Effect of Meloxicam on Behavior, Physiology and Pain Sensitivity in Holstein Calves Following Dehorning

The present work aimed to assess the effects of meloxicam on the behavior, feed intake, physiology and pain sensitivity in calves following dehorning. Calves were randomly allocated to four groups: dehorning by amputation with saline (ADNM) or meloxicam (ADM) and chemical disbudding with saline (CDNM) or meloxicam (CDM). Signicant differences in the frequencies of all behaviors and activity levels were not observed for the treatment*time interaction (p ≥ 0.05) but were observed for time points (p < 0.05), and the total intake, foraging, hay or milk intake, rectal temperatures, respiration and heart rates and mechanical nonreceptive thresholds (MNTs) in calves were all noted (p < 0.05). For the treatment*time interaction, signicant differences were showed between the CDNM and CDM groups in the total intake (at d 0 and 1), foraging (at d 1), hay or milk intake (at d 0 and 1), rectal temperatures (at 24 hr), heart rates (at 4 and 6 hr), and MNTs ( right horn at 2, 6, 24, 48 and 72 hr; left horn at 6 and 24 hr); and signicant differences were found between the ADNM and ADM groups in the rectal temperatures (at 6 hr), respiration rates (at 4, 6 and 24 hr), heart rates (at 2, 4 and 6 hr), and MNT (right horn at 2 to 72 hr; left horn at 24 hr). Our results indicated that meloxicam relieves pain after calf dehorning and helps restore feed intake. 1 . International veterinary clinical studies report that meloxicam is used for abdominal surgery in dogs 2 , osteoarthritis in cats 3 , delivery in cows 4 , castration in calves 5,6 , and dehorning in calves 7-9 . This drug has shown good analgesic effects and is safe, and a continuous analgesic effect after calf dehorning has been reported.


Introduction
Meloxicam is a non-steroidal anti-in ammatory drug (NSAID) belonging to the enol-amide group and was developed by the German company Boehringer Ingelheim. Meloxicam selectively inhibits the activity of cyclooxygenase-2 (COX-2) by blocking prostaglandin (PG) synthesis from arachidonic acid (AA) and reducing PG production to achieve antipyretic and analgesic effects. Meloxicam is widely used to control infection and non-infectious in ammation and painful diseases 1 . International veterinary clinical studies report that meloxicam is used for abdominal surgery in dogs 2 , osteoarthritis in cats 3 , delivery in cows 4 , castration in calves 5,6 , and dehorning in calves [7][8][9] . This drug has shown good analgesic effects and is safe, and a continuous analgesic effect after calf dehorning has been reported.
Dehorning is a routine procedure performed globally in the dairy cattle industry to ensure the safety of dairy cows and breeders and achieve maximum economic bene ts 10 . However, consumer interest in animal welfare, especially dehorning, has increased 11 . Amputation, cautery and chemical disbudding in calves are the main dehorning methods in dairy industry 12 . However, all of these methods cause trauma, stress, pain and in ammation in calves, thus violating animal welfare principles 13 . Dehorning is mainly performed with auxiliary local anesthetics and NSAIDs. Local anesthetics relieve acute pain through angular nerve block. NSAIDs inhibit COX-2 expression and block PG synthesis to achieve analgesic effects, effectively relieving chronic pain after surgery. However, the analgesic effect of the commonly used local anesthetic lidocaine lasts only 2-3 hr 14,15 , and the half-lives of ketoprofen, unixin meglumine and meloxicam are 2-4 hr 16 , 6-7 hr 17 , and 26 hr 18,19 , respectively. Ketoprofen and unixin meglumine are nonspeci c COX inhibitors.
Dehorning bene ts the long-term welfare of the animal, but all methods of dehorning cause acute pain and distress, which lead to physiological and behavioral responses. The degree of these responses varies with different dehorning methods. Pain is a subjective state that can only be measured indirectly, and a systematic pain evaluation method has not been developed for cattle after dehorning 20 . Of course, changes in blood physiological indexes, including the concentrations of serum cortisol and haptoglobin [21][22][23][24][25] , plasma substance P 26,27 , prostaglandin E 2 17,28 , and whole blood parameters 29 , can be measured. Other physiological indicators, such as heart and respiratory rates 7,30,31 , eye temperature 7 , electroencephalogram results 30 , and the mechanical nonreceptive threshold (MNT) 22,32 , can also be measured.
In addition, changes in behavioral indexes, such as head shake, kick, stamp, ear ick, tail ick, walk, stand, lie, head down, eat and drink [33][34][35][36][37] , can be measured. Average daily gain may also be affected by dehorning in calves 8,38 . Nevertheless, further investigations on the elimination of local anesthetics, such as lidocaine or bupivacaine 14,15 , the pain responses in recovered dehorned calves, and potential long-term pain alleviators are needed.
To our knowledge, the effect of meloxicam on the behavior of calves after dehorning have been assessed based on the frequency of each time point, whereas few investigations have focused on postoperative changes in diet and behavior. Moreover, the effects of meloxicam on pain-related behaviors and feed intake after calves undergo chemical disbudding or amputation dehorning with cauterization have not been examined. Therefore, the objective of this study was to assess changes in pain-related behaviors, feed intake, activity levels, physiological indexes and MNTs with different dehorning methods and to estimate the e cacy of s.c. meloxicam administered immediately prior to dehorning in Holstein calves of different ages.

