Comparison of Three Anaesthetic Options to Reduce Acute Pain Response in Kid Goats

ABSTRACT Three options for anesthetizing the skin around the horn bud of dairy goat kids were explored. Forty-five <10-day-old Saanen goat kids from were randomly split into five treatment groups (topical anesthetic cream (TA), vapocoolant spray (VS), local anesthetic applied by jet injector (JI), control – no treatment but painful stimulus applied (C), sham – no treatment and touching sites with a finger. The painful stimulus was multiple needle pricks on the skin around the horn bud. The outcome variables measured were heart rate movement, and vocalization during treatment application and administration of a painful stimulus around the horn bud. Heart rates were greater during application of a VS compared to TA.Neither the TA nor the VS appeared to have any effect on the response to the painful stimulus. Kids in the JI group had a 96% reduced odds of expressing a marked pain response in comparison to TA group and an 83% reduction in the odds of a high movement grade during a painful procedure in comparison to the combined results of the other three treatment groups.


Introduction
On commercial dairy goat farms, replacement does and breeding bucks are commonly disbudded using a hot iron in their first week of life.Disbudding is associated with significant pain and distress, shown through high-intensity vocalizations and struggling during the procedure (Alvarez, Nava, Ramírez, Ramírez, & Gutiérrez, 2009).Following the procedure, they show a number of behavioral changes such as head shaking, scratching, rubbing, self-grooming, and body shaking (Hempstead, Waas, Stewart, Cave, & Sutherland, 2017), isolation, and time spent motionless (Ingvast-Larsson et al., 2011) which all indicate pain and distress, as do elevations in cortisol and β-endorphin concentrations during and after disbudding (Alvarez et al., 2015;Hempstead et al., 2018).
Currently, in New Zealand, a person must not disbud a goat unless the goat is given pain relief that is authorized by a veterinarian for the purpose of the procedure (Animal Welfare (Care and Procedures) Amendment Regulations 2020, New Zealand).Although these drugs have been shown to be effective at reducing the signs of pain (Ajuda, Battini, Mattiello, Arcuri, & Stilwell, 2020;Hempstead et al., 2018;Ingvast-Larsson et al., 2011) and are practical for farm staff to administer, they only reduce the pain associated with inflammation not the acute pain caused by the destruction of nociceptors during disbudding.
General anesthetic can provide effective pain relief during the disbudding process (MN; Hempstead et al., 2018;Matthews, 2016); however, it is not a feasible approach for non-veterinarians as such drugs need to be used under direct veterinary supervision in most countries.Local anesthesia of the nerves that supply the horn (usually via a cornual nerve block or local injection in to the horn bud) is effective in calves (Bates et al., 2019;Stafford & Mellor, 2011), but in goat kid reductions in pain-related behaviors and cortisol do not appear to be as consistent as in calves (Alvarez et al., 2009;Hempstead, Waas, Stewart, & Sutherland, 2020).The reason for this difference between calves and goat kids may be the anatomy of the horn.In contrast to the calf where it is the cornual branch of the infratrochlear nerve that supplies the horn, in goat kids two nerves (cornual branch of the lacrimal nerve as well as the cornual branch of the infratrochlear nerve) supply the horn (Matthews, 2016).This means that two sites per horn need to be effectively anesthetized to prevent disbudding-related pain in goat kids.The need for repeated injections makes the process significantly more difficult to achieve anesthesia whilst the injections themselves causes distress in goat kids.
One potential solution to this problem is the use of jet injectors.These injectors deliver the drug by forming it into a high-speed jet capable of penetrating through the skin and into the underlying tissue.Injectors driven by electric motors have been developed which allow a high-level of control over the pressure, depth, and volume of the injection (Hogan, Taberner, Jones, & Hunter, 2015).Currently, 0.5 ml can be injected without a needle (Taberner, Hogan, & Hunter, 2012) or with a very small ~1.5 mm 30 g needle (Li, Ruddy, & Taberner, 2016).The use of a jet needle injector could provide pain-free application of local anesthetic thereby improving the practicality of providing local anesthetic for disbudding in goat kids.
Another simpler method of potentially providing anesthesia for the horn bud site is through topical anesthesia.To be effective, topical anesthetics must move into the superficial layers of skin and affect the nerve endings within the dermis.The thickness of the stratum corneum and the acid dissociation constant (pKa) of an anesthetic determine how well the topical medicine can penetrate the stratum corneum (Adriani & Dalili, 1971).
The most widely used topical local anesthetic product is EMLA cream (Astra Pharmaceuticals, Westborough, MA).EMLA is an oil-in-water emulsion of 2.5% lignocaine and 2.5% prilocaine.The duration of application, the size of the treated area, and the specific anatomic location treated determine the amount of lignocaine and prilocaine systemically absorbed from EMLA cream.Occlusion of veins and longer duration of application increase its penetration and efficacy (Tahir, Webb, Allen, & Nancarrow, 2006;Wahlgren & Quiding, 2000).Analgesia is achieved, to a depth of 3 mm, 60 minutes after application, and a maximum dermal depth of 5 mm is reached after 120 minutes in humans (Bjerring & Arendt-Nielsen, 1990;Evers, Von Dardel, Juhlin, Ohlsen, & Vinnars, 1985).EMLA has been tested in goat kids prior to disbudding but found to be ineffective (Hempstead et al., 2020).
An alternative to topical local anesthetics for controlling pain is the use of cooling (Hindocha, Manhem, Bäckryd, & Bågesund, 2019).In New Zealand, a vapocoolant (IceCool, Shoof, Hamilton, New Zealand) is marketed for use prior to amputation dehorning in animals for pain relief.This product is used on some commercial goat farms in New Zealand prior to hot iron disbudding, but there are no data on its efficacy.
Therefore, the aims of this study were to determine the effect of these three possible solutions for pain relief (jet needle electronic injection of local anesthetic, topical lignocaine, and a vapocoolant) on the response, in dairy goat kids to a needle prick applied to the skin around the horn bud.

