Effects of multimodal pain management strategies on acute physiological and behavioral response to cautery disbudding in neonatal goat kids

Dairy goat kids are commonly disbudded in the United States without pain relief. Our objective was to identify an efficient pain management strategy by monitoring changes in plasma biomarkers and behavior of disbudded goat kids. A total of 42 kids (5–18 d old at the time of disbudding) were randomly allocated to 1 of 7 treatments (n = 6/treatment): sham treatment; 0.05 mg/kg i.m. xylazine (X); 4 mg/kg subcutaneous buffered lidocaine (L); 1 mg/kg oral meloxicam (M); xylazine and lidocaine (XL); xylazine and meloxicam (XM); and xylazine, meloxicam, and lidocaine together (XML). Treatments were administered 20 min before disbudding. One trained individual, blinded to treat-ment, disbudded all kids; sham-treated kids were handled similarly except the iron was cold. Jugular blood samples (3 mL) were obtained before (−20, −10, and −1 min) and after (1, 15, and 30 min, and 1, 2, 4, 6, 12, 24, 36, 48 h) disbudding and analyzed for cortisol and prostaglandin E 2 (PGE 2 ). Mechanical nociceptive threshold (MNT) testing was performed at 4, 12, 24, and 48 h after disbudding, and kids were weighed daily until 2 d post-disbudding. Vocalizations, tail flicks, and struggle behavior during disbudding were recorded. Cameras were mounted over home pens; continuous and scan observations over 12 periods of 10 min each, in the 48 h after disbudding, captured frequency of locomotion and pain-specific behaviors. Repeated measures and linear mixed models assessed treatment effects on outcome measures during and after disbudding. Models accounted for sex, breed, and age as random effects, and Bonferroni adjustments accounted for multiple comparisons. At 15 min after disbudding, XML kids had lower plasma cortisol concentrations compared with L (50.0 ± 13.2 vs. 132.8 ± 13.6 mmol/L) and M kids (50.0 ± 13.2 vs. 145.4 ± 15.7 mmol/L). Cortisol was also lower in XML kids over the first hour after disbudding compared with L kids (43.4 ± 9 vs. 80.2 ± 9 mmol/L). Change from baseline PGE 2 was not affected by treatment. Behaviors observed during disbudding did not differ by treatment group. Treatment affected MNT such that M kids were more sensitive overall compared with sham kids (0.93 ± 0.11 kgf vs. 1.35 ± 0.12 kgf). None of the recorded post-disbudding behaviors were affected by treatment, but study activities did influence behavior over time, with kid activity levels declining in the first day after disbudding but largely recovering thereafter. We conclude that none of the drug combinations investigated here appeared to fully attenuate pain indicators during or after disbudding, but triple modality seems to have offered partial relief compared with some of the single-modality treatments.


INTRODUCTION
Disbudding of goat kids is a routine procedure in the US dairy goat industry (USDA-NAHMS, 2020) and is most commonly performed by thermal destruction of the cornual epithelium with a cautery iron in the first 2 to 3 weeks of life (Smith and Sherman, 2009b;USDA-NAHMS, 2020).Cautery disbudding provokes a welldocumented pain response, as demonstrated through an increase in blood cortisol concentrations (Alvarez et al., 2015;Nfor et al., 2016;Hempstead et al., 2018a) and increased vocalization during the procedure (Alvarez et al., 2015, Chandrahas et al., 2013) compared with kids undergoing sham disbudding.However, fewer than a third of US dairy goat producers report using any analgesics or anesthetics for the procedure (Valdmanis et al., 2007;USDA-NAHMS, 2020).
Despite the prevalence of disbudding among US dairy goat herds, relatively little is known about the efficacy of different pain management strategies for the procedure.A multimodal approach to pain management takes advantage of different modes and sites of action of various analgesic agents and should thereby provide superior analgesia compared with the use of single-modality agents (Hellyer et al., 2007).
Local anesthesia alone (e.g., lidocaine) acts at sodium channels to prevent generation and propagation of action potentials and can reduce behavioral and physiological responses to cautery disbudding in calves (Stock et al., 2013).However, local lidocaine administration does not appear to reliably reduce pain-related behaviors (Chandrahas et al., 2013;Ajuda et al., 2020) or blood cortisol concentrations (Alvarez et al., 2015;Hempstead et al., 2020b) in cautery-disbudded goat kids.It is possible that these results could be explained by the acidic nature of lidocaine, causing administration to be painful beyond the initial "prick" of injections; in humans, buffering lidocaine with sodium bicarbonate reduces reported pain (Orlinsky et al., 1992;Matsumoto et al., 1994).Surprisingly, a recent study showed that cornual injections of buffered lidocaine provoked heightened aversive responses in dairy calves (Adcock and Tucker, 2022).Nevertheless, limited research has explored the efficacy of buffered lidocaine in reducing disbudding pain in ruminants, and to our knowledge no published studies have explored its application in goat kids for disbudding.Additional technical challenges are associated with lidocaine administration in goat kids, as lidocaine must be injected at 2 sites per horn bud (Fubini and Ducharme, 2016), and toxicity is a greater concern in small ruminants such as goats (Smith and Sherman, 2009a;Venkatachalam et al., 2018).Collectively, these issues are likely to be a substantial barrier to adoption of local anesthesia by goat producers and veterinarians in the field.
Xylazine hydrochloride is an α-2 adrenergic receptor agonist that is reported to provide both sedation and analgesia in ruminants (Abrahamsen, 2008;Shah et al., 2013), and has been shown to reduce pain response both during and following cautery disbudding in dairy calves when used in combination with a local anesthetic block and a nonsteroidal anti-inflammatory (Reedman et al., 2021).Its use for disbudding goat kids has been described (Ingvast-Larsson et al., 2011) and is often used by veterinarians in the field (Fubini and Ducharme, 2016), but to our knowledge its use in goat kids has not yet been assessed in experimental or field trials.In adult goats, 0.1 mg/kg administered intravenously produces optimal sedation and analgesia for 29 ± 4 min and 41 ± 3 min, respectively (Shah et al., 2013).The advantage of xylazine over other sedatives or anesthetics is that it is economical, fast-acting, and noncontrolled, meaning its use does not require special licensure.
Finally, relatively little is known about the application of postoperative analgesics for disbudding in goat kids.Meloxicam (a nonsteroidal anti-inflammatory drug) has been shown to reduce behavioral (injected subcutaneously; Hempstead et al., 2018b) and acute cortisol responses to cautery disbudding in kids (injected intramuscularly; Nfor et al., 2016).Because meloxicam is available as an oral tablet, administration in the field may prove a practical option for producers.Although there is some suggestion that oral meloxicam may mitigate behavioral indicators of post-disbudding pain in goat kids (Hempstead et al. 2018b), administering oral meloxicam 60 min before disbudding did not seem to affect cortisol or behavior indicators in the 30 min after disbudding (Hempstead et al. 2020b).To our knowledge, the longer-term effects of oral meloxicam and its use in combination with other modalities has yet to be explored.
Ultimately, cautery disbudding in neonatal goat kids without the use of pain mitigation presents a welfare challenge.It is likely a management challenge as well, in addition to being a practice that conflicts with broader social values about animal welfare on farms (Robbins et al., 2015).Most of the current pain management strategies utilized by veterinarians in the field are not feasible for producers' implementation due to the use of controlled drugs or technical difficulty (Smith and Sherman, 2009b).This represents an important gap in knowledge, since 75% of producers perform these procedures themselves (USDA-NAHMS, 2020).Thus, a clear need exists to identify an optimal combination of sedation, anesthetic, and postoperative analgesia for the disbudding procedure in goats, and to identify pain management strategies that are implementable for producers in the field.The primary objective of this study was to determine the effect of multimodal pain management on the cortisol response to cautery disbudding.Secondary objectives were to determine the effect of multimodal pain management on the acute pain response, measured through prostaglandin E 2 (PGE 2 ) response, behavioral expression, and mechanical nociception.We hypothesized that multimodal pain management would result in a larger reduction in serum cortisol and other measures associated with acute pain, compared with goat kids who received single-modality pain management.

