From minutes to days—The ability of sows (Sus scrofa) to estimate time intervals

Accepted Manuscript Title: From minutes to days—the ability of sows (Sus scrofa) to estimate time intervals Authors: Natascha Fuhrer, Lorenz Gygax PII: DOI: Reference:

S0376-6357(17)30208-5 http://dx.doi.org/doi:10.1016/j.beproc.2017.07.006 BEPROC 3484

To appear in:

Behavioural Processes

Received date: Revised date: Accepted date:

3-5-2017 11-7-2017 16-7-2017

Please cite this article as: Fuhrer, Natascha, Gygax, Lorenz, From minutes to days—the ability of sows (Sus scrofa) to estimate time intervals.Behavioural Processes http://dx.doi.org/10.1016/j.beproc.2017.07.006 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.

From minutes to days – the ability of sows (Sus scrofa) to estimate time intervals

Short Title: Time interval estimation in sows

Natascha Fuhrera,b, Lorenz Gygaxa1

aCentre

for Proper Housing of Ruminants and Pigs, Federal Food Safety and Veterinary

Office FSVO, Agroscope, Tänikon, CH-8356 Ettenhausen, Switzerland bAnimal

Behaviour, Department of Evolutionary Biology and Environmental Studies,

University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Corresponding Author: Lorenz Gygax Centre for Proper Housing of Ruminants and Pigs, FSVO Agroscope, Tänikon CH-8356 Ettenhausen [email protected] Tel: +41 58 480 33 84 Fax: +41 52 365 11 90

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Current address: Albrecht Daniel Thaer-Institute of Agricultural and Horticultural Sciences,

Humboldt-Universität zu Berlin, 10115 Berlin, Germany, [email protected]

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Highlights     

time estimation ability of dry sows were investigated intervals of minutes and days were considered at the end of an interval lasting few minutes slight changes in heart rate occurred sows learnt to correctly choose a high food reward every fifth day time estimation in the range of minutes was weaker than in the range of days

Abstract Time estimation helps allocating time to different tasks and to plan behavioural sequences. It may also be relevant to animal welfare if it enables animals assessing the duration of a negative situation. Here, we investigated the ability of dry sows to estimate short and long time periods. We used a variant of the peak-interval procedure and the choice between 2 resources of different quality and replenishment rates to assess time periods in the order of minutes and days, respectively. In the minute-experiment, the sows were trained to expect an interruption while feeding at the end of an interval. Heart rate and heart rate variability slightly and continuously increased and decreased, respectively, towards the end of that interval. In the day-experiment, lasting about 60 days, the sows were increasingly more likely to open the door to a high food reward on the correct day when this food reward was presented every fifth day. We conclude that the sows learnt to estimate time intervals of 5 days after lengthy training but did not accurately learn intervals in the range of minutes. Therefore, they might re-visit replenishing resources after several days, but may not base short-term decisions solely on the passing of time.

Keywords: day; minute; peak-interval procedure; renewable resource; sow; time estimation

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1 Introduction The ability to estimate time intervals has both basic and applied implications. In respect to behavioural control mechanisms, time estimation helps to improve allocation of time to different tasks and to optimally plan, for example, foraging trips to renewable resources (Held et al. 2005; Janmaat et al. 2016). The ability has direct potential fitness consequences, too. In a study with hummingbirds, the variability in the ability to adjust visits to flowers according to nectar renewal-intervals led to 6.3-fold differences in energy gain (González-Gómez et al. 2011). On the proximate level, timing capacities are also necessary for the combined memory of ‘what, where, and when’ (www-memory), an ability on which mental time travel is built (Suddendorf and Corballis 2007, 2010). If animals can remember when specific events happened where, they need at least the capacity to time these events relatively. On the other hand, if an absolute capacity for time estimation had been selected for, the www-memory could have been built up as an extension of that capacity. The brain structures at the basis of timing abilities are phylogenetically old (Corballis 2013). The hippocampus, basal ganglia, and cerebellum are commonly thought to be involved in time perception (Buhusi and Meck 2005; Pouthas et al. 2005; DeVito and Eichenbaum 2010; Underwood et al. 2015). Because many mammals share these brain regions, they have, in principle, the prerequisites to develop analogues to the human ability to perceive and estimate time. Time perception capabilities may thus be widespread among mammals. To assess this phylogenetic spread comparative studies with additional species are needed. Timing abilities may also be relevant for applied questions. Animals were long thought to live in the present moment (Roberts 2002; Mendl and Paul 2008). However, if an animal perceived the length of time during which it remained in a negative or high-arousal situation, its suffering could be inflated (Lea 2001; Mendl et al. 2001; Meyer et al. 2010). This effect could be exacerbated if animals can remember and foresee such events (Lea 2001; Mendl 3