Materials and methods
All manipulations in this study were approved by the Ruakura AgResearch Animal Ethics Committee, Hamilton, New Zealand, application number 14,797.
This was a randomized clinical intervention study carried out on 45 Saanen female goat kids, <10 days of age from one commercial dairy goat farm.There were five groups of interest (four groups of 10 and one group of five) detailed in Table 1.There were three treatment groups (n = 10 per group), one control group (n = 10), and one sham group (n = 5).The study was carried out over 1 day (8am-4pm) in 2018.All goat kids had already been separated from their mothers.The goat kids were randomly split into 5 treatment groups by whichever goat kid came closest to the person enrolling them was picked up and provided with a neck band.Neck band selection was made by a technician choosing neck band colors with a random allocation sheet (Table 1).Animals in each group were identified by placing colored Velcro neck band with two letters coding the goat kid goat to the treatment group.For example, Green AA, yellow AO.As the trial occurred throughout the day, the goat kids were fed prior to any treatments being applied and after the trial was completed.
For each goat kid (regardless of group), the hair around each horn and around the sites for local anesthetic block of the cornual and infratrochlear nerve were shaved using an electric shaver prior to any treatment applications or painful stimulus but after the first baseline heart rate.

Painful stimulus application
A total of 40 goats underwent painful stimulus testing and five goats underwent "sham" stimulus testing (Table 1), with testing being completed in approximately 20 seconds, 15-30 minutes and 1 hour following application of the treatments (Table 1).The variation in timing depended on the product (e.g., EMLA recommendations were to apply the cream 1 hour before a painful stimulus).
One veterinarian (non-blinded) was responsible for applying the painful stimulus to the 40 goat kids by inserting an 18 g needle, approximately 1 mm into the skin on four different positions on the dorsal, ventral, and two lateral sides of the horn bud at positions that would mimic where the disbudding iron would be applied.On the left side, the needle pricks started dorsally and continued clockwise around the horn bud.On the right side, the needle pricks started ventrally and then continued clockwise around the horn bud (Figure 1).The order in which horn was pricked first alternated between each kid.The intention was to remove the bias that would come from the operator deciding where to start and also to account for some of the differences between the response to the prick at each site, which could have been influenced by hyperalgesia.
The "sham" painful stimulus for five goat kids was completed by touching the goat kids in the needle locations gently with a finger instead of a needle.