MATERIALS AND METHODS
All study activities were conducted following approval by the University of Minnesota Institutional Animal Care and Use Committee (protocol no.1808-36275A).

Kid Management and Allocation to Treatment Group
This study was conducted in March 2019 at the University of Minnesota, College of Veterinary Medicine's Large Animal Teaching Barn (St.Paul, MN).Goat kids (n = 42) of mixed sex and breed (Alpine, Saanen, Toggenburg) and between the ages of 1 and 14 d were obtained from a commercial dairy goat farm near St. Paul, Minnesota.All kids enrolled into the study were examined by a veterinarian upon arrival and administered a prophylactic treatment of tulathromycin (Draxxin, Zoetis Inc.) at 2.5 mg/kg.Management of goat kids adhered to standards outlined in the American Society of Animal Science Guide for the Care and Use of Agricultural Animals in Research and Teaching (ASAS, 2020).Kids were housed in 6 pens (2.1 × 3 m; 7 kids per pen; 1 kid per treatment group, as will subsequently be described in detail) in a temperature-controlled barn.Pens were equipped with 10 cm of sawdust bedding and free-choice water.Kids were individually bottle-fed pasteurized goat milk 3 times per day (0600, 1200, and 1800 h) at a rate of 20% BW/d.Kids were allowed to acclimate for 3 d before commencement of study-related activities (kids ranged in age from 5 to 18 d at the time of disbudding).Kids exited the study at 48 h after disbudding and were returned to their home farms 72 h after study exit.

Treatment Allocation and the Disbudding Procedure
At arrival, kids were blocked by sex and breed and randomly allocated (random number generator in Excel, Microsoft Corp.) to 1 of 7 treatment groups, identified by their individual numbered tag and a colored neck chain corresponding to treatment group.All study technicians and data collectors were blinded to treatment through the duration of the study.Treatment groups were as follows: (1) simulated disbudding (sham treatment); (2) sedation with xylazine (X; 0.05 mg/kg i.m.); (3) oral meloxicam (M; 1 mg/kg orally); (4) a nerve block with sodium bicarbonate-buffered lidocaine (L; 4 mg/kg subcutaneous); and then combinations of (5) xylazine and lidocaine (XL); (6) xylazine and oral meloxicam (XM); and (7) xylazine, oral meloxicam, and lidocaine (XML) at the previously indicated doses and routes of administration (described in detail subsequently).
All kids were shorn on their neck and over their horn buds at least 1 d before disbudding, to reduce handling stress on their day of disbudding.Kids underwent disbudding (or simulated disbudding, sham treatment) between 0830 and 1100 h, at least 2 h after their morning milk meal, on 1 of 2 sequential days.(Due to time limitations associated with study procedures, half the kids were disbudded on one day, and the remainder were disbudded the next day.)Disbudding order was randomized within pen such that a kid from each treatment group was disbudded in each pen "block."Disbud-ding was performed in an adjacent room to the home pens, to minimize disturbance to other kids.Treatments were administered by a single study technician 20 min before the disbudding procedure.Meloxicam (Meloxidyl oral suspension, 1.5 mg/mL, Ceva Animal Health LLC) at a dose of 1 mg/kg was administered orally before any other treatments.Xylazine (Anased injection, 20 mg/mL, Lloyd Inc.) was administered through the semimembranosus/semitendinosus muscles at a dose of 0.05 mg/kg.Lidocaine (lidocaine HCl 2%, MWI Animal Health) was buffered in a 1:9 dilution with 8.4% sodium bicarbonate (Neogen Vet), diluted to a 10 mg/mL solution, and administered a standardized dose of 4 mg lidocaine per nerve to block the cornual branch of the infratrochlear and lacrimal nerve of both horn buds for a total lidocaine dose of 16 mg/kid.Kids who received multimodal pain management were always administered oral treatments first, then intramuscular, then lidocaine, as indicated according to treatment.Thermal disbudding was performed by the same trained individual, blinded to treatment, for all kids, using a Rhinehart X50 disbudding iron (Rhinehart Development).Kids were restrained manually on the lap of the disbudder, and the hot iron was applied to each horn bud for no more than 3 s at a time per bud.The head was allowed to fully cool in between hot iron applications, and the procedure was considered complete when there was a copper ring of cauterized skin that did not rub off with a fingernail.Sham-treated kids did not receive placebo treatments but underwent the same handling procedures before and during disbudding, with the exception that the disbudding iron was cold.