and Paul 2008; Smallwood and O’Connor 2011). In addition, time durations are likely to be overestimated when subjects are in a situation they would rather avoid (emotion–time paradox; Droit-Volet and Gil 2009; Dirnberger et al. 2012; Wearden 2015). Moreover, ‘boredom’ could develop in intensively housed animals that have little need and few possibilities to perform behaviours (Wemelsfelder 2005; Meyer et al. 2010). All of these aspects impact animal welfare negatively and are based on the ability for time estimation. However, timing abilities could also be used to alleviate negative conditions by signalling their duration to the animal (Mendl et al. 2010). Finally, training to estimate time intervals as used here could represent a form of cognitive enrichment (Mendl and Paul 2008, Mendl et al. 2010). Time estimation in non-human animals has mainly been studied in pigeons and rats (Kohman et al. 2006; Onoda and Sakata 2006; Laude et al. 2016). The focus of these studies was on time intervals in the range of milliseconds to seconds, extending in a few studies to about a minute (Lewis and Miall 2009; Grondin 2010). These studies may shed light on neural mechanisms of timing (Grondin 2010). In addition, these short intervals seem important for motor control (Buhusi and Meck 2005). However, they seem too short to be relevant for many ecologically meaningful decisions. Nevertheless, the experimental approaches used in these studies can be transferred to longer intervals. Apart from discrimination studies in which animals are trained to differentiate between intervals varying in relative length using operant conditioning (Crystal 2002; Odum 2006; Zentall 2007; Kim et al. 2009; Heinrich et al. 2016), the so-called peak-interval procedure has been used (Zentall 2006; Sanabria and Killeen 2007). In this procedure, animals are trained to expect a specific event (reinforcer) after a fixed interval (absolute time), and the changes in etho-physiological variables in the course of and towards the end of this interval are observed. In the subsequent non-reinforced trials, the usual finding is an increase in the animals’ response rate followed by a decrease, with a peak at about the time when reinforcement would have happened in the reinforced trials (Roberts et al. 1989; Whitaker et al. 2003; Zentall 2006). In two studies, chickens were tested for their ability to estimate time durations in the ranges of 4

minutes and hours (Petherick and Waddington 1991; Taylor et al. 2002). The results indicated that the chickens could not do so with precision because the peak in their reaction was very wide and flat around the trained time point. Intervals lasting longer than hours have been studied in experiments investigating episodiclike memory (Clayton et al. 2001; Babb and Crystal 2005; Kart-Teke et al. 2006). Such experiments often involve feed and simulate the replenishment of a food source (Laughlin and Mendl 2000; Held et al. 2005; Noser and Byrne 2015; Janson 2016) or the deterioration of food over time (Clayton et al. 2001). Pizzo and Crystal (2007) have shown that rats could learn to time a 48-h interval in that they anticipated feed days that alternated with non-feed days. We wanted to address the ability to estimate time in a non-model species to add to the body of comparative data. At the same time, we chose time-intervals that are ecologically meaningful. We used sows as our subjects because they are long-lived, easily accessible, and results may be relevant also for their housing. In addition, there have been previous studies on pigs showing their memory capacities, which seem to be partly dependent on and interwoven with timing abilities (Held et al. 2002; Mendl et al. 2010). Apart from a first study on www-memory (Kouwenberg et al. 2009), however, no time estimation research has been done with pigs so far. Here, we investigated the ability of dry sows to estimate time intervals in the ranges of several minutes and days. For both interval durations, external timing cues related to the experimental approach were excluded as much as possible so that the sows had to rely on internal mechanisms. We used a procedure motivated by the peak-interval approach to see whether the sows would show an increase in arousal and attentiveness specifically towards the end of an interval of a given length once they had been trained to expect an interruption while feeding at the end of this interval. In addition, we used a setup resembling renewable resources to see whether the sows could predict the replenishment of such resources across several days. We expected that the sows are able to learn to predict renewable resources across 2 days but are unlikely to do so across 5 days because a rule5

of-thumb may be used for the shorter of these intervals, whereas the longer interval does seem difficult to estimate even for humans.

2 Material and Methods 2.1 General Aspects 2.1.1 Animals We used dry sows (Swiss Large White derivation) as our study subjects at the Agroscope research station (Tänikon, Switzerland). Dry sows had been inseminated and were grouphoused throughout their pregnancy until shortly before expected farrowing. The experimental sows remained integrated in the regular pig production cycle of the farm. Apart from the experiments, they were kept in the farm’s standard group housing pens for dry sows. Two dry sow groups of up to 16 sows each were available, and the housing pens consisted of an indoor lying area with straw bedding and an outdoor area. As common for dry sows, they were fed in a slightly restrictive way to avoid over-fattening. Every 3 weeks, an additional batch of 5 to 9 sows was introduced to these pens and available for experimentation. Each sow remained available for approximately 15 weeks before she left the dry sow group for farrowing. In both experiments, the experimental pen arrangement was in the same barn where the dry sow groups were kept. This setup allowed for simple retrieving of the involved sows from their groups. Eighteen sows aged between 2 and 5 years (birth years: 2011- 2014) were included in the 3 batches of the experiment that focused on time periods in the range of minutes (‘minuteexperiment’). Animals that could be touched to be equipped with measurement devices were selected. Three animals from the first batch had to be excluded from the data analysis. Two sows did not feed on the provided food throughout the experiment, and a timing mistake was made with a third sow. Therefore, 15 sows were left for evaluation. 6

Thirteen animals aged between 1and 4 years (birth years: 2012-2015) were included in the 5 batches for the experiment that focused on time periods in the range of days (‘dayexperiment’). One sow from the last batch was withdrawn from the experiment due to lameness. Therefore, the data of 12 sows were analysed. One sow had already participated in the minute-experiment. She had farrowed and raised a litter before participating in the dayexperiment about 14 weeks later, that is, she was back to being a dry sow with a parity increased by one. In the first few days after arrival of a new batch, 2 people were required to single out sows from the dry sow group and to bring them back. The first person walked behind the focal sow and directed her, if necessary, towards the pen door. The second person mainly prevented other animals from approaching the pen doors when the focal sow left the pen or helped if a sow did not want to go back to the pen after the trials. This was mainly achieved by blocking the sows’ path and by verbally and gesturally shooing the sows.

