This work was undertaken at the University of California Davis Dairy Teaching and Research Facility from June to September 2018. All experimental protocols were approved by and carried out in accordance with the University of California Davis Institutional Animal Care and Use Committee (protocol # 20505).
Treatments
We enrolled all female calves born between June 19 and September 1 2018, for a total of 28 Holsteins and 8 Jerseys. Our sample size was determined by the availability of calves being born in our herd of approximately 105 lactating cows during this period. Calves were blocked by birth order and randomly allocated to 1 of 3 treatments balanced for breed: disbudded the morning of (Day 0) or 21 days before (Day 21) the startle test, or sham-disbudded (Sham, n = 12/treatment). Among the control calves, half were sham-disbudded the morning of the test, whereas the other half underwent the procedure 21 days earlier. Birth weights were similar across treatments (mean ± SD; Day 0: 35 ± 5 kg; Day 21: 35 ± 6 kg; Sham: 36 ± 9 kg). The startle test occurred between 25 and 32 days of age for all calves. Thus, all Day 0 calves and half of the Sham calves were disbudded between 25 and 32 days of age, and all Day 21 calves and half of the Sham calves were disbudded between 4 and 11 days of age. This design meant all animals were at the same stage of cognitive and motor development during data collection. This was a priority for us because we expected age to strongly influence behavioural responses during the startle test. While it is also possible that disbudding at different ages may affect responses, previous research suggests disbudding has similar outcomes across this range13,15,20.
Animal husbandry and housing
Immediately after birth, calves were housed individually in outdoor enclosures consisting of a plastic hutch (2.0 m long × 1.5 m wide) and a wire-fenced pen (2.0 m long × 1.5 wide × 0.9 m high). The enclosures were spaced 0.5 m apart and bedded with sand approximately 15 to 20 cm deep.
Calves were bottle-fed colostrum twice a day for 5 days. From 5 days of age, calves received milk replacer (26% CP and 16% fat, 15% total solids; Calva Products Inc., Acampo, CA) in bottles at 0645, 1245, and 1845 h. At each meal, Holsteins were fed 1.9 L from 1 to 13 days, 2.4 L from 14 to 23 days, and 2.8 L from 24 days. Jerseys received 1.4 L from 1 to 13 days, 1.9 L from 14 to 23 days, and 2.4 L from 24 days. Water and starter (18.3% CP, 2.8% fat, 4% crude fat; Associated Feed & Supply Co., Turlock, CA) were provided ad libitum in buckets. As part of a separate concurrent study, 11 calves (3 Sham, 3 Day 21, 5 Day 0) received chopped mountain grass hay (34% CP) ad libitum.
Disbudding
Disbudding occurred between 730 and 1000 h. For the procedure, the calf was restrained in a head device in her home enclosure21. A 5 × 5 cm patch of hair was clipped with a size 40 electric razor blade on each side of the head to locate the horn bud. We used a 20 gauge × 25 mm needle to administer a cornual nerve block consisting of 5.5 mL buffered lidocaine (2% lidocaine hydrochloride diluted with 8.4% sodium bicarbonate in a 10:1 ratio). If the horn bud was not numb after 10 min, as assessed by pinprick, we gave an additional 2 mL of buffered lidocaine (13% of horn buds). An electric cautery iron (X50, Rhinehart Development Corp., Spencerville, IN) was fitted with a 1.3 cm tip and heated to 439 ± 15 °C (mean ± SD). It was applied to the horn bud for 17 ± 5 s (mean ± SD). Immediately before disbudding, the calf received approximately 1 mg/kg of meloxicam tablets in a gelatin capsule (3.5 g; Torpac Inc., Fairfield, NJ). For Day 0 calves, meloxicam was given after the startle test had occurred later that same day (maximum 12 h later) to ensure the calf was in a drug-free state during the test. Sham-disbudded calves received the same treatment, with the exception that the iron was ambient temperature and the gelatin capsule was empty. Sham calves did not receive meloxicam because the Animal Medicinal Drug Use Clarification Act limits nontherapeutic off-label use of this drug22. SJJA performed all disbudding procedures.
Arena
We tested calves individually in a single 10-min period in a shaded outdoor arena bedded with 10 to 15 cm of sand. The arena was divided into a waiting pen (2.0 × 1.5 m) and a test pen (3.0 × 5.5 m) constructed of 0.9 m high wire panels (MidWest Homes for Pets Foldable Metal Exercise pen, Muncie, IN). A rolling gate provided access between the pens (Fig. 1).
Aerial view of the arena used for startle tests, including the position of the milk bottle and speaker used to broadcast the startle noise. Figure is drawn to scale.
A bottle containing 500 mL of the calves’ regular milk replacer was secured to the panel opposite the entrance to the test pen. The bottle was fitted with a rubber teat positioned 80 cm above the ground. Between calves, a fresh bottle was placed in the arena and urine and feces were removed with a shovel.
Testing procedure
Calves were habituated to the arena for 15 min daily between 700 and 1100 h for 3 consecutive days before the startle test. Calves were brought to the arena in the same order each day, with order balanced across treatments. During habituation, no startle stimulus was delivered, but otherwise the same procedure followed on test days was applied.
The startle test occurred between 1530 and 1800 h (Supplementary Video S1). The calf was transported from her home pen to the waiting pen in a cart (Caf-Cart, Raytec, Ephrata, PA). The test began when the gate providing access to the test pen was opened and ended after 10 min. The gate was closed behind the calf after she had entered so that the waiting pen was inaccessible during the test. Three observers were seated quietly 3.5 m away from the pen during the test, and were partially concealed behind a tree branch. One observer remotely controlled the speaker broadcasting the startle noise, while the other two observers were present to respond if a calf escaped from the arena (only one calf jumped out, on the first day of habituation, and was promptly escorted back into the pen). Calves showed no apparent responses to the observers and had no visual contact with other animals.