Data Collection
Wound Sensitivity and Weight Gain.Mechanical nociceptive threshold (MNT) testing was performed via pressure algometry using a calibrated vonFrey anesthesiometer fitted with a 1-cm 2 rubber tip (Wagner Pain Test FPIX Digital Algometer, Wagner Instruments) at 4, 12, 24, and 48 h after disbudding at a single location directly caudal to the disbudding site of each horn bud (location 1 as described by Hempstead et al., 2018c).The algometer was applied once to each horn bud; readings from each bud were then averaged such that each kid had a single value per time point (n = 164 total observations).Kids were weighed daily with an electronic scale at 0600 h before the morning milk feeding.
Physiology.Blood samples were collected before, during, and after disbudding for all treatments.A 3-mL blood sample was taken from the jugular vein via venipuncture into a blood collection tube containing no anticoagulant before (at −20, −10, and −1 min) and after (at 1, 10, 20, and 30 min, and at 1, 2, 4, 6, 12, 24, 36, and 48 h) disbudding.Blood samples were refrigerated after collection and centrifuged at 3,000 × g for 10 min at 20°C, and plasma was separated within 3 h and stored at −80 until shipment on ice to the VetPhast Laboratory at Iowa State University College of Veterinary Medicine (Ames, IA).Processed plasma samples were analyzed in duplicate for cortisol using a commercially available radioimmunoassay (MP Biomedicals, Cortisol CT, cat.no.07221105R) and for PGE 2 concentration using a commercially available competitive ELISA (Cayman Chemical, Prostaglandin E 2 ELISA kit, monoclonal, item no.514010).The cortisol assay had a detection range of 1 ng/mL to 300 ng/mL, with the limit of detection estimated to be 1 ng/mL and the limit of quantitation to be 3 ng/mL.Quality control samples were run at 25 ng/mL and 150 ng/mL at the beginning and end of each assay.Analysis was repeated if a coefficient of variation (CV) >18% between the samples was determined.All curves were linear, with an average R 2 value of 0.9855.The inter-and intra-assay variabilities were 17.55 and 11.32%, respectively.All assays showed acceptable specificity, with an average binding of 40.6% and nonspecific binding of 1.326%.The PGE 2 assay had a detection range of 7.813 pg/ mL to 1,000 pg/mL, and analyses were repeated if the CV between samples was >20% or if the value was not on the standard curve.Samples were diluted or concentrated as necessary to appear on the standard curve.The inter-and intra-assay variabilities were 7.23 and 12.6%, respectively.All curves were linear and had an average R 2 value of 0.9959, with percent binding calculated at 85.01% over all assays.
Behavior During and After Disbudding.During the disbudding procedure, the incidence of vocalizations was recorded by a trained individual blinded to treatment, and 2 video cameras (Lorex 1080p, Lorex) placed to provide orthogonal views of goat kids during disbudding were used to continuously record escaperelated behaviors (struggles and tail flicks; see Table 1 for definitions of each behavior).The intraclass correlation coefficient to describe intraobserver reliability for video-recorded continuous data was 0.97 (95% CI: 0.93-0.98).Time of the disbudding procedure for each kid was determined from the first application of the iron to when the iron was removed for the final time via recorded video.In addition, one video camera was mounted at the front of each kid pen and continuously recorded video during the study period (Figure 1).From the continuously collected pen-level video data, a 10-min period before and after milk feeding at each of the 3 feedings per day was analyzed, starting 24 h after arrival and continuing until 48 h after disbudding.A single observer blinded to treatment performed continuous observations within these periods to record frequency of the following behaviors in all kids: play (jumping, parkour, gambol, perching, running), head-directed behaviors (head rubbing, head scratching, head shaking, ear flicking), social behaviors (allogrooming, ear biting, head butting, mounting, and mountain climb), self-directed behaviors (body shaking, self-grooming, elimination, and drinking), and other.See Table 1 for the experimental ethogram, adapted from Hempstead et al. (2017) and Duffield et al. (2010).Within those same recorded periods, instantaneous scan sampling was performed every 30 s, to describe time budgeting of postural and locomotive behaviors (ambulating, standing, laying socially, laying alone, and other).The intraclass correlation coefficient and Cohen's kappa for continuous and scan observations were 0.98 (95% CI: 0.96-0.99)and 0.91, respectively.