2.1.2 Statistics: general aspects For the analysis, generalised linear mixed-effects models implemented with the function (g)lmer (package: lme4; Bates et al. 2013) were used in R (version 3.3.1; R Core Team 2016). Statistical assumptions were checked based on a graphical analysis of residuals. Pvalues were calculated based on a parametric bootstrap approach for continuous outcome variables (package: pbkrtest; Halekoh and Højsgaard 2014). Due to convergence problems in the parametric bootstrap, a simple likelihood-ratio test was conducted for binary outcomes. We evaluated models that included at most one interaction. If the interaction reached a pvalue >0.05, it was dropped from the model and the p-values for the remaining main effects were calculated. Therefore, either the full model including the interaction or a main-effects model is presented.

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2.2 Experiment on time periods in the range of minutes: ‘minute-experiment’ In the minute-experiment, the sows were introduced individually to a test pen where they could feed for a defined interval of 4, 5, or 6 min (varied between but constant within sows). At the end of this interval, they were interrupted by a human entering the pen. After habituating the sows to the experimental pen and the measurement devices (habituation trials), they could learn the length of the interval (training trials) before they were tested in a final probe trial with no human entering the pen at the end of the interval. If the sows were able to estimate time intervals in the order of minutes, we expected them to show an increase in arousal and attentiveness towards the end of the interval when they expected to be interrupted while feeding. A video illustrating a trial of this experiment is available as supplementary material online.

2.2.1 Experimental design and procedure The minute-experiment was conducted with 3 batches consisting of 6, 8, and 4 sows. The whole experiment ran for 10 weeks between January and March 2016. One sow was lame on one leg during the time of the experiment and was treated with antibiotics for 5 days. Nevertheless, she was mobile and voluntarily walked to the experimental pen. All sows were between days 24 and 90 of pregnancy. During 3 experimental weeks per batch, each subject received 13 trials. One trial was conducted per day with a given sow and included entering a test pen once. In the first experimental week, Wednesday to Friday, 3 habituation trials took place. In the second week, Monday to Friday, 5 training trials took place. In the third week, Monday to Thursday, another 4 training trials took place and a probe trial on Friday. All trials were conducted between 9:00 a.m. and 12:00 p.m. After the habituation phase, each sow’s trials were usually conducted in the same sequence every day to keep the time of day constant. The order of testing was fixed based on how the sows were collected from 8

the dry sow group in the morning before the start of the experiment. The maximum variability in time of day when a trial started was 30 min for each sow. In the second batch, sows of the 2 dry sow groups were included and the sequence of the groups was switched after 3 training trials, leading to a single time change of up to 60 min for trials to start for a given sow. The experimental setup consisted of a test pen with an adjacent waiting pen (Fig. 1A). A wooden box (‘trough’) filled with hay and hidden feed items was available in the test pen. The feed was composed of about 800 g of the sows’ usual middlings (UFA 331-5), 5 slices of apple, 50 g of dry pasta, and 100 g of food pellets (UFA 463-6). Each third of the food pellets was sweetened with 25 cl microcrystalline sugar solution (about 60 weight-% sucrose, 40 weight-% water), 25 g maple syrup, or 25 g alpine herb syrup. The pellets were soaked in the sweetening solutions for about 12 h. Habituation trials showed that the amount of food allowed a single animal to feed for approximately 15 min leaving a small amount of leftover.

2.2.2 Habituation, training, and probe trials During habituation, the sows were trained first to singly enter the waiting pen (Fig. 1A). They were equipped with the measurement devices (see next sub-section) before the gate to the test pen was opened. In the first habituation trial, baiting with food at the gate was necessary for most animals so that they entered the test pen. For 5 animals, baiting was continued in the second trial. The habituation trials lasted 15 min during which the sow was allowed to feed at the trough without any disturbance. At the end of this period, the experimenter entered the test pen, removed the measurement devices, and let the sow return to the group. In the last habituation trial, all sows with one exception readily allowed placement of the measurement devices and voluntarily entered the test pen. Only 1 sow, for the remaining trials, had to be equipped with the devices when she was still in the dry sow group.

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The 9 training trials differed from the habituation trials only in their length and how feeding was interrupted: the experimenter entered the test pen after a delay of 4, 5, or 6 min (measured using a stopwatch) and shook a goad to interrupt the feeding sow and move her back to her group. The goad was a 1 m long stick with a plastic bottle filled with pebbles at its end and a plastic bag attached around it. Each sow was confronted with only 1 specific delay, but durations were varied between sows (5 sows per delay) in order to be able to increase the external validity (i.e. robustness) of our results in respect to the ability of time estimation in sows in the range of several minutes. The probe trial differed from the training trials in that the sows were allowed to feed for a time interval lasting 1.5 times their respective delay time (that is, for 6, 7.5, or 9 min) and that the experimenter entered the pen without the goad to stop the trial. Throughout all training trials and the probe trial, the experimenter was neither visible nor audible to the sow to minimise any influence by the experimenter.

