Comparative Study of Pain-Related Responses of Male Piglets up to Seven Days of Age to the Application of Different Local Anaesthetics and Subsequent Castration

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animals Article Comparative Study of Pain-Related Responses of Male Piglets up to Seven Days of Age to the Application of Different Local Anaesthetics and Subsequent Castration Franz Josef Söbbeler 1, *,† , Sören Wendt 2,† , Andreas Briese 3 , Julia Tünsmeier 1 , Karl-Heinz Waldmann 2,‡ , Sabine Beate Rita Kästner 1,† and Alexandra von Altrock 2, *,† 1 Clinic for Small Animals, University of Veterinary Medicine Hannover, Hannover, Foundation, 30559 Hannover, Germany 2 Clinic for Swine, Small Ruminants, Forensic Medicine and Ambulatory Service, University of Veterinary Medicine Hannover, Foundation, 30173 Hannover, Germany 3 eduToolbox, 31157 Sarstedt, Germany * Correspondence: franz.josef.soebbeler@tiho-hannover.de (F.J.S.); alexandra.von.altrock@tiho-hannover.de (A.v.A.) † These authors contributed equally to this work. ‡ Valuable contribution to this paper before he departed. Simple Summary: Since 2021, surgical piglet castration must be performed with complete pain elimination according to the Animal Protection Law in Germany. General anaesthesia by isoflurane inhalation, which can be performed by the farmer, or by injection of ketamine and azaperone, which must be performed by a veterinarian, are the options available. At present, local anaesthesia is still under debate because of the lack of proof of complete pain elimination and the pain on injection. Citation: Söbbeler, F.J.; Wendt, S.; We tested three local anaesthetics (procaine, lidocaine, and mepivacaine) at two different doses Briese, A.; Tünsmeier, J.; Waldmann, each. Because pain responses can be masked by reactions caused by handling, the piglets were K.-H.; Kästner, S.B.R.; von Altrock, A. given superficial isoflurane anaesthesia. The pain on injection to the testes was compared with

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e piglets were K.-H.; Kästner, S.B.R.; von Altrock, A. given superficial isoflurane anaesthesia. The pain on injection to the testes was compared with Comparative Study of Pain-Related intramuscular injection, and the effectiveness during castration was compared among the local Responses of Male Piglets up to anaesthetics. Nocifensive movements, respiratory rate, blood pressure, heart rate and its variability as Seven Days of Age to the Application well as electroencephalography (EEG) changes were studied in relation to the painful interventions. of Different Local Anaesthetics and Most indicators of nociception point to testicular injection pain being beyond intramuscular injection Subsequent Castration. Animals 2022, pain when an effective amount of local anaesthetic was used. However, complete pain elimination 12, 2833. https://doi.org/10.3390/ could not be achieved during castration under local anaesthesia. ani12202833 Academic Editor: Robert E. Meyer Abstract: To evaluate pain responses to intratesticular and subscrotal injection of three local anaes- thetics and their efficacy during castration a randomized controlled study was conducted. In groups Received: 23 August 2022 Accepted: 14 October 2022 of 20 piglets, procaine (2%), lidocaine (2%), or mepivacaine (2%) were administered subscrotal and Published: 19 October 2022 intratesticularly in two different dosages: 0.5 mL of the original substances or the maximum rec- ommended dosage according to body weight diluted with isotonic saline to a volume of 0.3 mL Publisher’s Note: MDPI stays neutral per each injection site. Two placebo groups received the equivalent volume of isotonic saline. A with regard to jurisdictional claims in control group was injected intramuscularly with 0.5 mL isotonic saline for

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valent volume of isotonic saline. A with regard to jurisdictional claims in control group was injected intramuscularly with 0.5 mL isotonic saline for injection pain comparison. published maps and institutional affil- iations. Electroencephalographic changes, respiratory rate, heart rate and its variability, blood pressure, and nocifensive movements were assessed in superficial isoflurane anaesthesia. While EEG-changes and linear measures of heart rate variability did not appear conclusive, the low frequency/high frequency (LF/HF) ratio corresponded best with the other pain indicators recorded. The injection of Copyright: © 2022 by the authors. 0.3 mL diluted local anaesthetic per injection site elicited significant fewer signs of pain compared Licensee MDPI, Basel, Switzerland. to intramuscular injection of saline. However, pain reduction, but not complete pain elimination, This article is an open access article during castration could only be achieved with 0.5 mL of the 2% local anaesthetics per injection site, distributed under the terms and whereby lidocaine and mepivacaine were the most effective. conditions of the Creative Commons Attribution (CC BY) license (https:// Keywords: castration; piglet; local anaesthesia; pain assessment; heart rate variability (HRV); creativecommons.org/licenses/by/ lidocaine; procaine; mepivacaine; minimal alveolar concentration (MAC); isoflurane 4.0/). Animals 2022, 12, 2833. https://doi.org/10.3390/ani12202833 https://www.mdpi.com/journal/animals Animals 2022, 12, 2833 2 of 24 1. Introduction Surgical castration of male piglets is a painful procedure. Primarily, castration prevents boar taint, which affects the consumer acceptability of pork and pork products. Therefore, castration is not a producer’s decision but a

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tion prevents boar taint, which affects the consumer acceptability of pork and pork products. Therefore, castration is not a producer’s decision but a market-driven choice [1]. In 2010, representa- tives of European farmers, the meat industry, retailers, scientists, veterinarians and animal welfare organisations voluntarily committed to abandoning piglet castration as of 1 January 2018 [1]. While surgical castration is still practiced in many EU countries, anaesthesia be- comes more and more mandatory. Since 1 January 2021, in Germany, the surgical castration of male pigs aged less than eight days is no longer allowed without effective pain elimi- nation (German Animal Protection Law (Tierschutzgesetz)). In comparable regulations of other European Union (EU) Member States, pain attenuation is considered as sufficient [2]. Anaesthesia for piglets can be either general or local. In Germany, general anaesthesia in piglets may be performed by inhalation of isoflurane or by intramuscular (i.m.) injection of ketamine in combination with the neuroleptic drug azaperone. Ketamine-azaperone anaes- thesia must be carried out by a veterinarian. The main disadvantage of this technique is the prolonged recovery period, during which the piglets must be separated to prevent them from being crushed by the sow. Since 2020 anaesthesia with isoflurane for castration of piglets can be performed by the farmer and permits a safe and rapid anaesthetic induction as well as a brief recovery [3]. Inhalation anaesthesia with isoflurane by the farmer requires an anaesthetic device, which delivers isoflurane over a set period of time. Due to the lack of individual adjustment, sufficient depth of anaesthesia is not always achieved [4,5]. Addi- tionally, isoflurane is a greenhouse gas, affecting

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lack of individual adjustment, sufficient depth of anaesthesia is not always achieved [4,5]. Addi- tionally, isoflurane is a greenhouse gas, affecting the environment and the operator. For economic and animal welfare aspects anaesthesia for routine piglet castration should meet the following criteria: short induction and recovery period, quick and easy to be performed, cause minimal stress, cost-effective, no residuals, and a large therapeutic range of used drugs [6,7]. Therefore, local anaesthesia seems to be a good option to eliminate pain during castration. Topical anaesthesia has failed to provide the required analgesia for all surgical steps of castration [8], the effect of infiltration local anaesthesia in connection with piglet castration is the subject of controversial discussion. Local anaesthetics reversibly block voltage gated sodium channels inhibiting neural conduction. According to their structure, they are categorized into esters or amides, and into short- (e.g., procaine), intermediate (e.g., lidocaine, mepivacaine)—and long-acting (e.g., bupivacaine) compounds [9]. Depending on their pKa local anaesthetics differ in their onset of action, making lidocaine (pKa = 7.8) and mepivacaine (pKa = 7.9) faster acting than procaine (pKa = 9.0) [10]. Procaine has been assessed by the European Medicines Agency (EMA) as a local anaesthetic, which can be used in food producing animals and is currently the only substance approved for pigs in Germany. Lidocaine has been licensed in swine for cutaneous and epilesional use since November 16, 2020 [11]. However, injectable lidocaine and mepivacaine can only be used for piglets in accordance with the cascade rule. Lidocaine is up to twice as effective as procaine [12]. Studies on the efficacy of both substances during

