovements (e,f) and Defence (sum of the aforementioned behaviours (g,h) in the first 10 min of restraint (K0_10; left graphs) or in the five minutes after restraint when all animals were standing (K15_20, right graphs). P-values refer to result of Wilcoxon-tests; values in italics if PFriedman>0.05<0.1. Spitting not shown due to low occurrence (three times in total). Note

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differing scales.

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Fig. 7: ExpR: Number of scans animals were lying (a), walking (b), standing (c), feeding (d), ruminating (e) or vigilant (f) in the period 30 – 120 min after start of restraint (maximum value 10). P-values refer to results of Wilcoxon tests (lying not tested); values in italics if PFriedman>0.05<0.1. Note different scales. Lying was not analysed statistically, ruminating was

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dichotomized, but methods did not differ.

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Fig. 8: ExpR: HR and the HRV parameters SDNN, RMSSD and SDNN/RMSSD in the first 10

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min of restraint in Standing (N=4), Ground (N=3) and Table (N=5). Mean±SE are depicted.

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Table 1: Definition of behaviours recorded during restraint with or without shearing (during, min 0 – 20) and after restraint (after, min 23-26, 50-55 and 67-75) during continuous observations. Behaviour Defence/Restlessness1 Body/head Flinch/twitch MoveHead Spitting LegMovements2 Escape attempts

during

Animal tries to escape from restraint, e.g. lifting both front legs at once or sudden steps to the side, with or without success Quick movement of one leg in an upward-forward or upward-backward direction Slight lift of the leg or flinch/twitch of the leg, Animal lies down or lets itself fall down during restraint/shearing

during

Quiet vocalisations with drawn-out sounds Loud, sharp calls, without previous growls Growling sound, sometimes with a shrill sound at the end, occurs mostly in the social context, accompanies threatening posture

during, after during, after during, after

Animal spits regurgitated stomach’s content towards a conspecific Animal lifts the head in in the typical threat posture (chin heightened) with ears backward Animal first shows threatening and then runs after the other in this posture Animal lifts its forelegs against another alpaca, often with crossing of necks and pushing, with or without biting Neck is bent in a U-shape with lowered head, tail upright Animal lays ears backward without another sign of threat or subordination

after

during during

during

Social interactions Agonistic3 Spitting

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Threatening Chasing

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Fighting

during, after after after

after after after

Animal nibbles at another or rubs the head or neck

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Affiliative Social grooming Comfort behaviour Wallowing

during during

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Vocalisations Moaning Screaming Growling

Ears backwards

Sudden brief muscle contraction of the skin including whole body parts of the trunk Each movement of the head including a flinch/twitch of the head Animal spits regurgitated stomach contents towards a human

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Moving leg Lie down

Submissive

observation during

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Kicking

definition

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Animal lays down and rubs or rolls its body on the after ground Grooming Animal scratches itself with the hind-leg or rubs its after body against an environmental structure 1All behaviours in this category were summed into “Defence” (i.e. sum of behaviours in Body/Head and LegMovements) 2Behaviours were merged into “LegMovements”; they can only be shown in periods when the animal or the respective body part is not fixated by ropes during restraint, i.e. for Ground and Table mostly in the time from the end of restraint until minute 20, or, in case of Moving leg, the intensity of behaviour differs between the fixated and not fixated situation 3All behaviours except ears backward were combined into “Agonistic”

34

Table 2: Number of animals in the three treatment groups that did show the behaviours for K0_10 (0_10) and K0_20 (0_20). Threatening is not shown – no animal ever threatened towards the humans.

Standing

Ground

Table

Standing

Ground

Table

0_10

0_10

0_10

0_20

0_20

0_20

Flinch/twitch

8

12

10

10

14

14

MoveHead

9

10

11

10

11

14

Spitting

3

4

4

4

5

5

Kicking

1

0

0

2

1

1

Moving leg

9

11

9

10

12

14

Lie down

2

0

0

5

3

0

10

0

0

12

5

1

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Escape attempts

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Behaviour

35