Optimization of analgesia for piglet castration under isoflurane anaesthesia with parenteral butorphanol, meloxicam or intratesticular lidocaine

INTRODUCTION
This blinded prospective study investigated analgesic effects of intramuscular (IM) butorphanol, meloxicam or intratesticular (IT) lidocaine for castration of 7-14 days old piglets under isoflurane anaesthesia. 66 piglets were randomly injected with: meloxicam IM (0.4 mg/kg; group M), butorphanol IM (0.2 mg/kg; group B), or both (group BM) 20 minutes prior to castration, or lidocaine IT (4 mg/kg (group ML4) or 8 mg/kg (group ML8)) together with meloxicam IM (0.4 mg/kg) under anaesthesia with 1.8% end-tidal isoflurane. Heart rate, respiratory rate, mean arterial blood pressure and end-tidal carbon dioxide were recorded. Anaesthesia quality was scored and postoperative behaviour assessed. As butorphanol caused unacceptable side effects, its use was stopped. Group M showed worse anaesthesia quality than ML4 and ML8 (higher incidence of movements: 11/17, 3/18 and 4/17, respectively). There were no significant differences between groups regarding parameters measured during castration. Postoperative behaviour did not differ between groups. For castration of 7-14 days old piglets under isoflurane anaesthesia, IT lidocaine provides an additional side effect free analgesia.


Summary
This blinded prospective study investigated analgesic effects of intramuscular (IM) butorphanol, meloxicam or intratesticular (IT) lidocaine for castration of 7-14 days old piglets under isoflurane anaesthesia. 66 piglets were randomly injected with: meloxicam IM (0.4 mg/kg; group M), butorphanol IM (0.2 mg/kg; group B), or both (group BM) 20 minutes prior to castration, or lidocaine IT (4 mg/kg (group ML4) or 8 mg/kg (group ML8)) together with meloxicam IM (0.4 mg/kg) under anaesthesia with 1.8% end-tidal isoflurane. Heart rate, respiratory rate, mean arterial blood pressure and end-tidal carbon dioxide were recorded.
Anaesthesia quality was scored and postoperative behaviour assessed.
As butorphanol caused unacceptable side effects, its use was stopped. Group M showed worse anaesthesia quality than ML4 and ML8 (higher incidence of movements: 11/17, 3/18 and 4/17, respectively). There was no significant difference between groups regarding parameters measured during castration. Postoperative behaviour did not differ between groups.
For castration of 7-14 days old piglets under isoflurane anaesthesia, IT lidocaine provides side effect free additional analgesia.

Introduction
In Switzerland, 1.3 millions male piglets are castrated yearly, 75% under inhalation anaesthesia (Enz et al., 2013). Isoflurane is a volatile agent providing fast and save induction of anaesthesia in pigs (Hodgson, 2007). As it lacks analgesic properties (Petersen-Felix et al., 1995) it does not reduce stress response to castration (Schulz, 2007). With additional administration of a non-steroidal anti inflammatory drug (NSAID) such as meloxicam, stress response is reduced (Schulz, 2007). Piglets receiving meloxicam further show less pain related behaviour during surgery as well as postoperatively (Zöls, 2006;Hansson et al., 2011).
To improve analgesia during castration under inhalation anaesthesia, local anaesthesia could be used. With local anaesthesia, blockade of nociceptive transmission should result in pain relief (Vandermeulen, 2000). It has been shown in awake piglets, that intraoperative vocalisation and defence movements are reduced using intratesticular (IT) or subcutaneous lidocaine, in comparison to no analgesia (Leidig et al., 2009;Hansson et al., 2011). In piglets castrated under halothane anaesthesia, mean arterial blood pressures raised significantly less with local anaesthesia and fewer pain-related changes in EEG were seen . Otherwise, literature concerning perioperative pain reduction using local anaesthesia for castration of awake piglets is controversial. Procaine IT in comparison to no analgesia for castration of awake piglets did not result in a reduction of neuroendocrine stress response post operatively, or even an increased stress response was described (Zöls, 2006;Zankl et al., 2007). In contrast, piglets receiving lidocaine IT compared to no analgesia or meloxicam only, showed a lower increase in postoperative plasma cortisol levels (Kluivers-Poodt et al., 2012). An even better outcome was seen in a recently published study using a combination of parenteral meloxicam and IT and subcutaneous lidocaine (Bonastre et al., 2016).
Another option to improve perioperative analgesia is the use of butorphanol, a central acting opioid (Riviere & Papich, 2009). Under injection anaesthesia, butorphanol (0.2 mg/kg IM) in combination with different drugs improved anaesthesia quality and provided optimised perioperative analgesia (Nussbaumer et al., 2011).
The aim of this study was to investigate the effects of additional IM butorphanol or IT lidocaine on the intraoperative response to castration in piglets under isoflurane anaesthesia including parenteral meloxicam as well as the effect on postoperative behaviour.