Sample Size Calculation.
A sample size of 6 kids per treatment group provided in excess of 95% confidence and 80% power to detect a 50% reduction in mean cortisol concentration immediately following the disbudding procedure between L and XML groups.This assumes a mean cortisol level of 60 mmol/L in the L group (extrapolated from Alvarez et al., 2015 andNfor et al., 2016), a standard deviation (SD) of 12 mmol/L, and a 1-sided test.
Descriptive Analysis.All continuous data were visualized using boxplots, histograms, and quantilequantile plots and tested for normality using a Shapiro-Wilk test.Data were analyzed using R statistical programming (version 4.0.2,R Core Team, 2020) and SAS (v.9.4 SAS, SAS Institute Inc.).Significance in all models was determined at P ≤ 0.05.
General Modeling.Model building was informed by study design and descriptive visualization of the data.The unit of analysis was the individual goat kid.All models controlled for kid breed and sex to account for differences attributed to those variables.Disbudding day (i.e., whether kids were disbudded on the first or the subsequent of the 2 disbudding days) was included in models when it was associated with the outcome.Residuals from all models were plotted against the predicted values to verify normality assumptions and homogeneity of variance.All data reported met these assumptions.The P-values for multiple comparisons in all models were adjusted with a Bonferroni adjustment factor.
Weight Gain and Wound Sensitivity.Final models to assess the difference in weight gain in the 2 d following disbudding between treatment groups controlled for kid weight at baseline and the random Kids were restrained for the disbudding procedure on the lap of the disbudder (who sat in a chair) such that the forelegs were pinned between the disbudder's legs and the hind legs draped over the edge of the person's legs, with a second handler restraining the hind legs.The disbudder also held the kid's head such that ears were pinned (preventing the possibility of also recording ear flicks).From this position, the left foreleg was visible above the elbow until a kid made an attempt to lift their leg(s) to escape.Each instance of an upward motion at the point of the left fore-elbow was counted as a single struggle attempt.Physiology.Cortisol concentrations were converted from picogram per milliliter to millimole per liter and were found to be non-normally distributed, so outliers greater than 3 SD from the mean were removed, leaving 576 total samples (from n = 586) for analysis.As no changes in cortisol concentrations occurred after 1 h following disbudding, one model was built to describe the effect of treatment on serum cortisol in the hour after disbudding that included treatment, time, the interaction of treatment and time, baseline cortisol concentration, the random effect of breed and sex, and repeated measurements over time (n = 7 obs/kid for the 1-h model; n = 287 total obs).The correlation between measurements over time was accounted for with a compound symmetry correlation structure.
For PGE 2 analysis, the percentage change in PGE 2 concentration for each sample relative to that for the baseline sample was calculated as follows: (sample PGE 2 − baseline PGE 2 )/baseline PGE 2 × 100 (Kleinhenz et al., 2018).Percent change in PGE 2 was found to be nonnormally distributed; therefore, outliers greater than 3 SD from the mean were removed from analysis, leaving 577 total samples (from n = 586).The final model to describe the effect of treatment on the percent change in serum PGE 2 over time included treatment, time, the interaction of treatment and time, the random effect of breed and sex, and repeated measurements over time (n = 14 obs/kid).The correlation between measurements over time was accounted for with a compound symmetry correlation structure.
Behaviors During and After Disbudding.Final models to assess the effects of treatment on vocalizations, struggle, and tail flick behavior during the disbudding procedure as well as total time of the procedure included treatment, breed, sex, and disbudding day.
Continuous and scan sampling behavior data were either summed (continuous) or averaged (scan) so that each kid had one value for each behavior on each of the following days relative to disbudding (henceforth referred to as "day"): baseline day (24 h before disbudding), d 1 (24-h period after disbudding), and d 2 (24-h period after d 1).These summed or averaged data were then used to build models for each recorded behavior.Continuous behaviors with very few observations (elimination, drinking, other) were not modeled.All models included treatment, day, the interaction of treatment and day, breed, and sex.Models included a repeated statement to account for repeated sampling days per kid (3 obs/kid), with correlation between days accounted for with an unstructured correlation structure.

Kid Enrollment and Baseline Characteristics
A total of 42 kids were enrolled into 7 treatment groups, with breed and sex approximately balanced among the 7 groups (see Table 2).There was no difference between treatment groups in age (9.5 ± 3.4 d; P = 0.79) or body weight (4.4 ± 0.7 kg; P = 0.73) at kid enrollment (data presented as μ ± SD).One kid (XL) was removed from the study at 24 h to be treated for excessive swelling at her disbudding site.Her data until removal were used for analysis.
In the 48 h after disbudding, we discovered no overall effect of treatment on percent change in PGE 2 from baseline concentration (F 6,33 = 2.36; P = 0.052).Interestingly, by 4 h after disbudding, XM and M (but not XML) kids achieved consistent PGE 2 concentrations below baseline for the duration of the follow-up period (Figure 4).

Behaviors During and After Disbudding
The entire disbudding procedure lasted approximately 1 min per kid [62.6 ± 17 s (μ ± SD)], with no difference in time of the disbudding procedure between treatment groups (F 6, 31 = 1.29;P = 0.29).No treatment effect was observed on any of the behaviors recorded during disbudding (vocalization, struggling, or tail flicks; Table 3).
We also found no effect of treatment on any of the continuous or scan behaviors recorded after disbudding.However, for continuous outcomes, an effect of day occurred relative to disbudding on play, head, and self-directed behaviors (Table 4), as follows.In the 2 d after disbudding, kids were observed to less frequently perform play behaviors (gambol, parkour, jumping, and running) compared with the day before disbudding (baseline).Similarly, kids both performed and received fewer head butts in the days after disbudding compared with the baseline day.Additionally, kids performed fewer head shakes and mounts in the observations on d 1 after disbudding compared with baseline, and performed more mountain climbs on d 2 after disbudding compared with baseline or d 1 after disbudding.Finally, kids performed less self-grooming and less perching during observations on d 1 after disbudding compared with baseline or d 2 after disbudding (Table 5).
We also detected an effect of day relative to disbudding on all scan behaviors observed (Table 6).In the first day after disbudding, kids were observed to ambulate and stand less frequently and to lie (both socially and alone) more frequently as compared with baseline.By d 2 after disbudding, kids were still observed to ambulate and stand less, and lie socially more compared with baseline, although lying alone rebounded to baseline levels (Table 7).

