Exploring the effect of pain on the response to reward loss in calves

Ethics Statement

Procedures were approved by the University of British Columbia Animal Care Committee under application A21-0111 and conducted in accordance with the Canadian Council on Animal Care guidelines.38. Reporting followed ARRIVE guidelines.

Animals and housing

The study was conducted at the Dairy Education and Research Center at the University of British Columbia. To our knowledge, no study has used a similar paradigm in calves. To establish an estimate of the sample size, we relied on welfare studies using analogous CNS paradigms but applied to other species: rats (six subjects per treatment9) and pigs (sixteen subjects per treatment groupten). Given this range and our own practical limitations, we opted for a sample size of ten subjects per treatment group. Thirty-five Holstein calves (all female) were initially enrolled in the study. Five calves were removed from the trial: three became ill (desquamation and fever), one showed an extreme stress reaction when moved outside of its enclosure and another did not suffer any damage. dietary restriction before a test. The remaining thirty had a mean (± SD) birth weight of 38.3 ± 4.1 kg and were enrolled at age 39.9 ± 4.1 days.

As standard agricultural practice, calves from the three treatments were mixed in indoor pens (4.9 × 7.3 m, covered with sawdust and each containing eight to ten calves). The calves had ad libitum access to water and hay (RIC; Insentec BV, Netherlands), as well as time-limited access to 12 L of whole milk via a teat (CF 1000 CS Combi; DeLaval Inc., Sweden). To avoid long delays during testing, small repetitions (average number of subjects per repetition = 3.5) were performed.

Device

The experimental apparatus was located in the same barn as the calf pen, approximately 10 to 30 m away. The apparatus was a 1.8 × 1.2 m starting box leading to a 3.6 × 2.4 m enclosure through a vertical door (Fig. 2A). Directly in front of the starter box was a baby bottle and rubber teat mounted on rails, with an algometer (FPX 25, Wagner, Greenwich, USA) installed behind the bottle to measure the maximum pressure applied to the bottle (Fig. 2B).

Figure 2
Figure 2

The calves were brought to the starting box, the vertical gate was raised and the calves could access a milk reward (0.5 L during training, 0.1 L during testing) in the test pen (A). The bottle containing the milk reward was mounted on rails with an algometer positioned behind the bottle to measure the maximum pressure applied by the calf (B). Illustrations by Ann Sanderson.

Training

The trial was divided into three phases over seven days: training (three days), treatment (one day), and testing (three days). During training, calves were subjected to an overnight feed restriction (from 10:00 p.m.) to ensure high motivation for milk rewards over repeated trials. Around 10:00 a.m., the calves were introduced individually into the apparatus, in no defined order, then placed in the starting box. The vertical gate was raised and calves were allowed to approach and drink a 0.5 L milk reward from the bottle (this amount was based on previous studies of motivational trade-offs in calves).37,39). The latency to contact the bottle (with mouth or tongue), the latency to complete the reward, the number of vocalizations and the maximum pressure applied to the bottle were recorded live. The calf was then returned to the starting box, the bottle was refilled and two more trials were carried out (i.e. for a total of three trials/day). Once these trials were completed, the calf was returned to its enclosure with full access to its daily milk intake of (12 L/d). Training took place over three consecutive days, for a total of nine training trials. During the first day of training (for all three trials), no signal was given to the calf for the first minute after the starting box door was opened. After one minute, auditory (calls/whistle) and tactile (finger pacifier) ​​signals were given by the experimenter from outside the test enclosure to attract the calf’s attention to the bottle. If these signals failed after an additional minute, the experimenter entered the test pen and led the calf toward the bottle.

During the second and third days of training, no signals were given. If the calf did not approach the bottle within two minutes, the trial was recorded as a non-approach (and zero pressure applied to the bottle). Once a calf approached the bottle, it had three additional minutes to complete the reward.