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piglets in accordance with the cascade rule. Lidocaine is up to twice as effective as procaine [12]. Studies on the efficacy of both substances during castration of piglets have shown a pain reducing effect [6,13–17]. The injection itself was repeatedly found to result in pain reactions [18–21]. Direct comparison of study results is almost impossible, as the injection site, injected volume and drug concentration differ, as do the variables used for pain detection. In two recent studies the effect of four local anaesthetics (4% procaine, 2% lidocaine, 0.5% bupivacaine, 2% mepivacaine) was compared [18,22]. It was shown that all four local anaesthetics reduced signs of nociception during castration, but it was also demonstrated that the intratesticular injection caused visible nociception indicated by increased limb movements [18]. The evaluation of nocifensive movements for pain recognition is useful in severe acute events [23]. Since nociceptive withdrawal responses can vary between pigs, it is reasonable to look at behaviour and physiology in combina- tion to fully assess the impact of a painful event on the individual [23]. Parameters, like respiratory rate (RR) and heart rate (HR), blood pressure (BP) and processed EEG variables Animals 2022, 12, 2833 3 of 24 like Narcotrend-Index (NI), total power (PTOT), median frequency (MF) and 95% spectral edge frequency (SEF95), are objectively measurable and most of the parameters mentioned were already used in numerous studies to identify pain reactions in pigs [6,15,18,21,24,25]. Physiological responses of HR and BP result from activation of the autonomous nervous system and may be triggered by stress from handling and restraining [26]. To associate physiological reactions with nociception during castration general

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tem and may be triggered by stress from handling and restraining [26]. To associate physiological reactions with nociception during castration general anaesthesia models can be used [6,18]. Heart rate variability (HRV) is a promising clinical tool to evaluate the autonomic response of different stressors in pigs [27,28]. It indicates the variation in the time interval between consecutive heartbeats and represents the interplay of the parasympathetic nerves, which slow HR, and the sympathetic nerves, which accelerate it. A recent systematic review concluded, that HRV is a good measure of autonomic reactivity to nociceptive stimulation in man [29]. Investigations into HRV to prove pain reactions in pig castration were already conducted [30,31]. The main aim of the study was to assess the suitability of local anaesthesia for piglet castration under 8 days of age fulfilling the requirements of the German Animal Protection Law with respect to the castration procedure and pain of intratesticular and subscrotal injection. In order to reduce stress-induced responses caused by handling and restrain- ing piglets were held in a standardized, minimum alveolar concentration (MAC)-based subanaesthetic isoflurane anaesthesia. Our main hypothesis was that local anaesthesia with mepivacaine, lidocaine or pro- caine will lead to significant reduction in SEF 95% changes in response to castration. Secondly, we hypothesised that testicular injection leads to more nocifensive responses than i.m. injection and thirdly, local anaesthesia with lidocaine and mepivacaine leads to less nocifensive and autonomic responses to castration than procaine. 2. Materials and Methods This study was reviewed and approved by the ethical committee for animal experimen- tation of the Federal State Office for

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e. 2. Materials and Methods This study was reviewed and approved by the ethical committee for animal experimen- tation of the Federal State Office for Consumer Protection and Food Safety of Lower Saxony, Germany (33.9-42502-04-19/3218). All animal procedures were performed according to the German Animal Protection Law (Tierschutzgesetz). 2.1. Animals All piglets included in this study were male, two to seven days of age, and weighing one to three kg of bodyweight. They were acquired from four different producer farms (Main Study: Farm A: 41 piglets, Farm C: 88 piglets, Farm D: 51 piglets; no piglet from Farm B was included in the main study) in Lower Saxony, Germany, and have not been pretreated at the farm of origin. No piglet was subjected to teeth clipping or tail docking. Health status and descendance of both testicles into the scrotum was confirmed by a clinical examination. Piglets with hernia scrotalis or inguinalis were excluded. After completion of the study the piglets were raised motherless in an artificial rearing system (rescue deck). Based on electroencephalogram (EEG) response to nociception data from Kulka, et al. [32], an a priori power calculation resulted in a sample size of 20 piglets per group to detect the absence of a change of 20% in SEF 95% with an effect size of 0.83, an alpha error of 5% and a power of 95% (G*Power 3.1.9.4; Heinrich Heine University, Düsseldorf, Germany). 2.2. Study Design and Procedure 2.2.1. Pre-Trial In a pre-trial, distribution of a local anaesthetic after intratesticular and intrafunicular injection was evaluated in ten piglets via computed tomography Anaesthesia was induced via a face mask with 5 vol% isoflurane in 100% oxygen and a fresh gas flow of 4 L min−1 . After reaching a sufficient depth of anaesthesia the

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induced via a face mask with 5 vol% isoflurane in 100% oxygen and a fresh gas flow of 4 L min−1 . After reaching a sufficient depth of anaesthesia the piglets were orotracheally intubated with a Murphy style cuffed endotracheal Tube I.D. Animals 2022, 12, 2833 4 of 24 2.5 mm (Rüschelit® Super Safety Clear, 2.5 mm, Teleflex Medical Sdn. Bhd., Kamunting, Malaysia). According to positioning in commercial castration racks the piglets were fixed in supine position in a radiolucent foam positioning device, so that the testicles were easily accessible. As a surrogate for the corresponding volume of a local anaesthetic, a 1:7 dilution of a non-ionic, water-soluble X-ray contrast medium (iobitridol, Xenetix® 300, Guerbet, Villepinte, France) with lidocaine hydrochloride (Lidor® 20 mg/mL, WDT, Garbsen, Germany) was used in order to obtain a comparable viscosity and pH value as the pure local anaesthetic solution. At each testicular side a total volume of 0.3 mL was injected with a 25G × 5/8 needle (Neoject® , Dispomed Witt oHG, Gelnhausen, Germany). Five different injection techniques were investigated: intrafunicular (0.3 mL—palpation and fixation of the funiculus with one hand while injection was performed in an approx. 45◦ angle from caudal with the other hand), intratesticular (0.3 mL—fixation of the testicle with one hand while injection was performed in a 90◦ angle), subcutaneous in the subscrotal tissue (subscrotal) (0.3 mL—lifting a skinfold with one hand while injection was performed in an approx. 45◦ angle from caudal with the other hand), a combination of subscrotal (0.15 mL) and intrafunicular injection (0.15 mL) and a combination of subscrotal (0.15 mL) and intratesticular injection (0.15 mL) was performed. Injections were performed without