DISCUSSION
Disbudding of dairy goat kids using thermal cautery is painful, and work investigating practical pain management is needed to assist those who routinely perform this procedure on farm.To our knowledge, this is the first study to investigate the use of xylazine and buffered lidocaine as single-and multimodal options for pain mitigation during the disbudding procedure.We found that the combination of xylazine, buffered lidocaine, and meloxicam attenuated the acute cortisol response to disbudding, but none of the single or combination therapies investigated fully attenuated the acute physiological and behavioral responses to the disbudding procedure examined in this study.

Kid Enrollment, Weight Gain, and Wound Sensitivity
Kids in our study were disbudded at an average age of 9.5 d due to logistical limitations associated with kid sourcing.Although this timing is later than recommended for cautery (nearer to 5 d of age; Smith and Sherman, 2009b), it is in line with other published work investigating pain management for kid disbudding (Alvarez et al., 2009;Hempstead et al., 2017) and likewise is near the US industry average of 14.6 d (USDA-NAHMS, 2020).It is likely that kid age, size, and breed affect pain response as well as procedure success, but this remains to be investigated.Age has been shown to have differential effects on pain response in cats such that younger animals (6 mo) had a reduced thermal threshold after hydromorphone injection as compared with adult animals (12 mo; Simon et al., 2019).We observed no effect of pain mitigation technique on weight gain following disbudding compared with a negative control (sham disbudding), similar to findings from Hempstead et al. (2020a).In the interest of adhering to the "Three Rs" principle of refinement (minimization of distress and pain in research subjects; see Fenwick et al., 2009), we chose to not include a positive control group (cautery disbudding with no pain mitigation), which has been previously shown to reduce body weight in the 14 d following the procedure (Chandrahas et al., 2015) compared with kids receiving a local anesthetic block and a nonsteroidal anti-inflammatory drug.However, goat kids are highly motivated to drink milk even under aversive conditions (Withrock, 2015), so it may be difficult to find a difference in weight gain in this species.
We did observe a difference in MNT in the 48 h after disbudding between kids receiving meloxicam and those who were sham-disbudded, with meloxicam kids having a lower MNT (i.e., more sensitivity) compared with sham kids.Although MNT has been used to assess goat kid sensitivity associated with different disbud-ding techniques (Hempstead et al., 2018c) and disbudding wound healing over time (Alvarez et al., 2019), to our knowledge this is the first report of an effect of pain management on wound sensitivity in disbudded goat kids.Our finding suggests that oral meloxicam alone at the dosage used in this study should not be relied on as the sole pain management strategy to mitigate the acute pain response associated with cautery disbudding in goat kids.Most of the similar work in dairy calves has examined the effect of meloxicam in combination with lidocaine (as compared with meloxicam alone) and reports that MNT over the first 4 h after disbudding is higher compared with calves receiving lidocaine alone (see Winder et al., 2018).