2.2.3 Measurements of outcome variables The measurement equipment consisted of a Polar Team2 breast belt (Polar Electro Oy, Kempele, Finland) to measure the heart rate and heart rate variability and an MSR145 3Daccelerometer (MSR Electronics GmbH, Seuzach, Switzerland; Fig. 1A) to measure activity. In addition, the behaviour of the sows was video-taped. These measurements and recordings were taken in the 2 last training trials and the probe trial. For data processing, R (version 3.3.1; R Core Team 2016) was used. The computer on which the programs Polar Team2 and MSR Reader operated was synchronised with the watch used to record the start time of the trials. The breast belt of the Polar Team2 system contained 2 integrated electrodes, which were placed on the left side of the abdomen directly behind the forelegs, and a Polar Logger to save the data. The Polar Logger was connected to the electrodes via 2 snap-fasteners and 10

registered the time between heart beats (RR-intervals). Signacreme® (Parker Laboratories, Fairfield, USA) electrode cream was used to increase conductivity. After the trials of a single day, the data were transmitted to a computer via a serial interface using the Polar Team2 program (version 1.4.3). The Polar Pro Trainer 5 program (Equine Edition, version 5.10.121) was used to check whether the error rate of a given trial was less than 10% (Langbein et al. 2004). If this was the case, the RR-intervals corresponding to this trial were corrected, otherwise they were discarded (for an exact report, see Statistics). Subsequently, the heart rate (HR; beats per min) and heart rate variability (HRV) parameters were calculated based on the RR-intervals. HRV parameters were the root mean square of successive RR-interval differences (rmssd, ms) as an indicator of parasympathetic activity, and the ratio of the standard deviation of RR-intervals (sdnn, ms) to rmssd (sdnn/rmssd, dimensionless) as an indicator of sympathovagal balance. An R script automatically calculated the corresponding values of 16 and 24 time segments of equal lengths for the training and probe trials, respectively, based on total trial duration. The RR-intervals in the last 15 s in each of these segments were used for comparisons (Langbein et al. 2004). The MSR145 3D-accelerometer was attached on the left foreleg. To fix and protect the logger, self-adhesive elastic bandages (Eurofarm GmbH, Bützberg, Switzerland) were used. The logger automatically stored changes in acceleration along the x-, y-, and z-axes executed by the left foreleg at 1 Hz and was set to log a maximum of twofold force of gravity (the maximum was reached 1 to 7 times per trial in 29 trial segments, or in 3.625% of all trial segments). The data were transferred to a computer via a USB interface and exported as .csv files in the MSR Reader version 5.24.02 for further analysis. An R script automatically calculated the sums of the absolute differences along all axes for 16 (training trials) and 24 segments (probe trial) of equal length based on total trial duration. This resulted in absolute segment lengths of 15, 18.75, and 22.5 seconds. The amount of activity was expressed as per minute.

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From the video, the proportion of time the sow spent in different areas of the test pen (Fig. 1A) was recorded as relative durations with regard to the duration of a trial. In the statistical analysis, only the proportion of time the sow spent at the trough (duration ‘at trough’) was used because the sows spent most of their time in this area. ‘At trough’ was defined as a sow standing (or walking) with at least her head being in or above the trough. The relative duration likely conforms to the logarithmic perception of time by a subject (i.e. the sows), with longer intervals estimated with higher absolute variability (Buhusi and Meck 2005; Lewis and Miall 2009). In addition, behaviours thought to indicate attention were recorded as number of occurrences per minute. These included ‘head lift’ (the sow lifts the head at least until the neck builds a straight line with the back [if ‘near door’, ‘in between’ or ‘near trough’], or the sow holds the head higher than the side walls of the trough [if ‘at trough’]) and ‘stamp’ (the sow lifts one of the hind legs and puts it back on the ground when ‘at trough’). After continuous recording of the behaviour throughout the trials, relative durations and occurrences were automatically calculated by an R script. Again we did so for 16 and 24 segments of equal length for the training and probe trials, respectively. Video analysis was done using the program ETHO that automatically scored the start and end times when the sow switched between pen areas and the times of occurrence of behaviours.

2.2.4 Statistics The behaviours ‘at trough’, ‘head lift’, and ‘stamp’, the activity measured at the leg, and the HR and HRV variables (rmssd, sdnn/rmssd) were used as outcome variables. For the activity measure and all HR and HRV parameters, a log transformation was used so that errors and random effects were normally distributed, whereas the relative duration was logit transformed. The frequencies of behaviours fitted normal distribution best without transformation. 12

Overall, 45 trials (15 sows × 3 trials) were performed in which data were collected. Most loss of data occurred in the HR measurements: 9 animals had error rates less than 10% in all 3 trials, 3 animals in 2 trials (once excluding the first, once the second training trial, once excluding the probe trial), and another 3 animals in 1 trial (always the first training trial). Overall, this resulted in 36 trials (80%) with evaluable data. The reason for the high error rates remained unclear. With regard to activity measurements, data were missing in the last 2 trials of a single sow (total of 43 evaluable trials, 96%), because she did not allow the experimenter to fix the device, i.e. kept pulling the leg away. In the activity data, 1 outlier (visible in the residual plot) was omitted (it occurred in 1 segment of the first training trial, and no reason for its occurrence could be detected). All 45 trials were available for the analysis of the behaviour. The analysis was conducted with 3 subsets of the dataset. Segments 1 to 8, segments 9 to 16, and segments 17 to 24 were analysed separately. This allowed for separating effects at the start and the end of the interval to be learnt. We expected signs of settling down at the start and signs indicating the anticipation of feeding interruption towards the end of each trial. Training and probe trials could be compared for the first 2 subsets (segments 1 to 8 and 9 to 16). This comparison was performed to find out whether the experimenter’s preparation to enter the test pen altered the response of the sows. Additionally, longer lasting reactions could be quantified only when no interruption occurred as was the case in the third subset (segments 17 to 24). The outcomes were evaluated in dependence of the fixed effects elapsed time (the sequence of the segments modelled as a spline with df = 3 allowing for an unrestricted but smooth curve, Hastie 1992) for all 3 subsets and, for the first 2 subsets, type of day (factor with 2 levels: training or probe) and the interaction of elapsed time and type of day. Because the dataset incorporated repeated measurements across time within sows, the day of testing was nested within the identity of the sows and used as random effect.