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injection (0.15 mL) and a combination of subscrotal (0.15 mL) and intratesticular injection (0.15 mL) was performed. Injections were performed without aspiration prior to injection. Two animals were used per injection technique. A 64-multi- detector-row CT scanner (Phillips Brilliance 64, Philips GmbH, Hamburg, Germany) was used for image acquisition. CT scans with a slice thickness of 0.64 mm were acquired two minutes after injection and repeated 4, 6, 8 and 10 min after injection. Acquired images were visually analysed for distribution of contrast media. For evalua- tion of temporal distribution, changes in distribution pattern over time were evaluated visually. 2.2.2. Main Study The main study was designed as a placebo controlled, blinded, randomized study with 9 parallel groups (Table 1). Randomization was performed with the aid of www. randomizer.org (accessed on 11 February 2020). Table 1. Treatment groups, injected volume, total dose and concentration of the local anaesthetic. Group P0.3 P0.5 L0.3 L0.5 M0.3 M0.5 S0.3 S0.5 IM procaine lidocaine mepivacaine mepivacaine saline saline saline drug procaine 2% lidocaine 2% 2% 2% 2% 2% 0.9% 0.9% 0.9% volume per site 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.5 (mL) 5 mg kg−1 4 mg kg−1 0.4 mg kg−1 40 mg 40 mg 40 mg total dose diluted in diluted in diluted in undiluted undiluted undiluted saline saline saline total volume 1.2 2 1.2 2 1.2 2 1.2 2 0.5 (mL) The three local anaesthetics procaine (Procamidor® 20 mg/mL, WDT, Garbsen, Ger- many), lidocaine (Lidor® 20 mg/mL, WDT, Garbsen, Germany) and mepivacaine (Mepidor® 20 mg/mL, WDT, Garbsen, Germany) and the placebo (NaCl 0.9%, B.Braun Melsungen AG, Melsungen, Germany) were compared each in 2 different volumes and doses. According to the results of the preliminary

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(NaCl 0.9%, B.Braun Melsungen AG, Melsungen, Germany) were compared each in 2 different volumes and doses. According to the results of the preliminary investigation, each local anaesthetic or saline was adminis- tered at four locations (intratesticular left, subscrotal left, intratesticular right and subscrotal right) with a volume of either 0.5 mL or 0.3 mL resulting in a total volume of 2 mL or 1.2 mL per piglet. In groups where 0.5 mL were administered at each location undiluted local anaesthetic at a concentration of 2% was injected (procaine—group P0.5 , lidocaine—group L0.5 , mepivacaine—group M0.5 and the placebo group with normal saline—group S0.5 ). In groups where 0.3 mL were injected at each location the maximally recommended dose according to summary of product characteristics” (SPC) (https://vetidata.de/ accessed 11 February 2020) was calculated for each individual piglet and diluted with normal saline Animals 2022, 12, 2833 5 of 24 to a total volume of 1.2 mL (procaine—group P0.3 , lidocaine—group L0.3 , mepivacaine— group M0.3 and placebo normal saline—group S0.3 ). The maximally recommended doses were 5 mg kg−1 for procaine, 4 mg kg−1 for lidocaine and 0.4 mg kg−1 for mepivacaine. The dose for mepivacaine was calculated from a dose stated for horses. To compare the nociceptive response caused by intratesticular and subscrotal injection with a common clin- ical nociceptive stimulus one group was included that received an intramuscular injection of 0.5 mL normal saline (group IM). Anaesthesia and Instrumentation Anaesthesia was induced by mask with 5 vol% isoflurane (Isofluran Baxter® , Baxter Deutschland GmbH, Unterschleißheim, Germany) in 100% oxygen with a flow of 4 L min− 1 . Upon loss of consciousness a 4 Fr feeding tube

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axter® , Baxter Deutschland GmbH, Unterschleißheim, Germany) in 100% oxygen with a flow of 4 L min− 1 . Upon loss of consciousness a 4 Fr feeding tube (Ernährungssonde CH 4, B.Braun Melsun- gen AG, Melsungen, Germany) was inserted into the ventral meatus nasi to determine inspiratory and expiratory isoflurane concentration and respiratory rate with the multi- gas module of a multiparameter anaesthesia monitor (Datex Ohmeda S5, GE Healthcare Finland OY, Helsinki, Finland). Isoflurane was reduced to maintain an FeISO of 1 Vol% [representing 0.8 MAC determined in a similar population of piglets [33]. Two attempts were made to catheterize the saphenous artery with an over the needle catheter (Introcan® 22G, B.Braun, Melsungen, Germany). Invasive measurement of arterial blood pressure and pulse rate was performed with the multiparameter anaesthesia monitor and a pressure transducer (Meritrans DTXPlus® Disposable Transducer, Merit Medical GmbH, Eschborn, Germany) connected via fluid filled low compliance extension lines to the arterial catheter. No intravenous fluids were given. In case of unsuccessful catheterization, the procedure was continued without invasive measurement of blood pressure and pulse rate. Adhe- sive electrodes were attached to each leg to record a lead II electrocardiogram (Televet® 100. Rösch & Associates Information engineering GmbH, Frankfurt am Main, Germany). Recording of the EEG signal was performed as described by Waldmann, et al. [21] with the Narcotrend® -Compact-Monitor Version 5.0. Finally, the piglets were placed in dorsal recumbency according to positioning in a castration cradle. Animals were warmed with the aid of a heating lamp to maintain body temperature above 38 ◦ C. After instrumentation and an equilibration period of 10

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Animals were warmed with the aid of a heating lamp to maintain body temperature above 38 ◦ C. After instrumentation and an equilibration period of 10 min at a FeISO of 1 Vol%, the injection of the test drugs was performed. Injection Technique In Group IM one intramuscular injection of 0.5 mL saline was performed with a 18G × 2” canula (BD Microlance 3, BD GmbH, Heidelberg, Germany) behind the base of the ear. The piglets in Group IM were recovered without castration. In all other groups half of the total injection volume was drawn up in a syringe, the scrotal skin was punctured, and half of the volume was administered intratesticularly and half subscrotal when the needle was withdrawn (0.5 mL or 0.3 mL of the solution per localisation). Injections were performed in each piglet always starting on the right side, followed by the left side. For intratesticular injection, the testicle was fixated with thumb and index finger of one hand and the injection was performed with a 25G × 5/8 needle (Neoject® , Dispomed Witt oHG, Gelnhausen, Germany) into the middle of the testicle without prior aspiration. Castration The castration was performed 5 to 15 min maximum after intratesticular and subscrotal injection, depending on the loss of sensitivity of the scrotal skin. Two incisions parallel to the raphe scroti were made and the testicles were exteriorized. Both testicles were removed at the same time by use of an emasculator. To ensure an adequate haemostasis compres- sion of the emasculator was maintained for 20 s. Meloxicam 0.4 mg kg−1 (Melosolute® 5 mg/mL, CP Pharma, Burgdorf, Germany) was administered at least 20 min before the surgical procedure i.m. After data acquisition, the piglets were recovered and placed in a nursing pen. Animals 2022, 12, 2833 6 of 24 Data