Physiology
In the present study, kids treated with the triple combination of xylazine, meloxicam, and lidocaine had reduced cortisol concentrations in the first hour after disbudding compared with kids treated with lidocaine alone.Cortisol concentrations were also lower in this group at 15 min following disbudding compared with kids who received only lidocaine or only meloxicam, suggesting that the multimodal pain mitigation examined here provided better short-term protection compared with the single-modality treatments in goat kids.Other investigations of dual-modality pain mitiga-tion corroborate that combining modalities can confer better pain relief: for example, dual-modality-treated kids (isoflurane + meloxicam) had lower plasma cortisol concentrations, particularly in the 15 min after   Kleinhenz et al., 2018] in the 48 h following cautery disbudding of goat kids.Blood samples were taken at baseline (−20 min) and before (−10, −1 min) and after (at 1, 10, 20, and 30 min, and at 1, 2, 4, 6, 12, 24, 36, and 48 h) disbudding.Represented are model-adjusted means and SEM.The x-axis represents time on the ln scale for ease of visualization.Significance was determined at P ≤ 0.05, and a Bonferroni correction was applied to adjust for multiple comparisons.This model controlled for time, treatment × time, random effect of breed and sex, and repeated measurements across time points.Treatment groups (n = 6/group) are as follows: SHAM = simulated disbudding; X = sedation with xylazine (0.05 mg/kg i.m.); M = oral meloxicam (1 mg/kg orally); L = a nerve block with sodium bicarbonate-buffered lidocaine (4 mg/kg subcutaneous); XL = combination of xylazine and buffered lidocaine; XM = combination of xylazine and meloxicam; and XML = combination of xylazine, meloxicam, and lidocaine at the previously indicated doses and routes of administration.disbudding, compared with cautery disbudding with no pain control or single-modality treatments, including meloxicam (Hempstead et al., 2018b(Hempstead et al., , 2020a)).To date, in the only other published study investigating 3 pain mitigation drugs (dexmedetomidine, meloxicam, and lidocaine), the authors reported these multimodal treatments kept plasma cortisol concentrations similar to sham disbudding immediately after the procedure, but by 30 min after disbudding, cortisol concentration in these groups were higher than in the sham treatment (Nfor et al., 2016).However, it is important to note that kids in the Nfor et al. (2016) study had their sedative dose reversed at 0.5 h (injection of an equivalent volume of atipamezole hydrochloride), so it is unclear how that may have affected cortisol or pain control in those kids.
In the present study, kids were allowed to "sleep off" their sedation, remaining sedated for approximately 40 min [43 ± 10 min (μ ± SD)] after disbudding.Others (Alvarez et al., 2009(Alvarez et al., , 2015;;Hempstead et al., 2020b) have observed increased cortisol in kids receiving li-docaine before disbudding, suggesting that lidocaine administration to goat kids is aversive in and of itself.However, we did not observe an increase in cortisol in our lidocaine group before disbudding (as compared with sham), possibly because we used buffered lidocaine in our protocol, which has been shown to be less painful in human studies (Lee et al., 2013).However, given the finding of Adcock and Tucker (2022), that buffered lidocaine may induce greater aversion in calves compared with non-buffered lidocaine, future work is clearly needed to discern whether buffering lidocaine is beneficial (or not) for pain mitigation in goat kids undergoing disbudding.In contrast, we observed no overall effect of treatment on percent change in PGE 2 concentrations from baseline in the 48 h following disbudding.This result was unexpected, as meloxicam inhibits cyclooxygenase-2 activity preferentially (Coetzee et al., 2009) and, thus, should reduce prostaglandin production when administered.Reduction of PGE 2 is positively correlated with Models controlled for treatment, day (0, 1, 2), treatment × day, breed, sex, and repeated measurements by day.Frequency of observed behaviors for each kid (n = 42) were summed per day for a 10-min period before and after milk feeding at each of the 3 feedings per day for baseline (24 h before disbudding), d 1 (24-h period after disbudding), and d 2 (24-h period after d 1). 2 Treatment groups (n = 6/group) are as follows: sham = simulated disbudding; X = sedation with xylazine (0.05 mg/kg i.m.); M = oral meloxicam (1 mg/kg orally); L = a nerve block with sodium bicarbonate-buffered lidocaine (4 mg/kg subcutaneous); XL = combination of xylazine and buffered lidocaine; XM = combination of xylazine and meloxicam; and XML = combination of xylazine, meloxicam and lidocaine at the previously indicated doses and routes of administration. 3 Numerator degrees of freedom are 6, 2, and 12 for treatment (TRT), day, and TRT × day, respectively.Denominator degrees of freedom for all models are 32, 34, 48 for TRT, day, and TRT × day, respectively.Denominator degrees of freedom approximated with Kenward-Roger approximation.
anti-inflammatory activity (Kleinhenz et al., 2018) and should therefore contribute to pain control associated with inflammation at the disbudding site in kids disbudded with cautery.As expected, kids in the meloxicam and xylazine-meloxicam groups had a negative percent change from baseline PGE 2 concentrations starting 2 h after disbudding.However, the triple-modality kids did not have similar changes from baseline.Inclusion of an LM group could have helped elucidate whether an interaction occurs between buffered lidocaine administration and suppression of PGE 2 at the disbudding site.We chose not to include an LM group in our study for reasons similar to our decision to exclude a positive control group: we hoped to minimize the number of kids who were injected without sedation, as injection itself has been shown to be aversive to kids (Hempstead et al., 2020b).Another possible explanation to the suppression of PGE 2 in some treatment groups (M, XM, L) is that the higher average endogenous serum cortisol in those kids reduced release of arachidonic acid and thus resulted in a reduction in PGE 2 serum concentrations (Perretti and D'Acquisto, 2009).

