Animals and housing

We conducted personality and cognitive assays between March and September 2013 on a captive population of red junglefowl reared at Linköping University, Sweden. All birds originated from two sources (Copenhagen Zoo and Götala Research station, Skara) held in captivity for over 12 generations (Schütz and Jensen 2001) and pedigree bred since 2011. Founders and study populations have been randomly bred and not subject to any intentional selection, and our study animals look and behave similarly to their wild ancestors (Schütz and Jensen 2001). All individuals were artificially incubated and reared together without exposure to their mothers, thereby reducing any influence that maternal effects and accumulated ontogenetic experiences may have on development of cognitive abilities and personality (Stamps and Groothuis 2010).

To be able to assay a large number of individuals across different cognitive tasks and at the same age, we used two batches of chicks, separated by 3 weeks, totaling 100 individuals (n females = 55, n males = 45, n families = 18). All chicks were marked with wing tags to facilitate recognition and housed in same-age, mixed-sex groups (1–3 groups per batch) in indoor enclosures (0.5–3 m2, size increasing with the age of the chicks). Not all chicks took part in all cognitive tasks (see below), and test individuals were chosen to represent all available families. Eight weeks post-hatching, following the first set of cognitive tasks and personality assays (see below), 87 individuals (n females = 45, n males = 42) were moved to an experimental chicken facility, at Linköping University, and kept in a single group until sexual maturity when birds were divided into two groups according to sex. All birds had ad libitum access to water, commercial poultry food, dust bath, and perches, but were deprived of mealworms (used as reward) between test trials.

Experimental setup

In all tests, individuals were tested singly, and between 8 and 18 local time (lights were on 7–19), following descriptions of previous testing in the same population (e.g., Zidar et al. 2017a, b; Sorato et al. 2018). Sex of subjects (because young chicks are monomorphic) and their personality (since personality was measured after cognitive testing) were not known to observers at the time of cognitive testing; thus, we aimed to minimize observer bias.

Cognitive testing of chicks

Discrimination learning

Chicks were handled from day 1 post-hatching and gently habituated to the test arena (28 × 18 × 37 cm, see SI, Fig. S1), and to temporary isolation from their pen mates. Three days post-hatching, the ability to learn a simple association was tested (n females = 35, n males = 27). Chicks had to associate a conditioned stimulus (CS), randomly assigned to be blue or green color, with an unconditioned stimulus (US), ca. one third of a mealworm (see Zidar et al. 2017b, for detailed information on the test setup). Vision is the primary sense in many bird species including the fowl, and novel colors are readily learned in chickens (Osorio et al. 1999; Zylinski and Osorio 2013). We measured reflectance of colors with a light spectrometer (Ocean Optics, Dunedin, FL, USA, USB2000 PX-2 pulsed xenon light source spectrometer), confirming that they are perceived as separate colors by domestic fowl, with the same intensities and color distances from the achromatic point in a two-dimensional color space describing photoreceptor stimulations in the chick eye (see Osorio et al. 1999). Chicks were allowed to make a discriminative choice by moving from a set starting point at one end of the arena to the other end where two bowls (one green and one blue) were placed (ca. 20 cm away, see Fig. S1). The reward in the bowl could not be seen by the chick until just above the bowl. The chick was placed with its head facing away from the bowls and a trial started as soon as the chick turned around and was facing the bowls (i.e., a “trial” was defined as when a chick walked from the starting position to the bowls). The trial ended as soon as the chick had made its choice and eaten a mealworm (i.e., even if a chick chose the incorrect bowl, it was allowed to inspect the correct bowl and consume the mealworm). To reduce individual variation in learning performance due to different levels of habituation or emotional state (i.e., fear, anxiety), each chick was allowed the amount of time it needed to make a choice (which usually occurred within seconds).

