Fig 1.
(A) aerial diagram of test pen and adjoining lobby area with positioning of doors and milk buckets (to scale); (B) three-dimensional projection of test pens and lobby area (not to scale). The coloured boards were fixed to the pen walls, positioned for calves to better distinguish the left and right side of the pen from memory. The colours are therefore a proxy for left vs right side of the experimental pen.
Fig 2.
Timeline illustrating the experimental procedure.
The top graph illustrates the volume of milk given to calves per trial in each experimental session. Numbers on the x-axis correspond to each experimental session. Session 1–4 represent the training and habituation phase. Session 5 corresponds to the pre-disbudding baseline session. Disbudding occurs between session 5 and 6. Session 6–11 correspond to the testing phase. The conditioned place preference test occurred immediately following session 11. Each session took approximately 20 minutes per calf. The layout of each session is also illustrated, with each session consisting of 6 separate trials, alternating between a cool pack (blue box) and ambient (red box). In this example, the cold pack was associated with the blue test pen and the ambient pack associated with the white test pen. These pen colour/treatment paired associations, as well as the pack temperature of the first trial, were pseudorandomised and allocated evenly across participants.
Fig 3.
Changes in response variables post disbudding.
Panel A) shows the change in latencies (from calf-specific baseline session 5) to approach cold or ambient packs as time since disbudding increases. Panel B) shows the change in post-drinking contact duration against time since disbudding. Error bars in each plot represent standard errors. In both plots, data-points at -1h post-disbudding represent session 5 (baseline).
Fig 4.
Latency by session for each trial.
Trial 1 shows no difference between treatments, whilst trial 2 and trial 3 demonstrate a marked increase in average latency from session 7 onwards for the ambient pack only. Session 7 occurs approximately 25h post-disbudding, which corresponds roughly to the duration of effect of the meloxicam anaesthetic [38]. Error bars in each plot represent standard errors.
Fig 5.
Plot of conditioned place preference.
Percentage of time spent within the pen associated with the cool pack. A conditioned place preference test was conducted to see whether calves preferentially spent more time in the side associated with the application of the cool pack. Calves were allowed 15 minutes free access to both test pens with the door between them open, following the conclusion of the last approach-aversion test on Saturday morning. Time spent in each half of the test pen was recorded over the 15 minute duration. Error bar represents standard deviation.