Behavior During and After Disbudding
To our surprise, we did not observe any treatment differences in frequency of vocalization, struggle, or tail flick behavior during the disbudding procedure.Howev- a-c Different subscript represent P-value ≤0.05 between day.
1 Models controlled for treatment, day (0, 1, 2), treatment × day, breed, sex, and repeated measurements by day.Frequency of observed behaviors for each kid (n = 42) was summed per day for a 10-min period before and after milk feeding at each of the 3 feedings per day for baseline (24 h before disbudding), d 1 (24-h period after disbudding), and d 2 (24-h period after d 1).
2 Day 0 = baseline (24 h before disbudding); d 1 = 24 h after disbudding; d 2 = 24 h after d 1. 3 Numerator degrees of freedom are 2 for day.Denominator degrees of freedom for all models are 34 for day.
Denominator degrees of freedom approximated with Kenward-Roger approximation.
er, we did notice some interesting numerical differences.
For example, M kids vocalized 28 times (1 vocalization every 2 s, on average), which was 6 vocalizations more than the next-highest group (XM).Additionally, L kids had an average of 2 struggles per disbudding event, numerically much lower than any of the other groups.Additionally, our sham group was not the numerically lowest in any behavior observed, in contrast to much of Day 0 = baseline (24 h before disbudding); 1 = 24 h after disbudding; 2 = 24 h after d 1. 1 Models controlled for treatment, day (0, 1, 2), treatment × day, breed, sex, and repeated measurements by day.Instantaneous scan sampling was performed every 30 s for each enrolled kid for a 10-min period before and after milk feeding at each of the 3 feedings per day for baseline (24 h before disbudding), d 1 (24-h period after disbudding), and d 2 (24-h period after d 1) to describe time budgeting of postural and locomotive behaviors (ambulating, standing, lying socially, lying alone, and other).
3 Numerator degrees of freedom are 2 for day.Denominator degrees of freedom are all models are 34 for day.Denominator degrees of freedom approximated with Kenward-Roger approximation.
the other goat work in this area (Alvarez et al., 2009(Alvarez et al., , 2015;;Nfor et al., 2016;Hempstead et al., 2020b).It is possible that, despite daily handling of the kids for weighing, the stress incurred from the amount of activity and handling on disbudding days may have obscured treatment effects.Finally, a key limitation of this study is that our sample size was calculated based on physiological measures and not on behavioral endpoints; with the variability in individual goat kid behavior, this probably hindered detection of behavioral differences.Nfor et al. (2016) found that kids sedated with 0.1 mg/kg dexmedetomidine hydrochloride (an α-2 adrenergic receptor agonist) showed no kicking or vocalization behavior during disbudding.During our study, we observed that kids receiving xylazine were sedated at the beginning of the disbudding procedure but came out of sedation sufficiently to display pain-related behaviors during disbudding.Xylazine has a dose range in goats from 0.01 to 0.2 mg/kg (Depenbrock, 2017), so it is possible our conservative dose of 0.05 mg/kg was insufficient to provide adequate sedation.This dosage was chosen to reduce the risk of hypotension and hypothermia, which are not uncommon in small ruminant neonates administered α-2 adrenergic receptor agonists (Galatos, 2011).We likewise observed no post-disbudding behavioral differences between treatment groups, but behavioral expression did shift from baseline measures after disbudding.In contrast to other studies examining continuous behavior after disbudding (e.g., Hempstead et al., 2017Hempstead et al., , 2018b;;Ajuda et al., 2020), we chose to sample behavior around feeding time so as to maximize observations of kids at their most active.It is possible that the stimulation of feeding and human activity in the barn may have altered behavior and suppressed possible differences between treatment groups that may otherwise have been detected had we chosen a different sampling methodology.Some types of pain mitigation (meloxicam) have been shown to increase the number of feeding bouts in disbudded kids compared with other treatments (sham disbudding and isoflurane, Hempstead et al., 2020a).
Pain mitigation (lidocaine and lidocaine in combination with flunixin) also increases exploration, play, and standing behavior (Ajuda et al., 2020) in the time after disbudding compared with kids disbudded with no pain control.In the present study, disbudding (or simulated disbudding) suppressed post-procedure expression of play behaviors in all kids, regardless of treatment, compared with baseline.It is probable that handling and disruption of the goat kids' daily time budget on the disbudding day were at least partly responsible for inducing a change from baseline behaviors.However, it is also possible that unmitigated pain from the procedure suppressed play behaviors in all disbudded kids (as seen in calves; Mintline et al., 2013) via negative play contagion, as has been described in calves (Größbacher et al., 2020).
Head-involved behaviors (e.g., head butting, shaking, and scratching) have been suggested as particularly promising behavioral indicators for post-disbudding pain in goat kids (Hempstead et al., 2017).All kids in our study exhibited fewer head-directed behaviors (e.g., head butting and head shaking) in the day following disbudding.Although others have not observed this (Hempstead et al., 2017), it seems plausible to propose that kids experiencing post-disbudding pain may attenuate their performance of head butting.Interestingly, in contrast to work in both calves (Faulkner and Weary, 2000) and goats (Martínez et al., 2022;Hempstead et al., 2017Hempstead et al., , 2018b) ) demonstrating that disbudding increased head shaking behavior, in our study kids exhibited a decrease in this behavior in the first day after disbudding.Head shaking has been reported in goat kids to increase the most in the first hour after disbudding, so it is possible that we missed the relevant window to distinguish a difference between treatment groups (Hempstead et al., 2018b).In the present study we suggest that head shaking was considered uncomfortable for disbudded kids so the behavior was suppressed in the 24 h following disbudding.
Kids were also observed to be lying in close social proximity to pen mates more frequently in the 2 d after disbudding.It is possible that this effect is simply an artifact of standing less, or because they were seeking social comfort from conspecifics after a period of stress (Rault, 2012).An increase in time spent lying has also been associated with pain for other conditions in cattle (Medrano-Galarza et al., 2012) and in other species (McGlone et al., 1993).Finally, it is also possible that kids were lying more in general to conserve energy after a stressful procedure, or to keep their painful heads from moving, as suggested by Hempstead (2017).

CONCLUSIONS
Cautery disbudding is commonly performed on dairy goat kids reared in commercial settings in the United States without the use of pain management.Our results support the growing literature demonstrating that disbudding induces, at minimum, acute pain responses in goat kids, which should be addressed to improve kid welfare.Our findings suggest that a combination of buffered lidocaine, xylazine, and meloxicam given 20 min before the disbudding procedure attenuates some of the physiological responses to disbudding.However, evidence of comprehensive relief using this combination was not found, nor were any of the single-modality pain mitigation approaches explored here sufficient to attenuate acute pain.Our results also suggest that further work is needed to more fully characterize behavioral expression of dairy goat kids undergoing disbudding.Although we did not observe any treatment effects on the behavioral outcomes measured, changes in play, head-related, and postural behaviors in the 2 d after disbudding suggest that disbudding changed the behavioral time budget of the kid.
effect of breed and sex.The final model to assess the effect of treatment on MNT included treatment, time, the interaction of treatment and time, sex, breed, and disbudding day.A repeated statement accounted for clustering by kid [4 observations (obs)/kid in the model].The correlation was accounted for with a compound symmetry correlation structure.

Figure 1 .
Figure 1.Image from video camera footage of housing for goat kids enrolled in a study to evaluate the effects of single-and multimodal pain mitigation on physiology and behavioral outcomes in the 48 h after cautery disbudding.Kids were housed in groups (n = 7) with each of 7 treatment groups represented per pen.
Figure 2. Results of a repeated measures linear mixed model to describe the overall effect of pain management on average mechanical nociceptive threshold after disbudding in neonatal goat kids.This graph represents model adjusted mean mechanical nociceptive threshold (kgf) with error bars (mean ± SEM are indicated above each bar).Measurements were taken at 4, 12, 24, and 48 h after disbudding.Significance was determined at P ≤ 0.05.This model controlled for treatment, time, treatment × time, sex, breed, and day of disbudding, and repeated measurements within kid.Treatment groups (n=6/group) are as follows: SHAM = simulated disbudding; X = sedation with xylazine (0.05 mg/kg i.m.); M = oral meloxicam (1 mg/kg orally); L = a nerve block with sodium bicarbonate-buffered lidocaine (4 mg/kg subcutaneous); XL = combination of xylazine and buffered lidocaine; XM = combination of xylazine and meloxicam; and XML = combination of xylazine, meloxicam, and lidocaine at the previously indicated doses and routes of administration.Statistically significant differences (P ≤ 0.05) are denoted by lowercase letters above each group such that groups without a common letter are significantly different.