Treatment administration
The treatments were applied 30-60 minutes after a second baseline heart rate measurement.
Approximately 15 minutes prior to the start of treatment application, all goat kids were fed milk to work in with usual farm practice as this was the time that they typically fed them.
The timing of application of each of the treatments prior to the application of the painful stimulus application is presented in Table 1.

Jet injector group
The injector used in this study was driven by a custom voice coil motor designed and constructed at the Auckland Bioengineering Institute.This device performed injections by using the motor to actuate a piston within a stainless steel ampoule with a maximum volume of 0.5 mL.The fluid within this ampoule was pressurized (up to 15 MPa) and forced through a tiny orifice (0.2 mm in diameter) at the end of the ampoule.The resulting high-speed jet of fluid traveled in excess of 120 m/s and was capable of delivering fluid to the underlying tissue.
While it was initially intended that this device be used to deliver anesthetic needle-free, after two goat kids received their four needle-free injections (two for each horn bud) out of the 10 goat kids in this group, the study team raised concerns about the ability of needle-free injection to consistently disperse the local anesthetic in such a small area with a lot of bone and limited soft tissue.This resulted in the decision to add a very short (1.5 mm long) 30 gauge needle to the tip of the device for the remaining eight goat kids in this group.For the remaining eight animals in the jet injector group injections were performed with the small needle attached to the end of the injector.This "needle-assisted" jet injection technique was able to avoid the inconsistency observed in the initial needle-free injections.
A custom-built controller-amplifier was used to control and power the injector (Ruddy, Dixon, Williams, & Taberner, 2017).This involved the controller momentarily (<0.4 seconds) supplying the motor with a voltage of around 150 V.The injection system (injector and controller) has been designed to fit medical electrical equipment safety standard IEC 60601-1.
A syringe and the filling adapter attached to the front of the ampoule were used to fill the device.Lignocaine (Bomacaine 2%, Bayer New Zealand Ltd) was diluted by 50% with sterile water.Once the device was completely filled, the syringe and filling attachment was removed from the injector and the controller was switched into injection mode.After charging the controller for ~30 s, the device was then placed in the specific sites (Figure 2) and the button was pressed to perform the 0.5 mL injection (Figure 3).The controller was then switched out of injection mode and the ampoule tip wiped clean.This process was repeated four times for each kid goat (i.e., two injections per horn bud).

Measurement of heart rates
The heart rate (HR) of the goat kids was measured on three separate occasions for the jet injector group, the vapocoolant group and the topical anesthetic group, and twice for the sham and negative control groups (Table 1).Approximately 10-20 minutes after the neck bands were placed, the baseline HR was measured.Two measurements were taken approximately 30 minutes apart so the mean of the result could be used.For each of these measurements, HR was measured for 10 seconds using a stethoscope in gently restrained kids that were standing on the ground.The next measurement of HR was a continuous measurement that started ~20 s before the application of the treatments and continued until ~20 s after the treatment application had finished.During this measurement, the goat kids were again gently restrained, and HR measured using a stethoscope.HR was recorded in 10 s blocks, with the 10s time frame being signaled by a small horn sound on a mobile phone.Treatment application HR was not recorded or required for the sham and control groups since they were not treated with anything.
The final measurement of HR was during application of a painful stimulus or sham stimulus.Continuous measurements in 10 s blocks were made from ~20 s-30 s before stimulus application to ~20 s after.

Measurement of vocalization and movement
Two technicians who were blinded to the treatments (with the exception of the cool spray, which was obviously applied just prior to pain testing) separately scored the subjective vocalization and movement response (0-3) of the goat (Table 2).
In order to reduce unnecessary suffering, goat kids were assessed for severity of vocalization and movement after the first two needle pricks on each side.After two needle pricks on the dorsal/ ventral or either lateral side of the horn bud, stimulation stopped on that horn bud if the goat kid responded with grade 2 or 3 vocalization and/or movement to the first two needle pricks.In this situation, the goat kid was noted as having a marked pain response, and did not undergo any further needle pricks.Goat kids that had a grade 0 to 1 movement and vocalization at the first two needle pricks were coded as not having a marked pain response and continued with the full needle prick methodology.