To decrease the possibility that a chick would make unilateral choices, i.e., that it may associate the reward with its position rather than the color cue, the stimuli changed place (left–right) between each trial. An additional side preference test showed no side bias at this age (days 6 and 17, see Fig. S2). After each trial and while the two cues were repositioned, the chick was held outside the arena, precluding the possibility to observe the positioning of the reward. A session lasted for a maximum of 15 min; the session could be shorter depending if a chick was not motivated in the test. Each chick was allowed a maximum of eight training sessions, with minimum 1 h of rest between sessions, over 2 days to learn the discriminative task. This was enough for all except one individual to learn the tasks. We assumed that a chick had learned the task if it met a criterion of five correct choices in a row in a single session. If a chick had learnt the task late in the afternoon, it was not tested any further in the day, but instead went through a “refresh trial” in the following morning (which was not included in our measure of trials needed to reach our learning criterion because it took place after our criterion was reached), where it again had to make five correct choices in a row before continuing to the next learning task. This was done to ensure that the association between the stimuli and reward was salient before moving on to reversal learning (see below). In simulations under the null hypothesis scenario of random choice, only an average of 3% of our putative learners could have passed this criterion by chance (Sorato et al. 2018). The total number of trials (choices) until the criterion was reached provided a measure of individual “learning speed.”

Reversal learning

After reaching the criterion for discrimination learning, chicks were assayed in a reversal learning task: the association between cue and reward was reversed, so that the previously unrewarded stimulus was now rewarded and vice versa. The reversal learning task was conducted in a similar fashion as the discrimination learning task with regard to the amount of time given, number of training sessions (with up to eight training sessions over 4 days), alteration of the side of stimuli, measure of learning speed, and learning criterion (Zidar et al. 2017b). Four birds did not learn to associate the reversed stimulus with a reward within the given time frame and were therefore not included in the analysis (sample size analyzed: n females = 33, n males = 24).

Spatial learning

At 5 weeks of age, the previously tested chicks (n = 62) were trained to learn the location of a food reward (a mealworm in a bowl) located 0.5 m after a turn in a U-shaped arena (76 × 114 cm, Fig. S3, Zidar et al. 2017a, b). The chicks could not see the reward from the starting position. This has similarities to spatial tasks used in rodents (Benus et al. 1990), and, although simple, we call it a spatial task. Chicks were trained to form a routine and move directly to the reward after release. If the task was performed five times in a row within the same session, without the chick stopping or turning around (Zidar et al. 2017b), the chick was assumed to have learnt. The number of trials needed to reach this criterion was used as “learning speed.” Six individuals did not engage in the task and were not able to learn the task and were therefore not included in the analysis (sample size analyzed: n females = 30, n males = 26).

For all chicks, each training session lasted ca. 15 min, unless a chick lost motivation for the reward (e.g., did not want to eat the mealworm, were distress calling, or tried to fly out of the test arena). If a training session ended before a routine had been formed, a new session commenced after approximately 1 h of rest.

Cognitive testing of adult female fowl

Discrimination learning

While adult females readily work for food rewards, sexually mature male fowl (≥ 5 months of age, Johansen and Zuk 1998) are harder to motivate to engage in a task using food as reward. Re-testing of learning speed therefore only included females (n = 45). To increase the sample size, we included both females that had been tested in the cognitive tasks as chicks (n = 27) and females (n = 18) that were not. Adult females were tested in a lab room adjacent to their home pen. To avoid memory effects, we used two novel cues consisting of two different color patterns (white background with black circle or black background with white circle; the amounts of black and white were 50–50%). Both novel colors and contrast patterns are readily learned in chickens (Osorio et al. 1999; Zylinski and Osorio 2013); thus, we did not expect differences in task difficulty for chicks and adults. The discriminative task for adults differed somewhat from that used in chicks and was adjusted because of the difference in behavior between chicks and adults. Females were trained to associate one of the two patterns with a reward (mealworm), hidden in a bowl underneath a patterned lid. Same as for chicks, females were randomly assigned to be trained on either of the two stimuli, while the left–right position of the two patterns now changed between trials in a random manner (role of a dice). Training took place on a table with females facing the tester and the two bowls. Three steps were used to facilitate learning: (i) reward was presented on top of the lid, (ii) reward was placed in the bowl half-covered by the lid so that the fowl could see the worm, and finally (iii) the bird had to remove the lid to reach the reward under it. Females were trained to remove the lid to obtain the reward and a choice was noted as “correct” if the rewarded stimulus was chosen. Females were only allowed one choice per trial and were not allowed to collect the reward if they chose the incorrect stimulus. A black shield was placed in front of the female so that she could not see when the stimuli changed places. Similarly, as earlier described for chicks, the criterion for learning was five correct choices in a row, and the number of trials needed until this was obtained was recorded as “learning speed.” All three steps of learning were included when measuring learning speed. All individuals got exactly 100 trials over six training sessions over 2 days to learn the association, enabling us to estimate learning curves (see Fig. S4a). Consistent with learning, individuals chose at random at the beginning of the discrimination test, and the proportion of correct choices increased steadily over trials towards 80% of correct choices (calculated in learning blocks of 5, Fig. S4a). Eleven females failed to learn the association and were not included in the analysis (where 7 were previously tested as chicks).