Figure 3 .
Figure 3. Results of a repeated measures linear mixed model investigating the effect of multimodal pain management on serum cortisol concentrations (mmol/L) in the 20 min before and 1 h after cautery disbudding of goat kids.Treatments were applied immediately after sampling at −20 min.Represented are model-adjusted means and SEM.Significance was determined at P ≤ 0.05, and a Bonferroni correction was applied to adjust for multiple comparisons.This model controlled for time, treatment (TRT) × time, baseline cortisol concentrations, random effect of breed and sex, and repeated measurements across time points.Treatment groups (n=6/group) are as follows: SHAM = simulated disbudding; X = sedation with xylazine (0.05 mg/kg i.m.); M = oral meloxicam (1 mg/kg orally); L = a nerve block with sodium bicarbonate-buffered lidocaine (4 mg/kg subcutaneous); XL = combination of xylazine and buffered lidocaine; XM = combination of xylazine and meloxicam; and XML = combination of xylazine, meloxicam, and lidocaine at the previously indicated doses and routes of administration.
Knauer et al.: PAIN MANAGEMENT IN DISBUDDED GOAT KIDS

Figure 4 .
Figure 4. Results of a repeated measures linear mixed model investigating the effect of pain management on percent change in baseline PGE 2 [(sample PGE 2 − baseline PGE 2 )/baseline PGE 2 × 100;Kleinhenz et al., 2018] in the 48 h following cautery disbudding of goat kids.Blood samples were taken at baseline (−20 min) and before (−10, −1 min) and after (at 1, 10, 20, and 30 min, and at 1, 2, 4, 6, 12, 24, 36, and 48 h) disbudding.Represented are model-adjusted means and SEM.The x-axis represents time on the ln scale for ease of visualization.Significance was determined at P ≤ 0.05, and a Bonferroni correction was applied to adjust for multiple comparisons.This model controlled for time, treatment × time, random effect of breed and sex, and repeated measurements across time points.Treatment groups (n = 6/group) are as follows: SHAM = simulated disbudding; X = sedation with xylazine (0.05 mg/kg i.m.); M = oral meloxicam (1 mg/kg orally); L = a nerve block with sodium bicarbonate-buffered lidocaine (4 mg/kg subcutaneous); XL = combination of xylazine and buffered lidocaine; XM = combination of xylazine and meloxicam; and XML = combination of xylazine, meloxicam, and lidocaine at the previously indicated doses and routes of administration.
Knauer et al.: PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Knauer et al.: PAIN MANAGEMENT IN DISBUDDED GOAT KIDS

Table 1 .
Knauer et al.: PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Knauer et al.: PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Ethogram of observed behaviors during continuous and scan behavioral sampling of neonatal goat kids before, during, and after disbudding; adapted from Hempstead et al. (2017) and Duffield et al. (2010) Tilting of the head so the rear foot scratches any part of the head (excluding neck).The rear fetlock must reach the shoulder and includes attempts (no head contact but foot is raised off the ground).Replacing foot to ground and then lifting again considered separate event.Head shaking Rapid tilting of the head from side to side a minimum of 3 times, concluding with a return to neutral position.Separation by >1 s is considered a separate event.Ear flicking Rapid movement of one or both ears to the front and back, independent of a head shake.Separation by >1 s is considered a separate event.
Ear bitingThe mouth comes in contact with a conspecific's ear, lasting > 1 s.A separate event is considered to occur after a break of >3 s.Head buttingEars pulled back while rearing or stepping backward, head lowered, followed by forward motion.Concludes with the top of head colliding with the body of a conspecific.A separate event is considered when kid removes head and then butts again.MountingKid rises on front legs and mounts either the front or back of a conspecific.1 Definition from Alvarez and colleagues' (2015) description of vocalization in goats at disbudding.2

Table 2 .
Knauer et al.:PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Baseline characteristics of goat kids enrolled in a study to investigate the effect of pain management on the acute behavioral and physiological effects of cautery disbudding

Table 3 .
Results of repeated measures linear mixed models investigating the effects of pain mitigation on frequency (n/disbudding event) of vocalizations, struggles, and tail flicks observed during cautery disbudding 1

Table 4 .
Knauer et al.:PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Results of repeated measures linear mixed models investigating the effects of pain management on video-captured behaviors after cautery disbudding in dairy goat kids 1

Table 5 .
Knauer et al.:PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Results of repeated measures linear mixed models investigating the effects of pain management on behaviors after cautery disbudding in dairy goat kids, stratified by day relative to disbudding 1

Table 6 .
Knauer et al.:PAIN MANAGEMENT IN DISBUDDED GOAT KIDS Results of repeated measures linear mixed models investigating the effects of pain management on scan behaviors after cautery disbudding in dairy goat kids 1 Numerator degrees of freedom are 6, 2, and 12 for treatment (TRT), day, and TRT × day, respectively.Denominator degrees of freedom for all models are 35, 34, and 48 for TRT, day, and TRT × day, respectively.Denominator degrees of freedom approximated with Kenward-Roger approximation. 3

Table 7 .
Results of linear mixed models investigating the effects of pain management on scan behaviors after cautery disbudding in dairy goat kids, stratified by day 1