Animals
The study was approved by the veterinary office of the Canton of Zurich (081/2016).
66 male mixed breed piglets aged between 7 and 14 days were included in this study and by means of an online randomization tool allocated to 5 groups.

Material and methods
Twenty minutes prior to castration, Group M was injected meloxicam 0. instrumented with ECG, pulse oxymetry (SpO 2 ), oscillometric blood pressure measurement device (Dinamap Carescape TM V100 vital sign monitor, GE Healthcare) with a cuff positioned over the right front limb metacarpal artery, and a capnograph tube, which was placed 0.5 cm inside the piglet's nostril. Two minutes after the onset of isoflurane the following parameters were recorded: heart rate (HR), respiratory rate (RR), mean arterial blood pressure (MAP), end-tidal CO 2 (EtCO 2 ) and end-tidal isoflurane (EtIso). Subsequently, the isoflurane vaporizer was adjusted to maintain EtIso of 1.8 ± 0.1%, the fresh gas flow of O 2 remained constant.
Piglets of group ML4 and ML8 were then injected lidocaine IT and meloxicam IM.
One minute thereafter, the blinded observer judged reaction to insertion of a 22G needle into each testicle (performed in duplicate) with a scoring system (table 1) described by Berchtold (Berchtold, 2015). Simultaneously, the vital parameters above mentioned were recorded.
If number of movements or intensity of movement was <2, castration was performed (see table 1). Otherwise an additional minute was deferred and the same procedure was repeated.
This was carried out for 10 minutes in one-minute intervals. If the piglet still showed a score ≥ 2, butorphanol 0.2 mg/kg IM was administered and the piglet castrated.
During castration the following steps were distinguished: 1. incision above both testicles, without opening the processus vaginalis (defined as "cut"), 2. exteriorization of testicles ("exteriorization") and 3. "emasculation" performed simultaneously in both testicles. During each step, monitored parameters and movements were recorded. If scores of movement were ≥ 2 in any of these three steps, an additional minute was deferred before the next step. The time from induction of anaesthesia until emasculation, as well as the time from the injection of lidocaine until cut, was recorded. At the end of the castration, the degree of postoperative bleeding (0 = no bleeding, 1 = mild bleeding, 2 = moderate bleeding) was judged. The piglets were weighted and placed back to the other male piglets. Time until full recovery (standing and 4 steps) was measured.
After 24 hours the piglets underwent a pain assessment adapted from Stark (Stark, 2014). For this purpose each piglet was observed during a 10-minute period and events such as playing (seconds), lying (seconds), time at the teats (seconds) and the amount of pain specific behaviours (quantity) were noted (table 2). Additionally a visual analogue scale (VAS) was accomplished to judge the general condition of the piglets.   (Stark, 2014).

Statistical analysis
A power analysis with a power of 0.8 showed, that to test if the incidence of defence movements during castration can be reduced with lidocaine from 50% to 0%, 11 piglets were considered necessary (WinEpiscope®).
All results were compiled in a spreadsheet (Microsoft Office Excel 2010) and extracted in NCSS 10 (NCSS Statistical Software, Kaysville, USA) for statistical analysis. All continuous parameters were tested for normal distribution. For nonparametric data a Kruskal-Wallis test was used. If significance was obtained, a post hoc analysis with a Mann Whitney U with a manual Bonferroni correction was performed to find the difference between groups.
Significance for the incidence of defence movement was tested with a fisher exact test. For this analysis, piglets were either classified as 0 = no movement, or 1 = movement during the castration. For scores of defence movement, all scores were summed up from the time-point 1 minute until emasculation and the total score was compared. Statistical significance was considered as p < 0.05. For the post hoc analysis p < 0.017 was considered significant, as three comparisons were performed.

