Individual Behavioral and Physiological Responses During Different Experimental Situations – Consistency over Time and Effects of Context

In a number of species, consistent behavioral differences between individuals have been described in standardized tests, e.g. novel object exploration, open field test. Different behavioral expressions are reflective of different coping strategies of individuals in stressful situations. A causal link between behavioral responses and the activation of the physiological stress response is assumed but not thoroughly studied. Also, most standard paradigms investigating individual behavioral differences, are framed in a fearful context, therefore the present study aimed to add a test in a more positive context, the feeding context. We assessed individual differences in physiological (heart rate, HR) and behavioral responses (presence or absence of pawing, startle response, defecation, snorting) of twenty domestic horses (Equus caballus) in two behavioral experiments, a novel object presentation and a pre-feeding excitement test. Experiments were conducted twice, in summer and autumn. Both experiments caused higher mean HR in the first ten seconds after stimulus presentation compared to a control condition, but mean HR did not differ between the experimental conditions. Interestingly, in the novel object experiment, horses displaying stress-related behaviors during the experiments also showed a significantly higher HR increase compared to horses which did not display any stress-related behaviors, reflecting a correlation between behavioral and physiological responses to the novel object. On the contrary, in the pre-feeding experiments, horses that showed fewer behavioral responses had a greater HR increase, indicating the physiological response being due to emotional arousal and not behavioral activity. Moreover, HR response to experimental situations varied significantly between individuals, and although we found HR to be significantly repeatable across experiments, repeatability indices were low. In conclusion, our findings show that horses’ behavioral and physiological responses differed between test situations and that high emotional reactivity, shown via mean HR and HR increase, is not always displayed behaviorally.


Introduction 44
The perception of a potential threat to homeostasis, caused by extrinsic or intrinsic 45 stimuli (stressors) results in the activation of the physiological stress response in animals 46 (Chrousus and Gold 1992;Moberg 1985). Regardless of the intensity of the stressor, 47 individuals respond both behaviorally and physiologically, and these responses aim to 48 counteract the effects of the stimuli and to re-establish homeostasis (Chrousus and Gold 49 1992). Physiological activation in response to a stressor causes a fast release of 50 norepinephrine in the brain, triggering the activation of the sympatho-adreno-medullary 51 behavioral responses were recorded for five minutes. Thereafter, the horses were fed by 158 placing the feeding bucket inside the box and their behavioral and physiological responses 159 were measured for the following ten minutes. 160 In the novel object test, the horses were exposed to two of three different objects in 161 their stable. The first object was formed of a main cylindrical hard body (approximately 30 162 cm in length) filled with gravel which was fixed to a soft foam rubber ball (about 15 cm in 163 diameter) and covered in fabric. The second object was formed of two cylindrical plastic 164 tubes fixed together to form an 'x'. Similar to the first object, the cylinders were 165 approximately 30cm in length, filled with gravel and covered with fabric. In addition, twelve 166 tennis balls of different colors and materials were pierced and attached to 4 strings (3 balls 167 per string) of approximately 50 cm in length. These were then tied to the main body of the 168 object and left hanging. The third object was an inflatable guitar of approximately one meter 169 in length. All objects were attached to a string, around four-meters-long, to allow their 170 retrieval from the stable. 171 The object assigned to each individual was randomized for each season as well as the 172 order of the horses tested. To avoid the horses seeing the object before testing, the objects 173 were covered from sight when carried around the yard. The novel object tests took place 174 between the hours of 0900 and 1300 and between 1500 and 1800 when the yard was quiet, 175 and the horses were fed. The testing procedure was based on the one used by Dai et al. placed over the box entrance, with the cord hanging over the stable door to keep the object at 178 the height of approximately one meter. The object was kept in this position for the following 179 five minutes and was then dropped to the floor (the objects filled with gravel created a 180 muffled noise). The horse reaction was recorded for the following five minutes. Thereafter, 181 the object was removed from the stable, whilst behavioral monitoring and HR measurement 182 continued for another 15 minutes. 183