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before the surgical procedure i.m. After data acquisition, the piglets were recovered and placed in a nursing pen. Animals 2022, 12, 2833 6 of 24 Data Recording • Nocifensive movements Animals 2022, 12, x FOR PEER REVIEW Nocifensive movements during injection at each location were scored as (0)—no 7 of 24 movement, (1)—minor purposeful movements (minor movements of one or 2 limbs) or (2)—major purposeful movements (more than 2 limbs or head). As any purposeful move- ment (movement associated directly related to the stimulus during injection or castration) POST-CAe post castration, 2 min after “CAe”. was considered not conform with the Animal Protection Law, for statistical analysis only The superscript “e” was added to differentiate these time periods/episodes from the purposeful movement yes or no was differentiated. All scores for nocifensive movements time points for MAP and respiratory rate. This also refers to EEG data. were performed live by the same investigator blinded to treatment. One minute after injec- The RR-interval sections were transferred to Kubios® RV version 2.0 (Biosignal tion followed by every 2 min sensitivity of the scrotal skin in the area where the injections Analysis were and Medical performed ImagingAGroup, was examined. University pean clamp (Peha®ofinstrument Kuopio, Kuopio, Finland). Pean Klemme, The ECG Hartmann, Heidenheim Germany) equipped with a rubber hose -to avoid severe tissue trauma- were recordings were checked visually, errors were edited manually, and the data was analysedtoafterwards. clamped the first ratchet lock for 1 s. Castration was performed if no aversive response Time domain was noted, HRV analysis or a maximum included of 15 min mean heart had passed. rate, standard A period of 5 mindeviation of the

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e response Time domain was noted, HRV analysis or a maximum included of 15 min mean heart had passed. rate, standard A period of 5 mindeviation of the heart after injection was rate (SDHR), always mean awaited RR-Interval to ensure (Meantime an adequate RR) and the standard for onset of actiondeviation of the RR-intervals of the injectate. (SDRR). Thecastration During LF/HF ratio was calculated nocifensive duringwere movements frequency scoreddomain as aboveanalysis for thewith the low timepoints frequency band (LF) defined at 0.02–0.15 Hz and the high frequency band (HF) “skin incision”, “exteriorization of the testicles” and “emasculation” which were performedaccording to respiratory with an intervalrate at s. of 15 0.25–1.4 Hz. •• EEG Variables Respiratory rate and blood pressure The following variables were recorded and values for every 5 s time interval were The following timepoints (Figure 1) were defined and analyzed: Baseline injection (BL- exported: INJ) valuesNarcotrend-Index were recorded every (NI), 15 stotal overpower (PTOT a period of 2), min median frequency before injection (MF) and and the 95% mean spectral was edge frequency calculated. Baseline (SEF 95). castration (BL-CA) value was as single value recorded directly Analysed timepoints (epochs) were: changes in response towards a short-lasting before skin incision. As cardiorespiratory • nociceptive stimulus BL-INJ only persist e: baseline beforefor a short period injection the highest value during a one-minute for 2 min period after start of injection (INJ-Max) and during castration (CA-Max) were recorded. • INJe: 1 min after start of injection After castration, respiratory rate and mean arterial blood pressure were recorded every • 2 min 15 s for BL-CA e: baseline 2 min

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er start of injection After castration, respiratory rate and mean arterial blood pressure were recorded every • 2 min 15 s for BL-CA e: baseline 2 min before castration (skin incision) (POST-CA) and the mean was calculated from the 8 individual values. A change• of CA e 10%: in cardiorespiratory castration, parameters start of skin compared incision until to baseline 20 s after was calculated for start of emasculation. absolute • values POST CA : post castration, 2 min after the end of emasculationof change would of respiratory e rate, heart rate and MAP, as this amount imply presence of nociception in a clinical setting. Figure1.1. Timeline Figure Timeline of ofthe theexperimental experimentalprocedure. procedure. Dotted Dotted arrows arrows indicate indicate time time segments segments with with variable variablelength. length.Abbreviations Abbreviationsofofanalyzed analyzedtimepoints timepointsare aregreen. green. •2.3. Heart rateAnalysis Statistical variability An ECG was recorded ® 100, Software Version 6.2.0 (Engel Engineering Data were analysedwith the Televet using R version 3.4.4. (The R Foundation for Statistical Services Computing,GmbH, Heusenstamm, Vienna, Austria). TheGermany) and RR distribution of interval data wasections wereShapiro–Wilk tested with extracted and Test the following time periods and Histograms. defined Normally for analysis: distributed data were analysed within groups with a paired t- e baseline 2 min before injection test BL-INJ and an ANOVA for repeated measurements. Wilcoxon sign Rank test was used to e INJ —1 min after start data of injection compare nonparametric within groups. In between groups an ANOVA for BL-CA e ” baseline 2 min before castration (skin incision) independent variables was used for normally

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c within groups. In between groups an ANOVA for BL-CA e ” baseline 2 min before castration (skin incision) independent variables was used for normally distributed data and the Kruskal–Wallis- e —castration, start of skin incision until 20 s after start of emasculation TestCAfollowed by Wilcoxon’s two sample test in case of significant differences was used to POST-CA e post castration, 2 min after “CAe ”. compare nonparametric data. Level of significance was set at 5%. Statistical analysis of e” was added to differentiate these time periods/episodes from the The superscript “ ordinal data was performed with the Chi-square-test followed by Fisher’s exact test in time case points of cell for MAP values and respiratory below 5. rate. This also refers to EEG data. 3. Results 3.1. Pre-Trial The 10 piglets used were between 2 and 7 days old and had a body weight between Animals 2022, 12, 2833 7 of 24 The RR-interval sections were transferred to Kubios® RV version 2.0 (Biosignal Analysis and Medical Imaging Group, University of Kuopio, Kuopio, Finland). The ECG recordings were checked visually, errors were edited manually, and the data were analysed afterwards. Time domain HRV analysis included mean heart rate, standard deviation of the heart rate (SDHR), mean RR-Interval (Mean RR) and the standard deviation of the RR-intervals (SDRR). The LF/HF ratio was calculated during frequency domain analysis with the low frequency band (LF) defined at 0.02–0.15 Hz and the high frequency band (HF) according to respiratory rate at 0.25–1.4 Hz. • EEG Variables The following variables were recorded and values for every 5 s time interval were exported: Narcotrend-Index (NI), total power (PTOT ), median frequency (MF) and 95% spectral edge frequency (SEF95 ). Analysed

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5 s time interval were exported: Narcotrend-Index (NI), total power (PTOT ), median frequency (MF) and 95% spectral edge frequency (SEF95 ). Analysed timepoints (epochs) were: • BL-INJe : baseline before injection for 2 min • INJe : 1 min after start of injection • BL-CAe : baseline 2 min before castration (skin incision) • CAe : castration, start of skin incision until 20 s after start of emasculation. • POST CAe : post castration, 2 min after the end of emasculation 2.3. Statistical Analysis Data were analysed using R version 3.4.4. (The R Foundation for Statistical Com- puting, Vienna, Austria). The distribution of data was tested with Shapiro–Wilk Test and Histograms. Normally distributed data were analysed within groups with a paired t-test and an ANOVA for repeated measurements. Wilcoxon sign Rank test was used to com- pare nonparametric data within groups. In between groups an ANOVA for independent variables was used for normally distributed data and the Kruskal–Wallis-Test followed by Wilcoxon’s two sample test in case of significant differences was used to compare nonpara- metric data. Level of significance was set at 5%. Statistical analysis of ordinal data was performed with the Chi-square-test followed by Fisher’s exact test in case of cell values below 5. 3. Results 3.1. Pre-Trial The 10 piglets used were between 2 and 7 days old and had a body weight between 1.9 and 2.4 kg. Figure 2A-C show examples of the distribution of the surrogate within 2 min after injection at the respective sites. After intratesticular injection (n = 8) the surrogate was visible in the testis and along the spermatic cord into the abdominal cavity. Only in one piglet the local anaesthetic was falsely injected which resulted in a mainly intrascrotal distribution.