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2.3 Experiment on time periods in the range of days: ‘day-experiment’ In the day-experiment, the sows were introduced individually to a test pen from where they could choose to enter 2 additional pens. In this experiment sows had one trial daily without interruptions. After habituation to the experimental pen configuration (habituation trials), the sows could learn that a standard-quality reward was offered on one side every second day and a high-quality reward on the other side every fifth day (training trials). After a series of free-choice trials sows received forced choice-trials. In the free-choice trials both pens with potential rewards could be visited. In the forced-choice trials, sows could only make one choice after which the entry to the other pen was blocked. If the sows learnt the intervals, we expected that on any one day with a high-quality reward, they would first choose the corresponding side. A video illustrating a trial of this experiment is available as supplementary material online.

2.3.1 Experimental design and procedure The day-experiment was conducted in 4 batches of 3 sows with 3, 6, and 3 weeks between the start dates of the batches; 1 additional sow was recruited 1 week after trials with the fourth batch had begun. The start dates were between March and June 2016. Recruitment occurred from days 9 to 97 of pregnancy. The sows participated in the experiment for 78 to 82 consecutive days with 1 daily trial. The first 7 trials served as habituation trials. Two sows were temporarily lame on one leg but voluntarily walked to the experimental pen and were therefore allowed to continue participating. The experiment took place between 8:30 a.m. and 12:00 p.m. As in the minute-experiment, the order of testing the sows was fixed after the habituation trials and remained the same until the habituation phase of the following batch. During habituation of a new batch, shifts in day time of the trials could occur for sows of a preceding batch, and when sows finished the

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experiment, shifts could occur for the remaining sows. Thus, the maximum variability in time of day when the trial started was 90 min for any given sow. The test pen configuration consisted of 1 central pen from which 2 pens, 1 on the left and 1 on the right, were accessible through doors which could be unlocked by a remote-control release (Fig. 1B). In each of the 2 adjacent pens, a wooden box (‘food box’) was presented at a distance of 2 m from the entrance (Fig. 1B). Two plastic boxes of the same size were fitted into the wooden box, one on top of the other. The content in the upper box was accessible to the animals and consisted of rewards according to the experimental setup. The lower box always contained food which the sow could smell but not access (see ‘smell agent’ below). Smell was therefore not available as a cue for the tested sows. The boxes were baited when the sows were still in their group housing at a distance of at least 5 m across the barn corridor. As boxes in both pens were always handled in the process of baiting, it is unlikely that the sows could get acoustic cues in respect to which boxes were baited in any given trial. To avoid inadvertent cueing, the experimenter sat at a small table in a narrow compartment between the right experimental pen and the barn wall during the experiment. All experimental pens were video-surveyed, and the video signals were simultaneously relayed to a screen on the table. Thus, by using the remote-control release for the doors and a stopwatch, the experimenter could do recordings without being visible to the sows. Two different food rewards were used that had been assessed in pilot trials. The reward with standard quality (‘standard reward’) consisted of 60 g of the usual middlings (UFA 331-5). The reward with high quality (‘high reward’) consisted of 60 g middlings in combination with 100 g food pellets (UFA 463-6) that were sweetened with 75 g alpine herb syrup. The pellets were soaked in the syrup for about 22 h. Usually, a sow needed about 1 to 2 min to eat the standard reward whereas she needed about 2 to 3 min for the high reward. For habituation, an ‘intermediate reward’ was used consisting of 60 g middlings and 50 g sweetened food pellets. Additionally, 50 g food pellets sweetened with syrup served as ‘smell agent’. 15

2.3.2 Habituation and training trials Sows entered the central pen individually. On the first 3 habituation days, the doors to the adjacent pens were unlocked. The experimenter trained the sow to open the doors and always started with the door to the right pen. The sow was lured with a small amount of middlings and was called. When the sow tried to reach the food, she would open the door after a few seconds. She was then guided to the food box and given time to eat the intermediate reward provided in the food box. Subsequently, the same procedure was repeated on the left side. Finally, the sow was directed back to the central pen, where the trial ended. On days 4 to 7 of the habituation phase, the rewards were no longer the same in the adjacent pens on both sides. On one side, a standard reward was presented, on the other a high reward, according to the side assignment of a given sow. The side of the high reward was balanced across sows, that is, 7 randomly chosen sows got the high reward on the left and 5 on the right side after 1 sow was withdrawn from the experiment. The smell agent was put into the lower box from this stage onwards. Whereas sows had free access to the pens until they had eaten all the food in the habituation phase, the maximum duration of each trial was shortened to 5 min per day from day 4 onwards. With that we wanted to impose a weak time constraint in order to sharpen the sows’ choices. After letting the sow enter the central pen, the experimenter went to his/her position for observation, pressed the release button to unlock the doors, and simultaneously started the time measurement. From now on, it was assured that the experimenter was neither visible nor audible to the sow throughout the trial. In each trial, the sow was free to visit all 3 test pens (Fig. 1B). If a sow had not visited both food boxes after 5 min, the experimenter went into the experimental pens, directed her to the non-visited box(es), and allowed her to eat the reward. This procedure ensured that the sow would learn that there 16

