These experiments were conducted at the University of British Columbia’s (UBC) Dairy Education and Research Centre, located in Agassiz, British Colombia, Canada (49°N, 121°W). The animals were cared for according to the guidelines outlined by the Canadian Council of Animal Care (2009). All procedures carried out in this study were approved by the UBC Animal Ethics Committee (A15–0117). All methods were performed in accordance with the relevant guidelines and regulations.

Animals and housing

We used 17 of the 21 animals tested by Lecorps et al. (2018), and added two additional animals housed in the same groups providing a sample of 19 calves. The four animals from Lecorps et al. (2018) that were not included were housed in another group that could not be subjected to the transportation treatment of the current study. Holstein calves were reared according to standard practice at the UBC Dairy Education and Research Centre. Calves were group-housed from 5 d of age. Before weaning, animals had access to 12 L of pasteurized milk as well as ad libitum access to grain, hay and water (see6 for additional details). Calves were weaned gradually by decreasing the amount of milk calves had access to from 12 to 0 L a day over a 2-week period ending at approximately 100 d of age. At the time of testing for the current study (when calves were approximately 4 months old), animals were group housed (n = 9 ± 1 heifers per group) on an open, sawdust-bedded pack measuring 7 × 5 m. Calves had ad libitum access of grain (Hi-Pro Medicated Calf Starter, Chilliwack, BC, Canada with an overall DM of 89.5%; chemical composition shown as % of DM, 90% DM; CP 21%, NDF 19%, ADF 11%), hay and water.

Individual traits

A full description of the assessment of Pessimism, Fearfulness and Sociability can be found in Lecorps et al. (2018). Briefly, animals were tested for judgment bias using a spatial learning task similar to that used on sheep16. Testing was conducted at 25 and 50 d of age. In both sessions, calves were presented with an empty bottle placed at three ambiguous locations located between the previously rewarded and punished training locations. We used the average latency to reach ambiguous locations as a measure of Pessimism.

Fearfulness and Sociability were assessed using four standardized tests (Open field, Novel object, Human reactivity and Social motivation test), commonly used in farm animals4,7. One test was performed per day.

Home-pen social behaviour

Social behaviours were assessed when the animals were 113 ± 8.3 and 118 ± 8.3 d of age, approximately 30 d after weaning off milk. Behaviours were recorded using two digital cameras (WV-CW504SP, Panasonic, Osaka, Japan) placed 5 m above the calves for two periods of 48 h (T1 and T2).

Calf behaviour (lying or standing) and position relative to any neighbouring calves were recorded using 5-min scan sampling for two sessions of 48 h separated by 3 d. Scans in which animals were feeding were excluded to avoid confounding proximity with competition for food. To be considered neighbours, animals were required to be less than one head length apart17. Individual proximity scores were calculated as follows:

$${\rm{Individual}}\,{\rm{proximity}}\,{\rm{score}}=\frac{Number\,of\,neighbours\,while\,resting}{Number\,of\,scans\,resting}+\frac{Number\,of\,neighbours\,while\,standing}{Number\,of\,scans\,standing}$$

Transportation

Each calf was individually transported on two 10-min journeys undertaken on consecutive days when they were approximately 120 ± 8.3 d of age. Calves were tested in random order and one at the time. The calf was gently moved out of the group by a familiar handler and loaded onto an open trailer (140 × 120 × 110 cm). After loading, the trailer remained stationary for 1 min, was then towed by a vehicle for 5 min, was again stationary for 1 min and then again towed for 5 min. The trailer then remained stationary for an additional 1 min before unloading. Total distance was 2 km of paved road including 12 turns at an average speed of 12 km/h and never exceeding 20 km/h. Although, the configuration of the trailer prevented animals from jumping out, the vehicle was immobilized in the rare cases where animals fell inside the trailer (2 times over 38 transportations), giving them sufficient time to stand up before transportation continued.

Physiological and behavioural measurements

Maximum eye temperature was measured with an infrared camera (T650sc, FLIR Systems USA, Boston, MA). Distance between the camera and the eye was kept constant at approximately 1 m. Measurements were taken at three time points: 1) 30 min before any kind of perturbation (Baseline), 2) just after loading in the trailer (Post-loading), and 3) at the end of transportation (Post-transportation). Video files were analysed with the FlirResearchIR software (Flir Systems USA, Boston, MA). For each picture, the ambient temperature at the time of transportation was set in the software during the analysis. A minimum of 1 and a maximum of 5 images were taken for each time point. Images were analysed only if they provided a clear and focused view of the eyes. Baseline values were used to calculate changes in maximum eye temperature after loading and after transportation. In addition, as maximum eye temperature measured at the beginning of unconditioned fear tests were recently shown to predict behavioural phenotype18, we also used maximum eye temperature after loading for further analyses. In addition, we counted the number of vocalizations from the moment the calf entered the trailer until it was unloaded.

Statistical analysis

All analyses were done with R (version 3.4.2). Linear regressions were calculated using the lm function and the associated p-value was calculated via permutation (Package pgirmess19). From the 19 animals used in the study 17 had been phenotyped for personality traits earlier in life. Consequently, when referring to personality traits, the results of this study are based on these 17 animals only.

Personality traits

Principle component analysis was used to define the personality traits, as described in Lecorps et al. (2018).

Social proximity score

We calculated an individual proximity score for each of the two 48 h sessions. Consistency over time was assessed using linear regression. We then averaged the proximity scores over the two time-periods to get one measure per calf and assessed the relationship between this measure and each of the personality traits.

Emotional response to transportation

When multiple pictures were available and fit the criterion (range: 1–5), we averaged eye temperature obtained to provide one measure of maximum eye temperature for the baseline, the post-loading and the post-transportation time-points. Changes in maximum eye temperature in relation to baseline values and maximum eye temperatures were used for the statistical analysis. The total number of vocalizations during the 10 min of transport were live-recorded. As two cohorts of calves were transported on different days with different weather conditions, we included group as an interactive term in all models to control for ambient temperature differences. We assessed consistency over time for all measures (i.e. over the two transportation challenges). For the rest of the analysis we used the averages for each measure taken during both transportations. Consistency over time, the relationship between behavioural and physiological measures, and the relationships with the different individual traits were all assessed using a linear model with P values extracted using a non-parametric permutation approach as described above.