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ic cord into the abdominal cavity. Only in one piglet the local anaesthetic was falsely injected which resulted in a mainly intrascrotal distribution. Intrafunicular injection resulted in less consistent distribution in the spermatic cord, in the scrotum and in the abdominal cavity or in the subcutaneous tissue in the inguinal area. All subscrotal applications (n = 12) resulted in a subcutaneous depot. In all localizations the distribution of the surrogate did not change over time. 3.2. Main Study Mean age of the 170 piglets included in this study was 4 days with a mean bodyweight of 1.95 ± 0.31 kg. 3.2.1. EEG Baseline values for NI before injection (BL-INJe ) were comparable among all investi- gation groups (Figure 3). Changes in EEG parameters NI, PTOT , MF, SEF95 in response to injection or castration did not follow a consistent pattern and did not show any significant differences among the groups. injection at the respective sites. After intratesticular injection (n = 8) the surrogate was visible in the testis and along the spermatic cord into the abdominal cavity. Only in one piglet the local anaesthetic was falsely injected which resulted in a mainly intrascrotal distribution. Intrafunicular injection resulted in less consistent distribution in the Animals 2022, 12, 2833 spermatic cord, in the scrotum and in the abdominal cavity or in the subcutaneous tissue8 of 24 in the inguinal area. All subscrotal applications (n = 12) resulted in a subcutaneous depot. In all localizations the distribution of the surrogate did not change over time. Figure 2. 3D-reconstruction of the CT-images showing the distribution of the surrogate after Figure 2. 3D-reconstruction intratesticular of the(B) (A), intrafunicular CT-images showing and subscrotal (C)the distribution injection.

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rogate after Figure 2. 3D-reconstruction intratesticular of the(B) (A), intrafunicular CT-images showing and subscrotal (C)the distribution injection. of the Orange capital letters:after surrogate intrates- A-anterior, ticular (A), intrafunicular (B) L-left, R-right, H-head, F-feet. and subscrotal (C) injection. Orange capital letters: A-anterior, L-left, R-right, H-head, F-feet. 3.2. Main Study 3.2.2. Response to Injection Mean age of the 170 piglets included in this study was 4 days with a mean • Nocifensive movements bodyweight of 1.95 ± 0.31 kg. Compared to intramuscular injection, intratesticular injection in group P0.5 resulted in markedly stronger nocifensive movements (score 2). Intratesticular injection of the smaller volume of 0.3 mL led to statistically significantly less nocifensive movements than the administration of a volume of 0.5 mL even in comparison to the control group (Figure 4). 3.2.1. EEG Baseline values for NI before injection (BL-INJe) were comparable among all investigation groups (Figure 3). Changes in EEG parameters NI, PTOT, MF, SEF95 in response to injection or castration did not follow a consistent pattern and did not show Animals 2022, 12, 2833 9 of 24 any significant differences among the groups. Figure 3. Baseline Narcotrend Index. Baseline before injection of Narcotrend Index (NI) for groups P0.3, P0.5, L0.3, L0.5, M0.3, M0.5, S0.3, S0.5 and IM. The box covers the interquartile interval, where 50% of the data are found. The whiskers indicate minimum and maximum and the band inside the box represents the median. 3.2.2. Response to Injection • Nocifensive movements Compared to intramuscular injection, intratesticular injection in group P0.5 resulted in markedly stronger nocifensive movements (score 2). Intratesticular injection of

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uscular injection, intratesticular injection in group P0.5 resulted in markedly stronger nocifensive movements (score 2). Intratesticular injection of the Figure 3. Baseline smaller Narcotrend Index. Baseline significantly before injection of Narcotrend Index (NI) for groups Figure 3.volume Baselineof 0.3 mL ledIndex. Narcotrend to statistically Baseline before injection less nocifensive of Narcotrend movements Index than (NI) for groups P P0.3, P0.5, L0.3, L0.5, M0.3, M0.5, S0.3, S0 5 and IM. The box covers the interquartile interval, where 50% 50% the , P , L administration 0.3 0.5 0.3 , L 0.5 , M of a 0.3 , M volume 0.5 , S of 0.3 , S0.5 0.5 and mL IM. evenThe inbox covers comparison the interquartile to the controlinterval, group where (Figureof of data 4). the the data are are found.found. TheThe whiskers whiskers indicate indicate minimum minimum andand maximum maximum and thethe and band band inside inside the the box box representsthe represents themedian. median. 3.2.2. Response to Injection • Nocifensive movements Compared to intramuscular injection, intratesticular injection in group P0.5 resulted in markedly stronger nocifensive movements (score 2). Intratesticular injection of the smaller volume of 0.3 mL led to statistically significantly less nocifensive movements than the administration of a volume of 0.5 mL even in comparison to the control group (Figure 4). Figure Figure Nocifensive 4. 4.Nocifensive movements movements toto intratesticular intratesticular injection injection forthe for IM,PP groupsIM, thegroups 0.0.3 3, P0.50.5 , L, 0L , P , L, 0L.30.3 , , .50.5 M , M M0.3,0.3M0.5,0.5 , S and S 0.3 S0.5. 0.5 S0.3 and . Number of piglets with a score of 0 are displayed in grey, a score of Number of piglets with a score of 0 are displayed in grey, a

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0.5. 0.5 S0.3 and . Number of piglets with a score of 0 are displayed in grey, a score of Number of piglets with a score of 0 are displayed in grey, a score of 1 in light 1 in light blue blue and and a score a score ofof 2 in 2 in dark dark blue. blue. The The # represents # represents a significant a significant difference difference comparedtotogroup compared groupIM. IM. • Cardiorespiratory changes No significant differences were seen between BL-INJ and INJ-Max after injection of local anaesthetic in respiratory rate, heart rate and RR-intervals, but groups with larger volumes administered showed a higher difference compared to IM. Difference values in MAP were statistically significantly greater in P0.3 and S0.5 compared to IM. A 10% increase in respiratory rate was seen in all groups but IM and P0.3 and in MAP for all treatments but Figure 4. Nocifensive movements to intratesticular injection for the groups IM, P0.3, P0.5, L0.3, L0.5, IM. L0.3 and M0.3 . There was no group with an increase in heart rate above 10%. For P0.5 M0.3, M0.5, S0.3 and S0.5. Number of piglets with a score of 0 are displayed in grey, a score of 1 in light difference of SDHR was significantly higher compared to IM and difference of SDRR was blue and a score of 2 in dark blue. The # represents a significant difference compared to group IM. higher compared to IM and M0.5 whereas the LF/HF-ratio increased statistically higher during injection in group P0.5 and S0.5 compared to IM (Figure 5A–G). volumes administered showed a higher difference compared to IM. Difference values in MAP were statistically significantly greater in P0.3 and S0.5 compared to IM. A 10% increase in respiratory rate was seen in all groups but IM and P0.3 and in MAP for all treatments but IM. L0.3 and M0.3. There was