Statistical analysis
There were three outcomes of interest: HR during treatment application, HR during painful stimuli, and vocalization and movement during painful stimuli.Goats had varying time periods for when HR was measuredin particular, from the application of the treatment to the measurement of the heart rate.As a result, all HR analyses used the median HR (beats/10 seconds) of all collected HR measurements for each measurement period (i.e., baseline, treatment application and during painful stimuli).The two goat kids that received needle-free injections were removed from all analyses, resulting in eight goat kids in the jet injector treatment groups available for analysis.
The association between treatment application and heart rate, and the association between treatment and heart rate during a painful procedure Data for the median HR (beats/10 seconds) before (baseline) and during treatment administration were descriptively analyzed.A linear model was used to assess the effect of treatment on HR; the sham and control groups were not included in this analysis as they did not have HR measured during treatment.As it was important to assess how HR changed during the time of administration, the baseline HR prior to treatment administration was included in the model, as was an interaction term between treatment group and baseline HR.The interaction term was removed from the model if the F-test between two nested models had a p > 0.05; baseline HR and treatment group were retained in the model, regardless, as predictors of interest.No other variables were included.Assumptions of linearity of residuals and heteroskedasticity were assessed graphically, and outliers, if present, were investigated.This analysis above was repeated for HR during painful stimuli; however, the sham and control groups were included in this analysis.

The association between treatment and vocalization and movement scores
Due to the perfect agreement between the two technicians for marked pain response (based on classification scores), only data from one technician was used for analysis for all vocalization and movement scores.As further painful stimulation was precluded if the first two needle pricks around each horn bud were considered to produce a marked response, there were missing data in locations three and four for both left and right horns.The data were therefore analyzed in two subsets: (1) Marked pain response.This was analyzed using logistic regression, with the outcome variable being whether a goat had a subjective marked pain response (any vocalization or movement score 2-3) recorded against it or not (all vocalization and movement scores 0-1).No clustering needed to be accounted for as the outcome was measured at the goat level.The only fixed effect risk factor assessed was the treatment group.Contrast analysis was conducted to assess whether differences existed between each group.The assumptions of logistic regression were assessed, and outliers investigated, if present.
(2) Vocalization and movement scores for sites 1 and 2 on each horn.
The scores for vocalization and movement were collapsed into two groups: low scores (0 and 1) and high scores (2 and 3).The outcome variables were vocalization and movement at each site; there were four measurements for each goat, two on each horn.Two mixed logistic regression models, one for vocalization and one for movement, were used to compare the difference in scores between treatment groups; repeated measures for goat was controlled for by including goat as a random effect.Potential fixed effect risk factors included site (1 or 2) and side (left or right), along with treatment group.As none of the sham group elicited a high grade for vocalization or movement, these had to be removed from the mixed model as the model could not converge if they were included.
All three of the potential risk factors were included in an initial multivariable model.Starting with the risk factor with the greatest Wald t-test p value, variables were sequentially removed if the loglikelihood ratio tests between two nested models were p < 0.05; treatment was retained regardless as a predictor of interest.A variable was retained as a confounder if coefficients or standard errors are altered by >20% with removal of the variable.
As the jet injector was a specific treatment of interest, the modeling process was repeated with the remaining treatments collapsed into one group and compared to the jet injector.

Results
Data were available from 43 goat kids.No data were collected from the two goat kids which were treated using the jet injector without a needle, as the process caused significant distress and the local anesthetic did not disperse in the tissue and remained on the surface.