Results
Based on the measured temperature and humidity, the THIs was calculated, and all calves were con rmed to not be in a heat stress environment during the experiment. Clinical examinations showed that all calves were in good health and without serious symptoms, such as death. No calves were eliminated from this study.

Behaviors
No signi cant differences in the frequencies of head shaking, head rubbing, ear icking, tail icking and kicking or in the treatment*time interaction were observed between the treatment groups, although signi cant differences were noted in the frequencies of all behaviors between time points (p < 0.05) except for head rubbing and ear icking (Table 1). In addition, no signi cant differences in the frequencies of head passing through the fence were observed between treatment groups (p ≥ 0.05).

Whole condition
Due to differences in the time points of the activity levels of calves on d -3, -2, -1, 0, and 1, the time points at d -3, -2 and -1 were regarded as controls. No signi cant differences in activity levels in all treatment groups were found for the treatment and treatment*time interaction (p ≥ 0.05) ( Table 2). In addition, signi cant differences in activity levels were observed between time points (p < 0.001). No signi cant difference in activity levels were noted between the time points of the controls at d -3, -2 and -1 (p ≥ 0.05), although signi cant differences compared with d 0 and 1 were observed (p < 0.05). Additionally, signi cant differences in activity levels were found between d 0 and 1 (p < 0.05). The feed intake of the calves at d -3, -2, -1, 0, and 1 was recorded by the researchers. No signi cant differences in total intake, foraging and hay or milk intake were identi ed for the treatment (p ≥ 0.05) except for foraging between the ADNM and ADM groups (p < 0.05) ( Table 3). Furthermore, signi cant differences in the total intake, foraging and hay or milk intake were observed between the time points or for the treatment*time interactions (p < 0.05). In terms of the treatment*time interaction, signi cant differences were observed between the CDNM and CDM groups in the total intake at d 0 (p = 0.007) and 1 ( p = 0.026), foraging at d 1 (p = 0.002), and hay or milk intake at d 0 (p = 0.007) and 1 (p = 0.035).

Physiology
The time of dehorning was designated 0 hr. Regarding the respiration and heart rates of calves from -24 to 24 hr ( Fig. 1), signi cant differences between treatments and time or for the treatment*time interactions were observed between the ADNM and ADM groups(p < 0.05). Moreover, a signi cant difference in the rectal temperatures and respiration and heart rates of calves was only identi ed for time points between all treatment groups. In terms of the treatment*time interaction, signi cant differences were observed between the ADNM and ADM groups in rectal temperatures at 6 hr (p = 0.002), respiration rates at 4 hr (p = 0.006), 6 hr (p = 0.015) and 24 hr (p = 0.002), and heart rates at 4 hr (p = 0.013), 6 hr (p = 0.001) and 24 hr (p = 0.032). In addition, signi cant differences in rectal temperatures at 24 hr (p = 0.022) and heart rates at 6 hr (p = 0.048) and 24 hr (p = 0.034) were found between the CDNM and CDM groups.  (Table 4). In addition, the different treatments had an effect on pain sensitivity at all locations (p < 0.05). Location 2 was the most sensitive compared with locations 1, 3 and 4 (p < 0.05).