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mpared to IM. A 10% increase in respiratory rate was seen in all groups but IM and P0.3 and in MAP for all treatments but IM. L0.3 and M0.3. There was no group with an increase in heart rate above 10%. For P0.5 difference of SDHR was significantly higher compared to IM and difference of SDRR Animals 2022, 12, 2833 was higher compared to IM and M0.5 whereas the LF/HF-ratio increased statistically 10 of 24 higher during injection in group P0.5 and S0.5 compared to IM (Figure 5A–G). A B Δ respiratory rate: BL-INJ – INJ-Max Δ MAP: BL-INJ – INJ-Max C D Δ heart rate: BL-INJ -– INJ e e Δ RR-Interval: BL-INJe - INJe 15 a a 10 a a a 10 a a a a a a a a Δ RR-Interval [beats min-1] a a a 0 Δ HR 5 [ms] a a 0 -10 -5 -20 IM P P L L M M S S IM P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 E F Δ SDHR: BL-INJe - INJe Δ SDRR: BL-INJe - INJe ab b 2 ab b ab ab ab ab ab ab ab ab ab ab [beats min-1] a 2 ab a a Δ SDHR Δ SDRR [ms] 0 0 -2 -2 IM P P L L M M S S IM P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 Figure 5. Cont. Animals Animals 2022, 2022, 12, x FOR PEER REVIEW 12, 2833 11 of 24 11 of 24 G Δ LF/HF-ratio: BL-INJe - INJe 15 ab ab 10 b Δ LF/HF-ratio b ab 5 ab ab a ab 0 -5 IM P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 (A–G)5. Difference Figure 5.Figure (∆) in respiratory (A–G) Difference rate, mean (Δ) in respiratory rate,arterial blood pressure, mean arterial heart rate, blood pressure, RR- heart rate, RR- Interval,Interval, SDHR, SDRRSDHR, and SDRRLF/HF-ratio betweenbetween and LF/HF-ratio baselinebaseline and injection in the experimental and injection groups. groups. in the experimental Bar diagrams Bar diagrams of (A) of (A) mean mean difference difference in respiratory in

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perimental and injection groups. groups. in the experimental Bar diagrams Bar diagrams of (A) of (A) mean mean difference difference in respiratory in respiratory rate minute rate [breaths [breaths −1minute ] between baselinebaseline −1] between and and maximum maximum respiratory respiratory rate duringrate injection, during injection, (B) mean (B)difference mean difference in meaninarterial mean blood arterialpressure blood pressure (MAP) [mmHg] (MAP) [mmHg] betweenbetween baseline baseline and maximumand maximum MAP during MAPinjection, during injection, (C) mean(C) mean difference difference in heart in heart rate [beats− minute 1 −1] between baseline and during injection, (D) mean difference in RR-Interval [ms] rate [beats minute ] between baseline and during injection, (D) mean difference in RR-Interval [ms] between baseline and during injection with the mean at the top of the bar and the whiskers as between baseline and during injection with the mean at the top of the bar and the whiskers as standard standard deviation. Boxplots of (E) difference in standard deviation of heart rate (SDHR) [beats deviation. Boxplots of (E) difference in standard deviation of heart rate (SDHR) [beats minute−1 ] minute−1] between baseline and during injection, (F) difference in standard deviation of RR-Interval between(SDRR) baseline[ms] and between during injection, baseline(F) difference and in standard during injection anddeviation of RR-Interval (G) difference in low (SDRR) frequency/high [ms] between baseline frequency andratio (LF/HF) during injection between and (G) baseline anddifference in low The during injection. frequency/high frequency boxes representing the first and (LF/HF)third ratioquartile betweenand baseline and during the whiskers injection. ranging

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n. frequency/high frequency boxes representing the first and (LF/HF)third ratioquartile betweenand baseline and during the whiskers injection. ranging The boxestorepresenting from minimum maximum. The the median first disthird indicated by quartile and band the the inside ranging whiskers the box. fromDifferent minimum letters (a, b, c) The to maximum. show significant median differences is indicated between the by the band experimental inside the box. Differentgroups letters(p(a,<b,0.05). Thesignificant c) show superscript “e” differentiates differences between time periods/episodes(“ the experimental groupse”) from time (p < 0.05). Thepoints (MAP“e” superscript andifferentiates respiratory time rate).periods/episodes(“ The red asterisk (*) e ”) marks from time an increase above points (MAP 10% from and baseline. respiratory rate). The red asterisk (*) marks an increase above 10% from baseline. 3.2.3. Response 3.2.3. Response to Castration to Castration • • ofOnset Onset action of action MedianMedian(Minimum; (Minimum; Maximum) Maximum) values of the values time periodof thebetween time the period between the intratesticu- intratesticular/subscrotal lar/subscrotal application and castrationapplication and castration for groups P0.3 , P0.5 , L for0.3 groups , L0.5 , M P 0.3 , 0,.3M 0.5, ,SL0.3and 0.5 0.3 , L0.5, M0.3, P S0.5 were , S015), M50.5(5; .3 and S07), 5 (5; (5;515), 5 (5; 510), .5 were (5; 5),(5; 117), (5;515), (5; 510), (5; 55),(5;115),(5;115), (5;and 15), 55 (5; (5;15) 5), minutes, 11 (5; 15), and 5 (5; 15)(Figure respectively minutes,6). respectively (Figure 6). • • Nocifensive Nocifensive movements movements Group Group P0.5and P0.5 , M0.5 , M0L .5 and L0.5 resulted overall in less nocifensive movements during 0.5 resulted overall

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nsive movements movements Group Group P0.5and P0.5 , M0.5 , M0L .5 and L0.5 resulted overall in less nocifensive movements during 0.5 resulted overall in less nocifensive movements during castration compared to placebo castration compared to placebo groups (S groups (S0.3 and S0.5) and M0.3. During emasculation less 0.3 and S0.5 ) and M0.3 . During emasculation less nocifensive nocifensive movements movements were seenwere seen in M in groups groups and ML0.5 and L0.5 compared compared to P0.5 2). to P (Table (Table 2). 0.5 0.5 0.5 Table 2. Table Number 2. Number of pigletsofshowing piglets showing nocifensive nocifensive movements movements during during skin skin incision, incision, exteriorization exteriorization of of testicles and emasculation (n = 20/group). testicles and emasculation (n = 20/group). P0.3 P0.5 L0.3 L0.5 M0.3 M0.5 S0.3 S0.5 P0.3 P0.5 L0.3 L M0.3 b,c M0.5 S0.3 S0.5c,d skin incision 6 a,c0.5 1 a 9 4 a 12 c,d 1a 10 14 d skin incision 6 a,c 1 aexteriorization 9 b,c 10 4b a 1 a 12 c,d 4a 12 aa 16 c10 c,d 1a 1418d c 15 b,c exteriorization 10 b 1 a emasculation 4a 162c a 8 16 9 b b c 11 aa 19 18 c c 3a b,cc 1518 17 c emasculation 16 c 8 b no reaction 9b 21 a 10 19 c 6 315a 0 18 c 17 172c 2 a, b, c, d: Different letters (a, b, c, d) show significant differences between the experimental groups (p no reaction 2 10 6 15 0 17 2 2 < 0.05). a, b, c, d : Different letters (a, b, c, d) show significant differences between the experimental groups (p < 0.05). • Cardiorespiratory changes in heart rate was higher compared to group M0.5 and RR-intervals shorter compared to M0.5 and L0.5 (Figure 8A,C). The difference in SDHR and SDRR were lowest for M 0.5 and L0.5. Groups S0.3, S0.5 and M0.3 had significantly higher differences in SDHR compared to M0.5, L0.3 and L0.5