Ethical statement 184
All applied methods were non-invasive, and the experimental procedure was 185 approved by Anglia Ruskin University's Departmental Research Ethics Panel. 186

Data processing 187
Raw HR data were purged with a moving average filter to remove biologically 188 implausible outlier values. The following HR variables were calculated: (1) mean HR in 189 beats per minute (bpm) for the ten seconds preceding and following the food presentation, as 190 well as preceding (control) and following the presentation, drop and removal of the object; 191 (2) HR increase in bpm following the food presentation and novel object presentation, drop, 192 and removal, calculated as difference between maximum value and three seconds average HR 193 before the presentation of the stimuli. 194 Behavioral responses of the horses were analyzed from videos using SOLOMON 195 Coder v. beta 17.03.22 (András Péter, www.solomoncoder.com). The behavior of the 196 individuals was analyzed for the five minutes prior to the presentation of the hard feed for the 197 pre-feeding excitement. For the novel object task, the five minutes following the presentation 198 and drop of the object and the two minutes following its removal were analyzed. Behavior 199 was recorded as continuous variables, e.g., feeding as duration of behavior in s per 200 observation period, or frequency of behavior per observation period, e.g., snorting. The 201 classification of the individual in high and low behavioral respondents was based on the 202 11 frequency and duration of vocalizations, pawing behavior, startle response and defecation 203 that the individuals performed during the experimental settings (Table 2) repeatability of the physiological and behavioral measurements was assessed using the 'rptR' 221 package (Stoffel et al. 2017). In particular, we assessed the repeatability of the ten second 222 average HR and HR increase with 1000 permutations for the physiological reactivity data 223 collected for the control, novel object and pre-feeding conditions. The significance level was 224 set at α = 0.05. 225

Seasonal variation and behavioral categorization 227
Horses had a significantly higher mean HR during summer compared to autumn 228 (GLMM1: z = 2.713, p = 0.007), but HR increase was not significantly different between the 229 seasons (GLMM2: z = -0.94, p = 0.348). Out of the 20 horses tested, only four individuals 230 were consistently categorized as high behavioral responders and one individual as low 231 behavioral responder, in both experiments (novel object, pre-feeding excitement) and seasons 232 (summer and autumn; see Table 1 for details). 233 234

Physiological and behavioral responses to experimental situations 235
Average HR of the horses was significantly higher during the novel object experiment 236 compared to the control period (Tukey: z = 5.205, p < 0.001; Figure 1A, Figure 2) and tended 237 to be higher during the pre-feeding excitement compared to the control period (Tukey: z = 238 2.197, p = 0.0662; Figure 1A, Figure 2). Heart rate between novel object and pre-feeding 239 excitement was not significantly different (Tukey: mean: z=-1.887, p=0.133; Figure 1A, 240 Figure2), despite horses showing a significantly lower HR increase in the pre-feeding 241 condition (GLMM2: z= -3.59, p < 0.001; Figure 1B). We found a significant interaction 242 between behavior and experiment affecting HR. Mean HR during the novel object 243 experiment was significantly higher in the group of horses showing a high behavioral 244 response compared to horses showing a low behavioral response (GLMM1: z = -3.139, p = 245 0.002; Figure 1A). This pattern reversed regarding HR increase in the pre-feeding 246 experiment, with the horses showing a low behavioral response having a higher HR increased 247 compared to individuals with a high behavioral response (GLMM2: z = 3.56, p < 0.001; 248 Figure 1B). Finally, the models including individual identity as random factor had a 249 significantly better fit compared to the models without the random effect (ANOVA: mean 250 13 HR: Deviance = 14.632, df = 1, p = 0.001; HR increase: Deviance = 7.984, df = 1, p = 0.005), 251 indicating that the random effect also significantly variance in the data.  High Presence of at least two of the following behaviors: pawing, startle response, defecation, snorting.