were different reward types, and that she could get a reward on both sides. The sow was also led to the other side if she went to one side first, the door closed behind her, and she did not re-open it within 5 min, which happened in 8 of 52 habituation trials. Sows could re-open the doors by pulling at the door when in the side pens. This happened twice in the habituation phase. From day 8 onwards, food boxes were baited at different time intervals: every second vs. every fifth day specific for the reward type and, thus, for the side. The high reward was used for the long time interval in order to increase the motivation of the sows to go first to this location. The days on which rewards were provided differed between the sows to prevent any information transfer between the animals tested in sequence. Given this temporal pattern, each sow encountered days without reward, with standard reward only, with high reward only, and with both rewards. The maximum time limit for visiting the food boxes remained at 5 min. In 11 of 427 trials (2.6%) that were all between days 1 and 33 of the experiment a sow had to be guided to a baited side after the 5 min delay in order to give her the opportunity to learn the different time intervals for baiting of the boxes on the 2 sides. This procedure of ‘free choice’ was continued until the day after the twelfth high reward. Because the first high reward could be given between days 1 and 5 of the experiment, each sow experienced 57 to 61 free-choice trials. Subsequently, the sows were allowed to enter only one of the side pens. This ‘forced choice’ was achieved by installing mechanical door closers which closed the doors after the sow had entered either the left or the right pen. The intention was to increase the motivation of the sows to make a correct initial choice about which side was worth or worth more visiting. Fourteen forced-choice trials were conducted with each sow. Thus, a sow could, in principle, receive another 3 high rewards.

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2.3.3 Measurements of outcome variables In the free-choice phase, data on the visits to the pens and boxes were collected directly from the video relay. These included the sequence in which the doors to the side pens were opened, the sequence in which the boxes were visited, and the latencies to visit the boxes. The latency to visit a box was defined as the duration from the time point when the doors were unlocked to the time point when the nose of the sow crossed the imaginary vertical line above the edge of the box. In the forced-choice phase, it was only noted which side pen the sow chose on a given day. A choice was defined as opening 1 of the 2 doors but not necessarily entering the respective pen. The door was considered to be opened when the click of the lock was heard. One of 5 people substituted for the principal experimenter (NF) 1 day per week on average. All these people were carefully instructed in respect to the experimental approach, how to fill the food boxes, and how to collect the choice and latency data.

2.3.4 Statistics The first side choice, i.e. whether the side pen with the high reward was visited first, was used as a binomial outcome variable. The latencies to visit the boxes were log transformed so that errors and random effects were normally distributed. Overall, 878 trials (12 sows × 71 to 75 trials) were performed. There was no loss of data. The first 14 days of the experiment were not analysed to allow for some learning of the time intervals. This resulted in 542 free-choice trials (12 sows × 43 to 47 trials) and 168 forcedchoice trials (12 sows × 14 trials).

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The outcomes were evaluated in dependence of the fixed effects day (continuous variable: days 15 to 61 and days 58 to 75 for the free-choice and forced-choice phase, respectively) and type of day (factor with 4 levels: no reward, standard reward only, high reward only, or both standard and high reward provided) and their interaction. Because the dataset incorporated repeated measurements within sows, the identity of the sows was used as the random effect.

3 Results 3.1 Experiment on time periods in the range of minutes With 1 exception (activity in segments 1 to 8), the interaction of the fixed effects type of day and elapsed time did not reach a low p-value for any of the outcome variables and was therefore omitted in the models (Fig. 2). Type of day did not reach a low p-value in any of the comparisons, giving no indication of a difference between training and probe days (Fig. 2). The HR decreased during segments 1 to 8, increased during segments 9 to 16, and seemed to peak in the middle of segments 17 to 24, though this peak was not supported by a low pvalue (Figs 2, 3). The rmssd was roughly constant during segments 1 to 8, decreased during segments 9 to 16, and remained constant during segments 17 to 24 (Figs 2, 3). The sdnn/rmssd showed a low point around segment 6, a peak around segment 11, and increased throughout segments 17 to 24, though this increase was not supported by a low pvalue (Fig. 2). Activity decreased from segment 1 to 6, starting at a higher value in the training trials compared with the probe trial, whereas it stayed roughly constant from segment 6 to 8 and throughout segments 9 to 24, with an increase from segment 17 to 19 (Fig. 2). The proportion of time the sows spent at the trough increased in the first 4 segments, then stayed constant from segment 5 to 16. The proportion of time at the trough was low in segment 17 19

after which it increased and levelled off in segments 19 to 24 (Fig. 2). The rate of head lifts peaked around segment 5, reached a low point in segment 7, and remained constant throughout segments 9 to 16. During segments 17 to 24, there was a steady increase in head lifts, although not supported by a low p-value (Fig. 2). The rate of foot stamps reached a low point in segment 7 and decreased again during segments 9 to 11, but neither the low point nor the decrease was supported by a low p-value (Fig. 2). Qualitatively, a large variability between individuals was apparent in respect to how quickly they went to the food trough and how they searched for the hidden food. As described above (Methods section), 2 sows were excluded from the analysis – one of them stopped feeding at the trough from training day 6 onwards and did not enter the test pen at all on the day of the probe trial. The other sow stopped feeding on the provided food from training day 7 onwards. These incidents indicated that shooing with the goad had a strong effect on the animals as intended.