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SDHR and SDRR were lowest for M 0.5 and L0.5. Groups S0.3, S0.5 and M0.3 had significantly higher differences in SDHR compared to M0.5, L0.3 and L0.5 and difference in SDRR compared to M0.5 and L0.5 (Figure 8E,G). The difference in LF/HF ratio was lowest for L0.5 and M0.5 with a statistically significant Animals 2022, 12, 2833 12 of 24 difference to S0.3, S0.5 and M0.3. In group L0.5 the difference was also significantly lower compared to P0.3 and L0.3 (Figure 8I). Figure 6. Scatter Figure 6. Scatterdotdot plot plot of the of the timetime period period in min inbetween min between the intratesticular/subscrotal the intratesticular/subscrotal application application and and castration forcastration groups P0.3for , Pgroups 0.5 , L0.3 ,PL0 . 0.53 , ,PM 0 . 5 , 0.3 L , 0M. 3 , L 0.5 , 0 . S 5 , M 0.3 . and 0 3 , MS 0 . 0.5 5 , . SThe 0 . 3 and S black0 .bar 5 . The black represents bar represents the median. the median. • Cardiorespiratory changes Overall the fewest changes of cardiorespiratory variables were noted in groups L0.5 and M0.5 during castration. The difference in respiratory rate was higher in S0.5 compared to L0.5 (Figure 7A). Further a 10% increase in absolute values of respiratory rate was observed in M0.3 , S0.3 and S0.5 for BL-CA-CA-Max and for M0.3 , S0.3 and S0.5 for BL-CA-POST-CA. Difference in MAP was significantly lower in groups L0.5 and M0.5 compared to S0.3 , S0.5 and P0.3 and in group L0.5 also compared to M0.3 (Figure 7C). A 10% increase in absolute MAP values was observed in P0.3 , M0.3 , S0.3 and S0.5 from BL-CA to CA-Max and from BL-CA to POST-CA. L0.5 was the only group with a decrease in heart rate during castration. There was no group with an increase in heart rate above 10% during or after castration. Difference in heart rate was lower

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art rate during castration. There was no group with an increase in heart rate above 10% during or after castration. Difference in heart rate was lower and difference in RR-intervals were higher in groups P0.5 , L0.3 , L0.5 and M0.5 compared to S0.3 and S0.5 . In groups P0.3 and M0.3 difference in heart rate was higher compared to group M0.5 and RR-intervals shorter compared to M0.5 and L0.5 (Figure 8A,C). The difference in SDHR and SDRR were lowest for M0.5 and L0.5 . Groups S0.3 , S0.5 and M0.3 had significantly higher differences in SDHR compared to M0.5 , L0.3 and L0.5 and difference in SDRR compared to M0.5 and L0.5 (Figure 8E,G). The difference in LF/HF ratio was lowest for L0.5 and M0.5 with a statistically significant difference to S0.3 , S0.5 and M0.3 . In group L0.5 the difference was also significantly lower compared to P0.3 and L0.3 (Figure 8I). Animals 2022, 12, x FOR PEER REVIEW 13 of 24 Animals 2022, 12, 2833 13 of 24 A B Δ respiratory rate: BL-CA – CA-Max Δ respiratory rate: BL-CA - POST-CA C D Δ MAP: BL-CA – CA-Max Δ MAP: BL-CA - POST-CA Figure 7. (A–D) Mean (SD) difference (Δ) in respiratory rate and mean arterial blood pressure Figure 7. (A–D) Mean (SD) difference (∆) in respiratory rate and mean arterial blood pressure between between baseline and during castration and between baseline and post castration in the baseline and during experimental castration groups. and between Bar diagrams of (A)baseline and post (Δ) mean difference castration in the experimental in respiratory rate [breathsgroups. minute−1] Barbetween diagrams of (A) mean difference (∆) in respiratory rate [breaths minute −1 ] between baseline and baseline and maximum respiratory rate during castration and (B) between baseline and maximum respiratory post

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breaths minute −1 ] between baseline and baseline and maximum respiratory rate during castration and (B) between baseline and maximum respiratory post castration rate as well as during castration bar diagrams of (C)and mean(B)difference between baseline in mean and postblood arterial castration as well pressure (MAP) as [mmHg] between bar diagrams of (C)baseline and maximum mean difference in mean MAP during arterial castration blood and pressure (D) between (MAP) betweenand [mmHg]baseline post castration. baseline and maximumThe barMAP duringthe indicates mean and castration andstandard deviation (D) between is represented baseline by the whiskers. and post castration. The Different letters (a, b, c, d, e) show significant differences between the experimental bar indicates the mean and standard deviation is represented by the whiskers. Different letters groups (p(a, < 0.05). b, The c, d, red asterisk e) show (*) marks significant an increase differences above between the10% from baseline. experimental groups (p < 0.05). The red asterisk (*) marks an increase above 10% from baseline. Animals 2022, 12, 2833 14 of 24 Animals 2022, 12, x FOR PEER REVIEW 14 of 24 A B Δ heart rate: BL-CAe - CAe Δ heart rate: BL-CAe - POST-CAe a a 15 a 15 ab a ac 10 ab ac ab 10 [beats min-1] bcd [beats min-1] cd bc Δ HR d 5 bc Δ HR 5 d c 0 0 -5 -5 P P L L M M S S P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 C D Δ RR-Interval: BL-CAe - CAe Δ RR-Interval: BL-CAe - POST-CAe E F Δ SDHR: BL-CAe - CAe Δ SDHR: BL-CAe - POST-CAe a 6 6 ab [beats min-1] [beats min-1] 4 4 Δ SDHR Δ SDHR ab ac bcd 2 d d 2 cd a a a a a a a a 0 0 P P L L M M S S P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 Figure 8. Cont. Animals Animals

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cd a a a a a a a a 0 0 P P L L M M S S P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 Figure 8. Cont. Animals Animals 2022,2022, 12, x FOR PEER REVIEW 12, 2833 15 of 24 15 of 24 G H Δ SDRR: BL-CAe - CAe Δ SDRR: BL-CAe - POST-CAe a 10 10 a Δ SDRR Δ SDRR ab [ms] [ms] 5 abc ad d 5 bcd cd a a a a a a a a 0 0 P P L L M M S S P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 I J Δ LF/HF-ratio: BL-CAe - CAe Δ LF/HF-ratio: BL-CAe - POST-CAe a a 10 40 a a Δ LF/HF-ratio Δ LF/HF-ratio 5 c bc a a a a a a 20 ac a 0 ab ab -5 0 -10 P P L L M M S S P P L L M M S S 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 0.3 0.5 Figure 8. (A-J) Mean difference in heart rate, RR-Interval, SDHR, SDRR and LF/HF-ratio between Figure 8. (A-J) Mean difference in heart rate, RR-Interval, SDHR, SDRR and LF/HF-ratio between baseline and during castration and between baseline and post castration in the experimental groups. baseline and during Bar diagrams castration of (A) and between mean difference baseline in heart rateand postminute [beats castration in the experimental −1] between baseline andgroups. during Barcastration diagrams and of (A) mean difference in heart rate [beats minute −1 ] between baseline and during (B) between baseline and post castration as well as bar diagrams of (C) mean castration andin difference (B) between baseline RR-Interval and post [ms] between castration baseline as wellcastration and during as bar diagrams (C) mean and (D)ofbetween difference baseline and post castration. The bar indicates the mean is displayed as the top of in RR-Interval [ms] between baseline and during castration and (D) between baseline and the bar and standard deviation post is represented castration. The