Reversal learning

Following the discrimination learning task, females that had succeeded in associating a stimulus with a reward (n = 34) were exposed to a reversal learning task. No pre-training was required and the task was conducted in the same way as for the discrimination learning task described above and for 100 trials, except that now the previously unrewarded stimulus was rewarded while the previously rewarded stimulus was not. Females showed a high proportion of errors (80%) at the beginning of the test (matching the rate at the end of the previous discriminative test), and rate of correct choices increased with test progression and reached a stable level of about 80% correct choices (calculated in learning blocks of 5) at the end of the test (Fig. S4b). Twenty-seven females successfully learned the task, while 7 females failed to learn the task and were excluded from the analysis (where 6 of the latter were previously tested as chicks).

Personality assays for chicks

All individuals were tested in three personality assays (a novel arena, novel object, and tonic immobility test, see below), at 4 and 6 weeks of age (Zidar et al. 2017a, b).

Exposure to a novel arena

To quantify variation in exploration and boldness, a novel arena test was performed (Forkman et al. 2007; Réale et al. 2007). Empty, familiar food, and water containers were placed in the arena (76 × 114 cm) to obscure the chicks’ full view and encourage exploration. The substrate was changed (first test occasion: wood shavings; second test occasion: shredded cardboard paper) and placement of containers altered to keep the environment novel between the test occasions (Fig. S6a, b). A chick was gently caught from its home pen and placed at one of the arena’s short sides during darkness. Behavioral measures of its responses started when the lights were turned on again. To prevent the chicks from escaping, a metal grid was placed over the arena. To be able to observe if the birds used the entire arena or only parts of the arena, we divided the arena into six equally sized (imagined) sections (i.e., the birds could not see these sections) and scored how many of these imaginary sections the bird visited during the test. Behaviors were scored live via video cameras connected to computer screens using an instantaneous recording rule every 10 s for the 10 min the test lasted. Latencies were on the other hand scored continuously (i.e., as exact latencies) and behaviors that occurred in very low frequencies were recorded continuously as number of occurring events (Table 1). Behaviors were based on previous personality studies in the fowl (Favati et al. 2014, b, 2016; Zidar et al. 2017a, b). Each chick was scored by one observer out of a total of four, and scoring was blind with regard to the bird’s scores on previous cognitive tests.

Table 1 Behaviors recorded in the novel arena and novel object test Full size table

Exposure to a novel object

To score variation in boldness and neophobia (Réale et al. 2007), a novel object (a spherical, brown/beige plush toy measuring 15 cm and with ca. 2-cm large yellow and black eyes) was placed in the arena. The novel object was placed along one of the short sides inside the arena directly after the novel arena test had finished. This occurred after birds had had 10 min to familiarize themselves with the test arena, which reduce confounding effects of a novel object being presented in a novel environment (Réale et al. 2007). Because the toy may resemble a potential predator, we aimed to capture both neophobia and boldness (Greggor et al. 2015). Lights were turned off while placing the novel object in the arena (birds were standing still in darkness), as far away from the chick as possible. The same behaviors as described above were scored. The novel object test lasted for 10 min starting when the object was introduced and the lights were turned on again.

Tonic immobility

Tonic immobility is a commonly used test of fear responses in birds (Forkman et al. 2007). Birds were calmly collected from their home pen and tested in a lab room adjacent to the home pen. To induce tonic immobility, a chick was placed on its back in a V-shaped wooden stand (20 × 10 cm). A light pressure was applied to its breast while also loosely holding a hand over its head for 15 s. Thereafter, the pressure was released and latency until the chick moved its head (“Latency to move TI”) was recorded. If the chick jumped up on its feet within 3 s following the removal of the pressure by the hand, the procedure was repeated a total of three times. If the bird was still not induced into tonic immobility after three attempts, the chick received a score of 0 s. If the chick stayed immobile more than 10 min, it received a maximum score of 600 s and the test was terminated. The observer avoided direct eye contact with the chick, and the same observer did all tonic immobility tests. The observer was blind to the birds’ learning scores in other tests. The tonic immobility test was performed after the novel arena and novel object tests, and on a separate day from these tests, with the aim that birds had similar initial stress level prior to the test.