3.2 Experiment on time periods in the range of days In the course of the free-choice phase, the probability that the sows first opened the door to the side pen with the high reward increased slightly on days with both rewards and more pronouncedly on days without rewards and when only the high reward was provided. It decreased on days when only the standard reward was provided (interaction between type of day and day: χ2 = 9.70, df = 3, p = 0.021; Fig. 4). In the forced-choice phase, the probability that the sows chose the side pen with the high reward increased on the days when only the high reward was provided and decreased on the days with a standard reward. This was similar to the free-choice phase. In contrast to the free-choice phase, the probability also decreased on days without a reward and massively so on days when both sides were baited in the forced-choice phase (interaction: χ2 = 7.42, df = 3, p = 0.060; Fig. 4). 20

The picture of the free-choice phase was supported by the latencies to visit the high (interaction; parametric bootstrap: p = 0.074) and low food reward (interaction; p = 0.006). This support was in the form of low and high latencies in the visits of the high and standard reward, respectively, corresponding to a high probability to open the door towards the pen with the high reward. In 39 of 542 free-choice trials (7.2%), the sows did not completely approach the food box in the pen of which they first opened the door. The box in this pen contained no feed and was in the pen on the right-hand side in 36 of these trials. Nevertheless, the general pattern found in the probability of first approaching the high reward was very similar to the probability of opening the respective door (interaction: χ2 = 13.67, df = 3, p = 0.003).

4 Discussion In two experiments, the capacity of sows to estimate time intervals in the range of minutes and days, respectively, was investigated. This capacity could be important in temporally organising behaviour and as the basis for www-memory. We found only slight ethophysiological changes towards the end of an interval when sows could expect being interrupted while feeding (minute-experiment). Over a period of two months, sows meaningfully adapted their visits to two pens offering either a standard reward every second or a high reward every fifth day (day-experiment). In the minute-experiment, no differences in the outcome variables were apparent in the data between training and probe days indicating that the sows did not use cues from the experimenter. Etho-physiological changes in the second half of the training interval (segments 9 to 16) and the prolonged probe trial (segments 17 to 24) could potentially indicate the sows’ expectation of being interrupted while feeding. Indeed, it seemed that a certain deactivation of the parasympathetic nervous system may have occurred throughout 21

segments 9 to 16: the HR of the sows increased whereas rmssd decreased. This change continued into segments 17 to 24 when the interruption was held off. HR peaked at a time point which was at approximately 125% of the learnt interval. The movement in the test pen in segments 17 to 24 further supported our expectation: the animals left the trough for a short time at just about the time of expected interruption but went back quickly to continue feeding. Finally, the rate of head lifts and foot stamps also increased slightly in segments 17 to 24, indicating that the sows remained expectant of an interruption throughout this last phase. An increase in HR has been related previously to increased arousal in potentially stressful situations in several studies with pigs (Goumon et al. 2013a, b; Henzen 2015; Goumon and Spinka 2016; Zupan et al. 2016). However, the reactions in heart rate and heart rate variability of our sows were weak and continuous. That is, on an absolute scale, the sows were only slightly more aroused when they could expect an interruption of their feeding. Accordingly, it may not be surprising that all other outcome variables did not reflect a consistent change in segments 9 to 16. The change in the cardiac measures was also incremental across several segments. In contrast, a sharp change would have been expected if the sows had estimated the length of the interval. Given the shape of this change, a simple rule of thumb could explain the pattern. For example, satiety itself could have been a cue. To account for that, feeding rate could be reduced in the probe trials of a future experiment e.g. by using obstacles in the feed trough (Held et al. 2005). Nevertheless, the continuous change found here is comparable to the outcomes of other time-estimation studies with shorter intervals. Commonly, the animals’ responses in peak-interval procedures increase steadily throughout the learnt interval and do not show a sharp change shortly before the time of expected reward provisioning (Zentall 2006; Sanabria and Killeen 2007). These patterns were found, for instance, in pigeons (Roberts et al. 1989), rats (Whitaker et al. 2003), and bumble bees (Boisvert and Sherry 2006). The peak at roughly 125% of the interval is consistent with Taylor et al. (2002), who tested chickens. The increase in head lifts and foot stamps until the end of the delayed interval contrasts with the usual finding in peakinterval procedures (Zentall 2006; Sanabria and Killeen 2007). All in all, our approach was 22

somewhat different from the classical peak-procedure which may account for the relatively weak pattern here. With the pigs, the number of trials for training and probing was relatively low. Moreover, they were not expected to become active at the end of the interval based on a reinforcer but encountered an aversive stimulus. In the day-experiment, the sows learnt to differentiate between the time intervals and the value of the corresponding food rewards in that the probability for a sow to visit first the side of the standard or high reward increased on days with a standard or high reward, respectively, in both the free-choice and the forced-choice phase. In the forced-choice phase, this pattern became apparent only after an initial drop of the corresponding probabilities, most likely due to the change in the experimental procedure. These results are similar to those of studies on episodic-like or www-memory. For instance, Janson (2016) showed that capuchin monkeys chose to visit the site with the greatest profitability in the majority of cases. To do so, the monkeys had to remember two different replenishment rates as in our experiment. Scrub-jays have been shown to remember and integrate information on when and where they cached a variety of food items, including degradation rates of different food types (Clayton et al. 2001). In our somewhat simpler circumstances, the sows were able to at least roughly learn when and where they get what food. They even seemed to outperform the rhesus monkeys in the study of Hampton et al. (2005): the monkeys failed to learn that their preferred food, unlike the non-preferred, was available after a short delay (1 h) but not available anymore after a long delay (25 h). The learning conditions were similar to the ones of the sows in that the monkeys had daily access to the experimental arrangement over a period of 60 days. It cannot be fully excluded in our experiment, though, that the sows used some simple rule of thumb of counting mechanism as they could have solved the tasks in counting circadian cycles, that is number of days between the reinforcements. The change in the probability to open first the door to the high reward was different in the free-choice and the forced-choice phases on days with no or both rewards. The probability was roughly constant or increased slightly in the free-choice phase but dropped markedly in 23