Results
All piglets survived the study. One piglet was euthanized due to inguinal hernia one day after castration. In the first 17 piglets that were castrated, unexpected adverse effects such as salivation (1), cyanosis (1), dyspnoea (1), vomiting (1), and paddling before (1), during (1) and after (3) castration as well as excitatory behaviour (2) was seen in 5 animals. It was speculated that butorphanol caused these symptoms. The dosage of butorphanol was then adjusted to 0.1 mg/kg IM. The same adverse effects were present in additional 5 piglets. The person responsible for drug administration revealed, that 10 out of 14 piglets, which received butorphanol, either alone or in combination with meloxicam, experienced side effects, while no piglet out of the other three groups (total of 18 piglets) showed similar symptoms.
Therefore, butorphanol was withdrawn from the study after the castration of 32 piglets and the remaining piglets from group B and MB were reallocated to the residual three groups.
Finally 52 piglets were included in the study, 17 in group M, 18 in group ML4 and 17 in group ML8.
Results are presented with median (interquartile range; range). There was no significant difference between groups in age, estimated weight, scaled weight, time from induction of anaesthesia until emasculation and time from injection of local anaesthesia until cut (table 3).
Also no difference was seen between the estimated and the scaled weight of the piglets (p=0.69). EtIso was maintained within predetermined range of 1.8 ± 0.1% during castration.
There was no significant difference in EtCO 2 between the groups at any time point.
The vital parameters HR, RR, SpO 2 and MAP were not significantly different between groups at any time point.
There was no significant difference between groups regarding degree of postoperative bleeding or time until full recovery (table 3).
The effectively used dose (based on the scaled weight) of lidocaine in group ML4 was 3.852 mg (3.15-4.286; 3.235) and in group 6.956). No adverse effects of lidocaine were seen.
No significant difference was seen in the VAS between groups. As the results of the postoperative behaviour assessment were inhomogeneous with wide distributions and the assessment method retrospectively did not seem appropriate, no more details are shown. Postoperative bleeding 1 (0-1; 2) 0 (0-1; 2) 0 (0-1; 2) 0.70