The association between treatment application and heart rate
A boxplot of the HR of the goat kids just prior to and during the administration of the treatments is presented in Figure 4. HR prior to treatment had a significant impact on HR during treatment (p < 0.001).After accounting for the HR immediately prior to treatment administration, HR was 3.3 beats/10 seconds faster (95% CI 0.4 to 6.1) during the treatment administration in goat kids receiving the cool spray compared to topical anesthesia.No difference was detected in the HR between topical anesthesia and jet applicator, with a difference of 1.4 beats/10 seconds (95% CI −1.22 to 4.03).There was no interaction noted between HR prior to treatment administration and treatment group (p = 0.18).

The association between treatment and heart rate during painful procedure
Neither treatment (p = 0.29) nor the interaction between treatment and baseline HR (p = 0.11), had a detectable effect on HR during the needle prick test.One beat/10 second faster baseline HR was associated with an increase of 0.40 (95% CI 0.17 to 0.62) beats/10 seconds faster HR during the painful stimulus.
For 33/43 of the goats, HR was lower during the painful procedure than baseline HR (Supplementary Figure 1).

The association between treatment and vocalization and movement scores during painful procedure
The raw data for the number of goats for the presence or absence of a marked pain response at any stage of painful stimuli administration is presented in Table 3.There was a difference in marked pain response between the treatment groups (p = 0.035; Table 4).Compared to kids treated with topical anesthetic, kids treated using the jet injector had a 96% (95% CI 50.5 to 99.8%) reduced odds of expressing a marked pain response.No statistical difference was detected between jet injector and cool spray or jet injector and no treatment; however, numerically there were fewer animals with a marked pain response with the jet injector compared to these two other groups.The summary table for the vocalization and movement grades on both the left and right sides for the treatment groups is presented in Table 5.No effect of treatment on movement (p = 0.13) or vocalization (p = 0.86) was detected.However, compared to the first site, kids had a 5.2 times (95% CI 2.2-12.1)greater odds of having a high vocalization grade and a 4.6 times (95% CI 2.4-8.6)greater odds of having a high movement grade when the second site had the painful stimulus applied to it.
When the contrast analysis for the jet injector treatment group was compared to the other three treatment groups, kids treated with the jet injector had an 83% (95% CI 22-96%) reduction in the odds of a high movement grade and a 63% reduction in the odds of a high vocalization grade (though the confidence interval of that effect was wide; 95% CI 97.5% reduction to 5.0 times increase).