the forced-choice phase. The drop on days when both rewards were provided contradicts the findings in the study of Held et al. (2005), in which pigs retrieved the larger of two rewards in trials when they were allowed to retrieve only one reward. Here, it seems that the sows preferred the high over the standard reward if the standard reward could be retrieved afterwards as in the free-choice phase, but they clearly preferred the more easily predictable standard reward over the high reward if forced to make a choice. Because the sows chose the door towards the standard reward more consistently, this could be taken as a sign for a win–stay rather than a win–shift strategy, the latter of which had previously been found in pigs (Laughlin and Mendl 2000). The probabilities of 0.2 (i.e. 1/5) to open first the door to the side with the high reward on days with no reward or a high reward at the start of our experiment are eye catching given that the high reward was provided every fifth day. Nevertheless, the observed initial probabilities of about 0.2, 0.5, 0.2, and 0.4 on days with no, standard, high, and both rewards, respectively, do not coincide with the actual frequency of days with these reward combinations. This actual frequency was 0.44, 0.33, 0.11, and 0.11, respectively. Therefore, the absolute level of these initial probabilities remains somewhat elusive. In conclusion, the results of the day-experiment show that the sows were able to estimate time intervals of 5 days after a lengthy training whereas they did not seem to learn a time interval in the range of minutes in the minutes-experiment. Therefore, they might take advantage of replenishing resources based on these capabilities, but the ability does not seem strong in respect to short-term planning. The experimental approaches that we used could be part of a cognitive enrichment because the sows eagerly participated in the tasks. The consequences of the sows’ capabilities to estimate time intervals as found herein remain unclear in respect to animal welfare.

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Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Compliance with Ethical Standards

Ethical approval All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Conduction of the experiments was approved by the Veterinary Office of the Canton Thurgau (application: TG 05/15).

Conflict of interest Both authors declare that they have no conflict of interest.

Acknowledgements We would like to thank B. Wechsler and two anonymous reviewers for constructive comments on this manuscript. We also thank A. Hagenbüchle and B. Ammann-Koeberl, who were always willing to lend a helping hand and who took care of the pigs. We are additionally thankful to A. Hagenbüchle, B. Ammann-Koeberl, H. Weigele, C. Raoult, and U. Marolf for helping NF with the daily experimental chores in the second experiment. U. Marolf also helped with camera installation, J. Lutz with explanations concerning Polar Team2 and the MSR145, R. Weber with the script for recording behaviour in the first experiment, A. Henzen with providing material and helpful insights, H.-R. Ott with constructing a trough and food boxes, R. Weilenmann with IT support, and S. Schönenberger and B. Kürsteiner with the installation of the electric door opener. We are grateful to Agroscope for the possibility to use the animals and run these experiments.

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Figure Headings: Fig. 1 Test pen configuration in the experiments on time periods in the range of minutes (A, minute-experiment) and days (B, day-experiment) and illustrations of the sows when in the test. Note the heart rate measurement belt and the 3D-accelerometer on the leg in (A) Fig. 2 Changes in heart rate (HR), heart rate variability (rmssd), sympathovagal balance (sdnn/rmssd), activity, proportion of time spent ‘at trough’, and rates of head lifts and foot stamps across the first half (segments 1–8) and the second half (segments 9–16) of the test period and after the time when an interruption of feeding behaviour could be expected by the sows (segments 17–24) on training and probe days in the minute-experiment. Lines reflect model estimates of the main-effects models including type of day and a spline function for the segments (activity in segments 1–8 additionally includes the interaction between these 2 main effects) with 95% confidence intervals based on a parametric bootstrap approach. Pvalues for the different periods are also indicated; i: interactions, t: type of day, s: spline function of the segments Fig. 3 Changes in heart rate (HR, top) and heart rate variability (rmssd, bottom) across the first half (segments 1–8) and the second half (segments 9–16) of the test period and after the time when an interruption of feeding behaviour was expected by the sows (segments 17–24) on training and probe days in the minute-experiment. Box plots reflect raw data (minimum, lower quartile, median, upper quartile, maximum) and lines model estimates with 95% confidence intervals based on a parametric bootstrap approach. Several minimum and maximum values are indicated as numbers Fig. 4 Probabilities of sows opening first the door towards the side pen with the high reward during free-choice (days 15–61, solid lines) and forced-choice trials (days 58–75, dotted lines) in trials with no reward, with the standard reward, with the high reward, or with both rewards in the day-experiment. Lines reflect model estimates including the fixed effects of 31

type of day and day and their interaction, with 95% confidence intervals based on a parametric bootstrap approach. Actual choices of the sows are indicated by the small vertical lines at the Y values of 0 (standard reward) or 1 (high reward)

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