Discussion
The objectives of the present study were to examine the effect of meloxicam on calves considering physiological, behavioral, feed intake and pain sensitivity indexes. Calves underwent two dehorning methods: CD or dehorning by amputation with vascular cauterization. CD in calves is now common in China because it is simple, convenient, and affordable for farmers, although there is currently little research on this method. By simulating false dehorning, the stress response caused by actual dehorning can be eliminated to a certain extent. The calves in the control group were also included in the treatment group to exclude the effects of individual differences. The administration of saline in this experiment was injected in exactly the same way in the same sites and with the same volume as meloxicam and served as the 'control' to evaluate the impact of the sham operation and eliminate the effect of the injection itself. Therefore, the following discussion will focus on the changes in physiology, behavior, feed intake, and pain sensitivity associated with meloxicam administration before and after calf dehorning to determine its analgesic effect.
An interesting phenomenon is the apparent circadian pattern of pain-related behaviors 32 , which appear to occur less frequently early in the day, frequently from mid-afternoon until evening feeding, and then less frequently thereafter. Therefore, the frequencies of behaviors displayed by calves in each treatment group on d -1, 0, and 1 included the frequencies of behaviors observed at 7:00 am, 12:00 pm and 7:00 pm. The purpose was to explore the effects of meloxicam on feed intake and pain-related behaviors during feeding in dehorned calves. Recent research has found that meloxicam-treated, dehorned, 6-to 12-week-old Holstein calves displayed less ear icking for 44 hr and head shaking for 9 hr after dehorning than calves not treated with meloxicam 32 ; moreover, meloxicam signi cantly increased the time spent eating following castration and/or dehorning in 3-month-old Holstein calves 29 . In another study, meloxicam signi cantly reduced the head turning frequency and increased weight gain following castration and dehorning in 6-to 8-month-old beef calves 37 , although clear observations of the effects of meloxicam on other behaviors displayed by 6-to 8-mo-old dehorned beef calves have not been reported 9 . Overall, the changes in behavior suggested that meloxicam was effective in reducing post-surgical pain and distress associated with calf dehorning. This experiment indicated that the pain relief process after calf dehorning is not associated with the analgesic effect of meloxicam and may be closely related to hunger. In addition, changes in behaviors caused by dehorning were not clearly observed, which may be because behavior was observed only during feeding and starvation may cause calves to ignore pain for a short time. The total intake, forage, silage and hay and milk intakes in all groups were different after dehorning, which may be due to pain intensity caused by differences in the dehorning methods and tolerance levels due to differences in calf ages. This experiment indicated that meloxicam promoted the resumption of intake in 1-to 3-wk-old calves, which may be because milk is easier for calves to consume. Thus, meloxicam can effectively relieve pain in 1-to 3-wkold calves after CD and promote feed intake recovery.
Currently, most studies have explored walking time to describe the relationship between calf activity levels and pain. Studies have found that ketoprofen 38 , lignocaine 36 , and meloxicam had no effect on walking time after calf dehorning, and even meloxicam caused a signi cant increase in walking time 37 . Therefore, it is feasible to use mobile phone software to observe data from a pedometer attached to the right hind leg of calves to record daily activity levels. In the present study, there were signi cant differences in the activity levels of calves in all groups after dehorning (p ≤ 0.05) but no signi cant difference between the treatment groups (p > 0.05), indicating that the change could have been caused by dehorning; however, meloxicam did not show an obvious analgesic effect. The activity level of calves before dehorning was clearly higher than that after dehorning, suggesting that postoperative pain existed. Generally, the activity level in the ADM and CDM groups decreased more than that in the ADNM and CDNM groups, respectively. In addition, the average activity levels in the CDNM and CDM groups were higher than those in the ADNM and ADM groups, which may have been caused by fear associated with the age differences in the calves and different pain levels caused by the different dehorning methods. Thus, meloxicam may promote a certain pain relief effect by potentially reducing the postoperative activity level.