As soon as the calf’s mouth was within a tongue’s reach of the teat, a 0.4 s, 105 ± 2 dB burst of white-noise was emitted through a wireless speaker (OT4200 Big Turtle Shell, Outdoor Tech, Laguna Hills, CA) mounted directly behind the bottle. The noise was created using an online signal generator23. We measured the sound level using a decibel meter (BAFX Products, Milwaukee, WI) held 30 cm in front of the bottle, approximating the distance of the calf’s ears to the source.
Behavioural data collection
Tests were recorded with a camcorder (HC-V180, Panasonic, Kadoma, Japan) positioned on a tripod approximately 3 m away from of the pen. One trained observer, blinded to the treatments, scored behaviours in all videos taken of the startle test and the third day of habituation (Table 1). Videos were analysed using BORIS (Behavioural Observation Research Interactive Software24). Intra-observer reliability was calculated using 12 randomly selected videos of the startle test (Intraclass correlation coeffcient ≥ 0.95).
Accelerometers (Hobo Pendant G Acceleration Data Logger, Onset Computer Corporation, Bourne, MA) were used to assess the magnitude of the startle response. On habituation and test days, we fitted calves with a triaxial accelerometer set to record acceleration in the x-, y-, and z-axis every 0.05 s. The accelerometer was placed in a pouch, strapped around the right hind leg, and secured with Vet Wrap (Co-Flex, Andover Coated Products Inc., Salisbury, MA) while the calf was in the waiting pen of the arena, immediately before the gate to the test pen was opened. Data were downloaded using HOBOware Pro Software (Onset Computer Corporation, Bourne, MA). To calculate the magnitude of the startle response, we summed total acceleration in all 3 axes over the startle duration for that calf. Total acceleration was calculated as the square root of the sum of squared acceleration in each axis25. No startle response was recorded for one calf who did not approach the bottle on the test day.
All calves were weighed the morning of the startle test (mean ± SD; Day 0: 56 ± 10 kg; Day 21: 55 ± 9 kg; Sham: 55 ± 11 kg).
Wound healing and sensitivity
We measured sensitivity via mechanical nociceptive thresholds around the horn bud area 1 to 2 h after the startle test using a digital algometer fitted with a 4-mm-diameter round rubber tip (ProdPlus; TopCat Metrology Ltd., Little Downham, UK). The calf was restrained in the head device in her home pen and blindfolded to reduce responses to visual cues. We then applied an increasing amount of force to the edge of the disbudding wound, or intact horn bud for sham calves, as described previously13. The test ended when the calf moved her head or a maximum cut-off point of 10 N was reached. We repeated the test if a fly landed on the head, a loud noise occurred, or the calf urinated or defecated. If a test was interrupted 3 times, it was abandoned (0% of tests).
Wound sensitivity was tested at the lateral and caudal edges of each wound or the equivalent location on sham calves. The order of test sites was: left lateral, left caudal, right caudal, and right lateral. To ensure force was applied at a consistent rate, personnel operating the algometer were trained and met a set of rigorous criteria before performing the tests13. We calculated the rate that force was applied in each test from video recordings (0.29 ± 0.10 N/s; 2% of videos missing). If force was increased at a rate < 0.1 or > 0.6 N/s or video was missing, the data were excluded (3% of tests). Due to the nature of the tests, the operator of the algometer was not blind to treatment.
We took digital photographs of the wound with a DSLR camera (D5300; Nikon Corp., Tokyo, Japan) after sensitivity testing was completed. Photos were taken 15 cm from the wound. One person scored the photos for tissues present in the wound bed using a 0/1 scoring system13. Due to the clear differences in Day 0 and Day 21 wounds, the scorer was not blind to treatment.
Statistical analysis
Due to the presence of zeros in the data, we used zero-inflated beta regressions to assess the effect of treatment (Sham, Day 0, Day 21) on the proportion of time suckling on the third day of habituation and during the startle test. A zero-inflated beta regression is a mixture of two models: a beta model for estimating non-zero proportions and a logistic model for estimating the probability of zeroes26. This approach allowed us to infer treatment effects on both the occurrence and duration of suckling. General linear models were used to test the effect of treatment on the duration of the startle response and its magnitude as measured from the accelerometer data.
We analyzed the effects of treatment on latency to approach the bottle and latency to return after startling using parametric survival regression models with a log-logistic distribution. Days on which the calf did not perform the behaviour within the allotted time (15 min for habituation, 10 min for startle test) were handled as right-censored data.
We ran a general linear model to test the effect of treatment on wound sensitivity. A preliminary analysis indicated that there was no effect of side (left vs right) or location (caudal vs lateral) on wound sensitivity, so we averaged data for each calf into one score.
Data were analysed in R, version 3.5.227. General linear models were fitted using the “lm” function in base R. We confirmed homogeneity of variance and normality using residuals vs fits plots and Q-Q plots, respectively. Beta and survival regressions were performed with the “glmmTMB” function in the glmmTMB package version 1.0.028, and the “survreg” function in the survival package version 2.3829, respectively. If treatment effects were identified in any of the models (P < 0.1), we calculated pairwise contrasts using Tukey’s method with the emmeans package, version 1.4.530.
Source: Ecology - nature.com