Discussion
This study was designed to test whether some practical and easy-to-apply solutions could provide effective pain relief around the horn bud in dairy goat kids.The study demonstrated that available topically applied products such as a coolant spray or EMLA cream were simple to apply but did not appear to provide any benefit in terms of pain relief.In contrast, local anesthetic applied with a jet injector did decrease some of the pain responses, suggesting that it is a potentially useful method of providing pain relief which should be tested in further larger studies.
The assessment of pain in goats is often challenging (Malavasi, Greene, Gay, & Grubb, 2016), so multiple assessment techniques were used in this study.Of the measures used, HR appeared to be the least useful as it was lower during the needle prick procedure than at baseline.This is probably because the goat kids had not been handled prior to the baseline HR measurement, so that first handling was more stressful than the third handling and needle prick tests.This is one of the challenges of using a stethoscope as opposed to an electronic measuring device that does not require any handling.Nevertheless, we were still able to detect the impact of using the vapocoolant on heart rate.It is likely that this heart rate response occurred because 1) vapocoolants cause pain at the time of application (Griffith, Jordan, Herd, Reed, & Dalziel, 2016) and 2) in order to protect the face, especially the eyes, from the vapocoolant, the kids treated with the vapocoolant had a towel placed over their face.
Grading vocalization and movement gave a much clearer picture than heart rate.The sham group (digital pressure rather than needle pricking) had no marked vocalization or movement responses (grade 2 or 3); in contrast, 23% and 43% of goat kids which did experience the needle prick procedure had a marked vocalization or movement response, respectively.Neither the vapocoolant nor the topical anesthetic reduced the proportion of marked vocalization or movement responses compared to the no treatment group, whereas the number of marked vocalization or movement responses in the jet injector were numerically lower (Table 5).When the jet injector was compared to the other treatment groups that received the painful stimulus, the odds of a marked movement and vocalization response were 63% and 83%, lower for the jet injector group, respectively.
The apparent effect of treatment on the vocalization and movement responses was consistent with the apparent effect of treatment on the pain response to the needle prick, i.e., there was a lower proportion in the jet injector group than the control, vapocoolant, and topical anesthetic groups (Table 3).
Although, as expected, there were no differences detected between the left and right horn bud of a goat kid, there was a clear relationship between site 1 and site 2 around each horn bud.Regardless of whether the direction was clockwise or anticlockwise, this difference indicates that the goat kids were "anticipating" the painful stimulus and were more responsive to subsequent painful stimuli.Another possibility could be that the nerve structures in the medial and lateral locations may have elicited a more pronounced reaction than those in the dorsal or ventral locations.
The data suggest that neither the vapocoolant nor the topical anesthetic showed any potential as methods for reducing the sensitivity around the horn bud.The absence of an apparent pain reduction result with EMLA cream were similar to Hempstead et al. (2020) who concluded that EMLA cream may have intensified the pain associated with disbudding when investigated on 10 doe kids in comparison to other pain relief methods.In addition, the application of the vapocoolant Table 5. Descriptive results of the subjective vocalization grade (0-3) and movement grade (0-3) for the first two needle prick or digital pressure sites (for positive control only) out of four on each horn bud (left or right).Each goat has four measurements (two for each horn bud).The results are grouped into treatment.seems to have been stressful for the kids.We recommend that neither product should be used in further investigation of methods to reduce the pain associated with disbudding.
In contrast, our data suggest that the jet injector may be a useful farmer-suitable way of reducing the pain associated with disbudding.This conclusion has several caveats.Firstly, this was a small study, with only 10 kids in the treated group, so the jet injector needs to be tested on a much larger scale and before a disbudding procedure before any recommendations can be made.Secondly, despite its apparent success in reducing the response to the needle prick test, the use of the jet injector did not result in vocalization or movement scores equivalent to those seen after digital pressure alone.Thus, even if these results were to be repeated in a larger scale study, work would still be needed to improve the effectiveness of the jet injector.Finally, there were numerical differences between the left and right side of the jet-injector in the proportion of kids with a marked vocalization or movement response when using the jet injector.This numerical difference aligns with what was happening practically as it was a lot easier for the person applying the injector, who was righthanded, to administer the jet-injector on the left side.This needs addressing if this method is to be used at a large scale.
We are unsure of the potential effect of feeding prior to the treatments and application of a painful stimulus, however this could not be avoided as feeding was necessary due to the young age of the kids and the length of time the trial was run over.

Conclusion
Data from this study suggest that neither vapocooling nor topical anaesthetic creams appeared to reduce pain associated with a pin prick test and therefore, would be unlikely to be useful prior to disbudding.In contrast, there was sufficient evidence that application of local anesthetic using a jet injector reduced the pain sensitivity of the horn bud to suggest that the jet injector should be tested in a larger scale study with goat kids undergoing disbudding not just a needle prick stimulus.

Figure 3 .
Figure 3. Image of the micro-injector use to inject the goat kids with lignocaine.

Figure 4 .
Figure 4. Boxplots of the raw heart rate data (beats per 10 seconds) for goat kids before treatment administration and after treatment administration.The red square represents the arithmetic mean for each group, and the horizontal black line within the box representing the median of each group.

Table 1 .
Treatment group description, number of goat kids in each group and timing of application of treatment.Lignocaine and Prilocaine, AstraZeneca Pty Ltd, Australia) applied at a rate of approximately 1 g/10cm2.Applied around the horn bud using a 1 cm sized blob and gloved finger, rubbing the cream over the area that would be burnt by disbudding irons in typical practice in circles.

Table 2 .
Description of vocalization and movement grades following a painful stimulus applied by an 18 g needle around the horn buds.

Table 3 .
Number, and percentage, of goats that had a marked pain response recorded for each treatment group at the time of painful stimuli on four sites of the skin near each horn bud; positive control goats only received digital pressure over the skin surrounding the horn bud.

Table 4 .
Results from a generalized linear mixed model outcome of marked pain response to a painful stimulus.Unit of measurement is the goat (n = 38).