Dehorning can cause traumatic stress, pain and local in ammation and affect a calf's normal physiological parameters, including rectal temperature and respiration and heart rates. Previous research mostly focused on the effect of meloxicam on the physiological condition of calves following cautery disbudding, and the results suggested that meloxicam reduces the respiratory rate, heart rate, and heart rate variability 7,23 but not the eye temperature 39 . The heart rate was signi cantly reduced by the combination of xylazine, although orphanol and bupivacaine 31 . Moreover, local in ammation can cause fever. The relationship between rectal temperature and meloxicam in calves following dehorning has not been previously reported. In the present study, signi cant differences were not observed in rectal temperature or respiration and heart rates before dehorning between the treatment groups (p > 0.05). In addition, all the treatment groups showed an overall increasing trend followed by a decreasing trend after dehorning. The effective time and e cacy of meloxicam were signi cantly different between the ADNM and ADM groups. In general, meloxicam can signi cantly reduce the rectal temperature and respiration and heart rates of calves undergoing dehorning relative to other methods of relieving postoperative pain.
A pressure algometer is effective for evaluating the effects of analgesics on dehorning pain and can determine the minimum amount of pressure required to elicit a pain response 32 . Meloxicam has been reported to increase the MNTs of the LH and RH in calves after dehorning, indicating that meloxicam can alleviate pain to some extent 22,39 . Therefore, it is necessary to handle calves in advance to adapt them to the operational procedures to avoid errors as much as possible. In the present study, all calves showed signi cantly decreased tolerance to pressure following dehorning. The effect of meloxicam on the MNT measured by the algometer was observed. However, the MNTs of the LH and RH in calves were signi cantly different. Between the ADNM and ADM groups, the MNTs of the LH after dehorning did not indicate the same e cacy of meloxicam e cacy as observed for the RH, and between the CDNM and CDM groups, the MNTs of the RH after dehorning more obviously indicated the e cacy of meloxicam e cacy relative to those of the LH. However, the MNTs of the LH and RH before dehorning did not show obvious differences in all groups. When using a pressure algometer to detect the MNT value of the horns, the RH precedes the LH; thus, because the pain caused by the pressure algometer induces fear in the calf, more obvious evasive psychology is observed when detecting the MNTs of the LH. In addition, there were signi cant effects on the mean MNTs according to the measurement sites (1)(2)(3)(4) in the LH and RH before and after dehorning. Location 2 was the most sensitive compared with locations 1, 3, and 4 (p < 0.05), which is in accordance with research by Heinrich et al. 23 Since local anesthetic was not used to block the cornual nerve, the MNT at 2 hr decreased dramatically after calf dehorning, which is consistent with the results of Allen et al. 39 , who used a local anesthetic. However, the average value of the MNT was slightly different from those in other related reports 23,32,39 , which may be attributable to differences in toleration according to the ages of the calves. Similarly, the pressure algometer used in this experiment contained a round iron tip instead of a round rubber tip, which may have caused discomfort at the sites of the wounds. With increasing time intervals and measurement times, the MNT of each measurement in the LH and RH will be affected by the interactions between the calves and experimenters, especially in 1-and 3wk-old calves. Overall, this nding indicates that meloxicam can relieve pain after calf dehorning.
Most previous studies explored the analgesic effects of meloxicam in calves with different ages, calves receiving different routes of administration and calves undergoing different dehorning methods. Dehorning by amputation is highly invasive and stressful and produces greater pain than cautery disbudding due to extensive damage to the horns. CD has the following advantages: it is simple, convenient, and affordable for farmers; causes less damage to horns; and causes less pain in calves than amputation. However, limited research is available on this method. The purpose of this study was to explore the analgesic effect of meloxicam alone in calves of different ages undergoing different methods of dehorning, and we found that meloxicam can relieve pain and promote the recovery of feed intake after calf dehorning. Thus, performing CD in 1-to 3-wk-old calves is bene cial.
In conclusion, the feed intake and behavior during feeding displayed by calves can re ect pain and tolerance at that point in time. Remotely observing a calf's daily activity with a pedometer can directly re ect the degree of pain better than walking time. Meloxicam effectively alleviated the pain caused by CD in 1-to 3-wk-old calves, and the effect was more obvious than that in 1.5-to 6-mo-old calves following dehorning by amputation. Therefore, meloxicam is recommended to relieve pain caused by CD. Although research is still scarce, meloxicam should still be recommended to farmers.