Treatments

Calves were pseudo-randomly assigned to one of three treatments (Disbudding, Disbudding+Analgesia, or imposture ; ten calves each). Treatment allocation was balanced according to age and birth weight ( Disbudding: 40.7 ± 4.3 days, 38.7 ± 3.9 kg; Disbudding + Analgesia: 39.2 ± 7.0 days, 38.0 ± 5.6 kg; Fake: 39.7 ± 6.0 days, 38.3 ± 2.4 kg). On the day of treatment, calves were not subject to any dietary restrictions and underwent treatment in their group pen at approximately 10:00 a.m. Regardless of treatment, calves were weighed and received a multimodal pain mitigation strategy including sedative, local anesthesia, and analgesia. The sedative was used to facilitate monitoring of injections and disbudding (xylazine 0.2 mg/kg subcutaneous, Rompun 20 mg/mL, Bayer, Leverkusen, Germany). Once sedation was achieved (decubitus and eye rotation, approximately 10 min), a local anesthetic was injected in the form of a corneal nerve block to alleviate the acute pain of the procedure (5 ml on each side, lidocaine 2%, epinephrine 1:100,000, Lido-2, Rafter8, Calgary, AB, Canada), an NSAID was administered to minimize inflammation (meloxicam 0.5 mg/kg subcutaneous, Metacam 20 mg/mL, Boehringer Ingelheim, Burlington , ON, Canada), and the horn bud area was shaved with an electric clipper. Ten minutes after the lidocaine injection, a pinprick test was performed on the horn buds to test the pain reflex. For calves in the Disbudding And Disbudding + Analgesia treatments, a preheated electric dehorner (X30, 1.3 cm tip, Rhinehart, Spencerville, IN, USA) was applied to both horn buds until a dark, consistent ring formed around each bud (requiring approximately 10 to 15 s). The calves of FakeThe group was treated identically but instead of being disbudded, only pressure on the horn buds was applied with the plastic handle of the dehorner. Once the procedure was completed, the calves were placed in sternal recumbency and allowed to recover in the pen. The magnitude and duration of the effects of NSAIDs after disbudding remain uncertain. 18the calves of Disbudding+AnalgesiaThe group received an additional injection of NSAID (ketoprofen, 3 mg/kg, subcutaneous, Anafen, 100 mg/mL, Boehringer Ingelheim, Ontario, Canada) 1 h before each of the three testing sessions to provide additional control pain at the time of the test. Based on a previous study on the effectiveness of ketoprofen after disbudding29we expected ketoprofen to produce analgesic effects for up to 2 hours after treatment.

Trials

Within three days of treatment, calves were tested for sensitivity to reward loss. The tests were similar to training: calves were brought individually to the apparatus after an overnight food restriction and allowed access to a milk reward three times in a row (for a total of nine trials), but during tests, the reward was reduced to 0.1 L. The time given to the calves to approach and drink the reward was adapted to their performance during training. The maximum pressure applied to the bottle, the number of vocalizations and the approach latency time were recorded. The calves ofDisbudding+AnalgesiaThe group received an additional injection of NSAID (ketoprofen, 3 mg/kg, subcutaneous, Anafen, 100 mg/mL, Boehringer Ingelheim, Ontario, Canada) 1 h before each of the three testing sessions. After each session, the calves were returned to their pen and again had access to their full milk allocation (12 L). After the three days of testing, the calves were returned to routine care on the farm.

statistical analyzes

A mixed model was carried out on each result (maximum pressure, vocalizations and approach latency) during the test phases (post-processing) using the lme4 package in R.40. For pressure and latency, data were log transformed to match model assumptions of linearity, normality, and homoscedasticity. For the number of vocalizations, we used a mixed Poisson model. The fixed factors were treatment (2 df), test day (1 df), daily trial (1 df), and their interaction (3 df). The daily trial, nested within the day and calf identification, was included as a random factor. Significance and trend thresholds were set atP.≤ 0.05 andP.≤ 0.10, respectively. Data (additional information 1) and R code (additional information 2) are available in supplementary materials.


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