Discussion
As with the current Swiss standard anaesthesia for piglet castration with isoflurane and meloxicam 14-34% of piglets show purposeful movement in response to surgery (Enz et al., 2013;Steigmann, 2013;Schwennen et al., 2016), the current study investigated further analgesics in order to reduce unnecessary suffering of piglets undergoing castration. In the present study, the additional use of IT lidocaine reduced pain-related defence movements during castration under inhalation anaesthesia from 65% to 17-18% independent of dose rate (4 mg/kg and 8 mg/kg) used. Incidence of movements of group M in the present study was higher than previously published (Enz et al., 2013;Steigmann, 2013;Schwennen et al., 2016).
This discrepancy may have occurred, as the authors of the present study recorded any kind of movement and the piglets were not fixed at all, whereas other authors judged mild movements as unconscious reflexes and not pain related and piglets were fixed with a commercially available castration system. The results of our study are in agreement with previous studies that showed a beneficial analgesic effect of IT lidocaine for piglet castration (Hansson et al., 2011;Kluivers-Poodt et al., 2012). Studies with procaine however concluded, that local anaesthesia was not beneficial for piglet castration, as increased cortisol concentrations thereafter were detected (Zöls, 2006;Zankl et al., 2007). The administration of procaine IT to conscious pigs was additionally deemed painful (Rittershaus, 2009). Also human data showed that the injection of lidocaine is painful (Wago et al., 2016). Therefore, in the present study, IT lidocaine was applied only under inhalation anaesthesia. Lidocaine is rapidly distributed to the spermatic cord when administered IT and reaches peak concentrations 3 minutes after injection in piglets . In the present study median time necessary from injection of lidocaine until start of castration was 120 seconds. Therefore we recommend to wait at least 2-3 minutes from lidocaine injection to castration. A study investigating the plasma concentration of lidocaine, which induces toxic effects marked by seizures in newborn piglets showed, that plasma levels of 40 mg/L lidocaine cause seizures. This corresponds to 30-60 mg of lidocaine for piglets of 1.6 kg administered intravenously (Satas et al., 1997).
Even though these dosages are higher than the dosages used in the present study, we wanted to test two different dosages of lidocaine to ensure safety of 8 mg/kg lidocaine and to detect any toxic effect. No signs of toxicity were observed in the present study and the application of either dose tested IT is considered safe.
Anaesthesia induction was performed with a fresh gas flow of 2 L/min O 2 and 5% isoflurane vaporizer setting, which is the dose used in the commercially available inhalation anaesthesia devices. In piglets 1 MAC isoflurane is 1.4% EtIso (Lerman et al., 1990). MAC is a number that gives an estimate of dose rates of inhalant anaesthetics necessary to maintain anaesthesia.
1 MAC is the number at which 50% of test animals do not react with purposeful movement to a defined stimulus (Levionnois et al., 2009). It is a concentration of inhalant anaesthetic not sufficient to perform surgery in any case and certainly not for surgical castration, therefore the concentration of inhalant anaesthetic was determined, where 95% of patients would not react to a surgical incision, which was found to be 5-40% higher than 1 MAC for humans (de Jong & Eger, 1975). For this reason we chose to provide 1.3 MAC, which corresponds to 1.82% EtIso for maintenance of anaesthesia during castration, as it was described to be sufficient plane of anaesthesia for a surgical stimulus in pigs (Hodgson, 2006).
The administration of butorphanol led to adverse effects and was therefore withdrawn from the study. To date, pronounced adverse effects of 0.2 mg/kg butorphanol, as noted in the present study, have not been reported in piglets. Nussbaumer et al. described mild excitatory behaviour in some cases during the induction of anaesthesia using a combination of ketamine, azaperone and butorphanol IM for castration in pigs of different ages (Nussbaumer et al., 2011). Bettschart-Wolfensberger et al. reported paddling in two out of 28 nine weeks old pigs receiving azaperone, butorphanol, ketamine and meloxicam (Bettschart-Wolfensberger et al., 2013). None of these authors related these side effects to butorphanol. Other authors used butorphanol in a combination with various drugs in pigs of different ages and did not report any adverse effects (Nussbaumer et al., 2008;Heinonen et al., 2009). As the described symptoms in the present study appeared in piglets that had not received any other drug, we suspected butorphanol to be the cause and therefore first reduced the dose and then withdrew butorphanol from the study. The symptoms were not observed again. Contradictory to that, Courboulay et al. used butorphanol (0.1 mg/kg) in combination with meloxicam for castration of awake French piglets and did not report any adverse reactions (Courboulay et al., 2015).
Genetic varieties could explain the discrepancy. In a separate study investigating the effect of butorphanol (0.2mg/kg IM), it was clearly shown, that butorphanol causes restlessness, vocalization and panting, as well as excitatory behaviour (Cap et al., 2017). In other species adverse effects of butorphanol are well described. While dysphoric behaviour is described in cats (Lascelles & Robertson, 2004), horses become ataxic and have increased locomotion (Sellon et al., 2001;Knych et al., 2013), whereas sheep become dysphoric and ataxic (Waterman et al., 1991). These adverse reactions to butorphanol administration warrant further investigation in piglets.
For the postoperative evaluation 24 hours after castration, the piglets were observed for ten minutes as described by Stark (Stark, 2014). When the study was designed this was the only pain assessment described for this age category of piglets. In each litter observation was randomly done for a group of three piglets at the time. It was quickly discovered during the trial, that it depended which ten minutes were evaluated. Sometimes these 10 minutes were spent with lying or drinking whereas 10 minutes later the litter was active and played. Some of the pain specific behaviour was also observed in not castrated piglets of the litter and therefore we conclude that the scoring system used was not appropriate to properly detect differences in postoperative pain between the groups. A newly available method to score postoperative pain, the piglet grimace scale, is an alternative that could be implemented in further investigations (Viscardi et al., 2017).
The authors conclude, that for castration of 7-14 days old piglets under isoflurane anaesthesia and parenteral meloxicam, perioperative analgesia is optimised by additional application of IT lidocaine. For optimal results at least two minutes have to elapse between local anaesthesia application and castration. No toxic side effects were seen and it is concluded that lidocaine can be used safely in daily practice. Last but not least I would like to thank the best possible team on earth -the A-Team! I could learn so much from each of you and you pushed me to the limit, which made me a better anesthesiologist. Thank you for this great time!