Methods
All experimental procedures were approved by the Sichuan Agricultural University College of Veterinary Medicine before the initiation of the study. All animal operations and procedures were conducted according to the approved guidelines and were in accordance with the international Guide for the Care and Use of Laboratory Animals.

Animals, experimental design and treatments
The study was conducted in 60 Holstein calves from a dairy farm in Qionglai, Sichuan, China. At 1.5 to 6 months of age, Holstein calves (n = 30) weighing 148 ± 32.68 kg were allotted to pens measuring 30.0 × 4.9 m (15 calves/pen), and hay, water and maize silage were provided daily. At 1 to 3 weeks of age, Holstein calves (n = 30) weighing 51.27 ± 5.97 kg were allotted to pens measuring 10.0 × 6.5 m (15 calves/pen). The calves were fed milk without antibiotics twice daily, and small amounts of maize silage and hay were provided daily. All calves underwent clinical examination 4 days (d) prior to the o cial start of the trial and were included in the study sample if they were determined to be healthy. In addition, the daily feed intake was calculated by calculating the total feed intake of all calves in the rst 4 days. Based on this condition, the quantity of feed offered was adjusted daily to obtain daily leftovers of 5 to 10% of the total amount provided in order to ensure random intake.
The experiment consisted of a 2 × 2 factorial design where the main factors included the dehorning method and medication treatment. The two dehorning methods and two medication treatments were as follows: 1) Dehorning by amputation (AD): physical dehorning was performed using Barnes dehorners and electrocauterization was performed at 600 ℃ for approximately 15 -30 sec to control any hemorrhaging. The treatment was applied to calves at 3-6 months of age.
2) Chemical disbudding (CD): sodium hydroxide ointment in an amount equal to the area of approximately 5 coins was evenly applied to each bud. The treatment was applied to calves at 1-3 weeks of age. The calves were divided into 4 groups, equally distributed by weight into pens and randomly assigned to treatments. Over a period of 7 d, the four pens of calves were treated with their respective treatments on each day. Calves were restrained in a squeeze chute to perform all treatments. In all calves, both horns were desensitized using a cornual nerve block at least 10 min prior to dehorning by injecting 5 ml (100 mg) of 20 mg/ml Lidocaine (Lidocaine Hydrochloride Injection; Shandong Hualu Pharmaceutical Co., Ltd) to a depth of approximately 1 cm under the temporal ridge around each cornual nerve. Treatments included the following: (1) ADNM (n = 15); (2) ADM (n = 15); (3) CDNM (n = 15); and (4) CDM (n = 15). In all calves the horn bud was prepared rst by shaving the hair around the site using electric clippers. All calves underwent a simulated dehorning procedure on d -1 without medication injection, electrocautery or sodium hydroxide ointment application. The simulated dehorning procedure is consistent with the true dehorning process, the test indicators are the same and the time points correspond to each other. The purpose of a simulated dehorning procedure is to eliminate the stress response caused by the experimenter's operation and ensure the accuracy of the test indicators. To keep ies away, the dehorning wounds were sprayed with 0.5% permethrin.
Behavior data collection The measured behaviors of calves included "head shaking, head rubbing, ear icking, tail icking, kicking and head passing through the fence" (Table 5). To encourage the calves to adapt to the experimenters and ensure that the experimenters could skillfully and accurately record various behavioral frequencies, a simulation of behavioral frequencies was carried out 3 days before the formal test. When the calves ate or drank at 7:00 am, 12:00 pm and 7:00 pm on d -1, 0 and 1 as usual, four trained experimenters recorded the behavior frequencies of the calves at 9 time points. The feeding process of calves can be fully guaranteed by 10 min of behavioral frequency measurement time. Repeated movement of the head from side to side in a rapid motion.
Head rubbing Lifting of a hind leg to scratch the top of the head with the foot or head rubbing against the side of the pen.
Ear icking Repeated movement of one or both ears in a quick motion independent of head shaking.
Tail icking Movement of the tail vertically.