Personality assays for adult fowl

At 5 months of age, following sexual maturity, all individuals were again tested in novel arena, novel object, and tonic immobility tests. Novel arena and novel object tests followed each other, as previously described. The arena (2 × 2 m) had peat as substrate and as for chicks, empty, familiar food and water containers were placed within to encourage exploration. To reduce the risk of between-individual differences in stress level prior to the test, the birds were collected from their home pen in a calm manner and with the light switched off. The novel object had the same shape and size as used for chicks, but a different color (black/gray/white). The tonic immobility test was performed on a separate day in a lab room adjacent to the home pen. Behaviors were scored by direct observations. All three tests were otherwise performed and scored in the same manner as described above for chicks (Zidar et al. 2017b).

Statistical analyses

All analyses and model selections described below were conducted in R version 3.2.2.

Individual consistency of responses

Rank order consistency in learning speed across cognitive and behavioral tasks within the same age class, and over time, were explored by the Spearman rank correlations. We adjusted p values for multiple comparisons using the false discovery rate procedure for multiple testing (Benjamini and Hochberg 1995).

Behavioral responses in personality assays

Some behaviors occurred in very low frequencies or showed little variation between individuals (freezing, standing, preening, laying down, foraging, other behaviors (e.g., flapping wings, shaking body)) and were not analyzed further. For the remaining behavioral variables (novel arena: “Locomotion,” “Vigilance,” “Latency to move,” “Latency to explore all areas”; novel object: “Vigilance” and “Number of escape attempts”, Table 1), we used the mean of behavioral responses obtained at 4 and 6 weeks, if correlations of responses obtained at 4 and 6 weeks showed the same direction for males and females. This resulted in pooled data for all behavior, except for “Latency to move TI” (Table S1). Accordingly, this variable was analyzed separately using only data from the behavioral responses obtained at 4 weeks of age (because this was obtained closer in time to cognitive testing).

Behavioral variables from novel arena and novel object tests were reduced by the use of a principal component analysis (PCA) with the R-package “FactoMineR.” Two distinct principal components with eigenvalues ˃ 1 were obtained for chicks (Table 2, Fig. S7) and adults (Table 2, Fig. S8), interpreted as describing individuals that are more or less exploratory (PC1, see Table 2) and shy (PC2, see Table 2).

Table 2 Principal component analysis of behavioral responses of red junglefowl chicks and adults in personality assays (novel arena, NA, and novel object, NO). The first principle component (PC1) is interpreted as primarily describing more or less exploratory individuals; the second (PC2) as mainly describing more or less shy individuals Full size table

Relationship between learning speed and personality

Across learning tasks, some birds failed to learn the task (between 1 and 11 individuals, dependent on task). Several of these birds failed to learn because they did not engage at all in the task, and as a consequence never had the possibility to learn the task. There are also potential problems with arbitrarily given maximum values (e.g., “ceiling effects,” Stamps and Groothuis 2010); therefore, we only analyzed learning speed of individuals that learned the tasks.

To investigate if there were any biases in learning that were due to the type of cue associated with the reward, we performed the Mann–Whitney U tests to assess whether learning speed differed according to the color (chicks) or pattern (adult females) that was matched to the reward.

To explore whether learning speed in the various tasks related to personality, we first evaluated normality by visual inspection of histograms of model residuals. To obtain homogenous variances on non-normally distributed count data (learning speed in discriminative learning and spatial learning), we used the function Box-Cox to find suitable transformations for our response variables. This resulted in log transformation of learning speed in the discriminative learning tasks for both chicks and adult females, and in the spatial learning task. Learning speed in the reversal learning task was approximately normally distributed for both chicks and adult hens and was therefore left untransformed. For chicks, models had Gaussian distribution with identity link and contained the variables “Sex” (i.e., male or female), “Exploration” (PC1), “Shyness” (PC2), “Sex*Exploration” (PC1), “Sex*Shyness” (PC2) as fixed effects, and “Family” added as a random effect. For adults, models did not include “Sex” because no adult males were tested. Models investigating the effect of variation in tonic immobility included the variables “Sex” (i.e., male or female), “Latency to move TI,” and “Sex*Latency to move TI” with “Family” as random effect. We explored full models containing all predictor variables. We centered factors so that zeros would correspond to the average value of the predictor. Results obtained following this approach were robust, because analyzing the data with other distributions (negative binomial or Poisson distribution) gave statistically similar results.