Kicking
Lifting of a hind limb and contacting the head or kicking backward or towards the belly.
Head passing through fence Inserting the head through the bars of the pen towards the food and water buckets. This behaviour was recorded at feeding time at only 6 hr following dehorning. a) Points are behaviours without a measurable duration and are quanti ed by the frequency.
Whole condition data collection Whole conditions measured included feed intake and activity level. The researchers observed and recorded the activity level and feed intake of calves on d -3, -2, -1, 0, and 1. The feed intake of all calves was the same as usual three days before the formal test. In addition, all calves wore pedometers three days before the experiment to ensure that the adaptive stress response of the calves was eliminated. The experimenters attached a pedometer (Huawei e3 pedometer, Huawei Technology Co., Ltd.) to the tarsal joint of each calf's right hind leg to monitor daily activity levels with a mobile phone. The Huawei e3 pedometer uses an extremely sensitive three-axis acceleration electronic shock sensor combined with built-in software to output steps and has no special requirements for placement position and direction. The temperature and humidity were measured with a thermo-hygrometer (Deli 9010 In-outdoor Thermo-hygrometer, Deli Group Co., Ltd.) at 7:00 am, 12:00 pm and 7:00 pm daily. Furthermore, the experimenters recorded the daily intake of all calves and weighed them at 6:00 am on d 0.
The experiment schedule was adjusted according to temperature-humidity indexes (THIs), and the calves with poor health were eliminated by clinical diagnosis.

Physiological data collection
Dehorning can affect the physiological status of calves. Three researchers participated in the collection of physiological data from all calves and observed the respiration rates, heart rates and rectal temperatures of calves at -24 hr, -6 hr, -4 hr, -2 hr, 0 hr, 2 hr, 4 hr, 6 hr, and 24 hr with a stethoscope (multifunctional stethoscope, JiangSu YuWell Medical Equipment Co., Ltd.) and a thermometer (soft head electronic thermometer BT-A21G, Dongguan Fudakang Industry Co., Ltd.).

Pressure algometry
Pain sensitivity was measured at -72 hr, -48 hr, -24 hr, -6 hr, -4 hr, -2 hr, 0 hr, 2 hr, 4 hr, 6 hr, 24 hr, 48 hr, and 72 hr for the sham and actual dehorning groups using a pressure algometer (Digital Force Gauge SL-30, Dongguan Jingyou Mould Hardware Co., Ltd.), and the same trained researcher performed all measurements (Fig. 3). The device was equipped with a round iron tip measuring 1 cm in diameter. The amount of pressure that a calf tolerated was measured in kilograms of force (kgf) and considered the mechanical nonreceptive threshold.

Statistical analysis
All data were analyzed using IBM SPSS statistics 21. GraphPad Prism software (Version 8.02; GraphPad Software Inc., San Diego, CA, USA) was used for graph plotting of the experimental data. The data on passing a head through a fence between treatment groups were analyzed by Wilcoxon rank sum test. Furthermore, the MNTs at each location surrounding the horn buds before and after the operation were compared by a one-way analysis of variance (ANOVA), and the other data were analyzed by a generalized linear mixed model (GLMM). The treatment, time, and treatment*time interaction were used as xed effects, whereas calf was the subject of the repeated measures. Data are presented as the mean ± SEM. For all statistical calculations, p values < 0.05 were considered statistically signi cant.

Data availability
The data used to support the ndings of this study are available from the corresponding author upon request.