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R-Interval [ms] between baseline and during castration and (D) between baseline and the bar and standard deviation post is represented castration. The barbyindicates the whiskers. Boxplots the mean of (E) difference is displayed as the in topstandard of the bar deviation and standard rate (SDHR) of heartdeviation is [beats minute −1] between baseline and during castration and (F) between baseline and post represented by the whiskers. Boxplots of (E) difference in standard deviation of heart rate (SDHR) castration, (G) difference in standard deviation of RR-Interval (SDRR) [ms] between baseline and [beats minute−1 ] between baseline and during castration and (F) between baseline and post castra- during castration and (H) between baseline and post castration and (I) difference in low tion, (G) differencefrequency frequency/high in standard deviation (LF/HF) ratioofbetween RR-Interval (SDRR) baseline and[ms] between during baseline castration and and during (J) between baselineand castration and(H) postbetween baseline castration. and represent The boxes post castration the first and(I)third and lowthe quartileinand difference frequency/high whiskers range frequency (LF/HF)to from minimum ratio betweenThe maximum. baseline median and during castration is indicated by the bandand inside (J) between the box.baseline post andletters Different (a, b, c, d) castration. show The boxes significant representdifferences between the first and thirdthe experimental quartile and the groups whiskers 0.05).from (p <range The superscript minimum “e” differentiates to maximum. time periods/episodes(“ The median is indicated by the e”) from time points (MAP and respiratory rate) band inside the box. Different letters (a, b, c, d) show significant differences between the

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ed by the e”) from time points (MAP and respiratory rate) band inside the box. Different letters (a, b, c, d) show significant differences between the experimental groups (p < 0.05). The superscript “e” differentiates time periods/episodes(“e ”) from time points (MAP and respiratory rate). Animals 2022, 12, 2833 16 of 24 3.2.4. Post-Castration Phase • Cardiorespiratory changes Differences in respiratory rate between BL-CA and POST-CA were lower in P0.5 , L0.3 , L0.5 and M0.5 compared to M0.3 , S0.3 and S0.5 (Figure 7B). Groups P0.5 , L0.5 and M0.5 showed statistically significant lower differences in MAP compared to all other groups except L0.3 (Figure 7D). The differences in heart rate were lower and in RR-intervals greater in groups L0.3 , L0.5 and M0.5 compared to S0.3 and S0.5 . The difference in heart rate increased further after castration and the RR-intervals became shorter in both procaine groups. 3.3. Missing Data Catheter placement for arterial blood pressure measurement was successful in Group P0.3 in n = 14, in group P0.5 in n = 14, in group L0.3 in n = 14, in group L0.5 in n = 14, in group M0.3 in n = 16, in group M0.5 in n = 16, in group S0.3 in n = 16, in group S0.5 in n = 15, and in group IM in n = 15 piglets. Due to technical problems and movement of piglets during injection and castration with displacement of the nasal feeding tube, measurement of respiratory rate was not determined during castration in group L0.5 in n = 1, during exteriorization of testicles in group S0.3 in n = 1 and in group S0.5 in n = 1, during emasculation in group S0.5 in n = 1, and post operatively in group P0.5 in n = 1 as well as in one piglet in group IM during the whole procedure. Furthermore, ECG recordings from two animals in group P0.3 and data for baseline

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= 1 as well as in one piglet in group IM during the whole procedure. Furthermore, ECG recordings from two animals in group P0.3 and data for baseline castration, castration and POST-CAe in one animal in group M0.3 were excluded due to technical errors and artifacts. 3.4. Supplementrary Material Absolute values for respiratory rate and mean arterial blood pressure for the time- points baseline injection (BL-INJ), injection (INJ-MAX), baseline castration (BL-CA), cas- tration (CA) and post castration (POST-CA) as well as absolute values for heart rate, RR-Interval, SDHR, SDRR and LF/HF-ratio for the timepoints baseline injection (BL-INJe ), injection (INJe ), baseline castration (BL-CAe ), castration (CAe ) and post castration (POST- CAe ) can be found in the supplementary materials. 4. Discussion Our main hypothesis was that local anaesthesia with mepivacaine, lidocaine or pro- caine will lead to significant reduction in EEG responses (SEF 95% changes) during castra- tion. At the beginning of the examination, the NI could confirm that all piglets were in a consistent, light plane of anaesthesia across the treatment groups. However, in contrast to observations made by Waldmann et al. [21] and Haga and Ranheim [6] in pigs as well as Otto and Mally [34] and Otto [35] in sheep, we were not able to detect consistent arousal re- actions in response noxious stimuli by desynchronisation or synchronisation with increase or decrease in EEG parameters. One cause may be the time-delayed display of the stages and index values of the Narcotrend® monitor. Pilge et al. [36] described a time delay of 30–65 s and Klesper et al. [37] of 20–175 s. In the present study, stimuli were set in a period of 60 s to 120 s; accordingly, an almost simultaneous adjustment of the display is

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37] of 20–175 s. In the present study, stimuli were set in a period of 60 s to 120 s; accordingly, an almost simultaneous adjustment of the display is necessary for interpretation. In other studies in dogs [38], cattle [39] and pigs [25,40] consistent EEG changes could also not be observed after painful stimuli. Therefore, EEG indices are not further discussed. Independently of the EEG examinations, it was possible to detect the painful interventions with the help of the other variables collected. Our second and third hypothesis, that testicular injection leads to more nocifensive responses than i.m. injection and that, local anaesthesia with lidocaine and mepivacaine leads to less nocifensive and autonomic responses to castration than procaine were confirmed. 4.1. Sub -MAC Isoflurane Concentration for Pain Assessment In this study, the basis for studying the pain responses of the piglets during castra- tion was a constant, reproducible, superficial anaesthesia that prevented stress-induced Animals 2022, 12, 2833 17 of 24 responses due to handling and fixation, but still allowed nociception induced changes in vital signs to be detected, as well as nocifensive movements. It was essential to keep all piglets under the same conditions to avoid bias in the results. To quantify and standardize depth of anaesthesia, the concept of minimum alveolar concentration (MAC) is useful in animal studies because of a small inter-individual variance [41]. The MAC of inhaled anaesthetics is defined as the alveolar gas concentration at sea level required to prevent purposeful movements in 50% of patients in response to surgical incision. Age has been shown to affect MAC [42]. Therefore, MAC determined in a pre-trial in piglets of the same age group (age: 2–7 days of life) was used to

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. Age has been shown to affect MAC [42]. Therefore, MAC determined in a pre-trial in piglets of the same age group (age: 2–7 days of life) was used to define superficial anaesthesia. In this pre-trial, MAC of 1.2 ± 0.3 vol% isoflurane was determined by electric stimuli [33], a similar value (1.20 ± 0.43 vol%) was determined by the clamping technique in 2–17 day old piglets [43]. A higher MAC value of 1.41–2.00% is given by the manufacturer of the isoflurane prepara- tion used [44]. To allow nociception assessment, end-tidal concentration was adjusted to 0.8 MAC, which corresponds to 1.0 vol% isoflurane according to our previous study [33]. The EEG recordings showed that all animals were at comparable depths of anaesthesia at baseline. Subanaesthetic isoflurane concentrations have little or no antinociceptive ef- fect [45] and withdrawal movements triggered by noxious stimulation can be elicited at sub-MAC anaesthetic concentrations [46], while reactions due to handling are suppressed. To measure nociception of piglets during injection and castration, Saller et al. [18] chose individual MAC isoflurane anaesthesia for their minimal anaesthesia protocol determined by single reactions to a toe pinch. Although the applied mean end-tidal concentration, which was finally used, was not mentioned, it seems to be higher than the MAC we chose, as they started with 1.69 ± 0.3 vol% with a flow of 3 L/min oxygen and adapted anaesthetic depth in steps of 0.2%. Similar to our own observations they could see in blood pressure and heart rate as well as limb movements, associated with nociceptive stimuli. Haga and Ranheim [6] made corresponding observations using 1.4 x MAC of halothane anaesthesia. 4.2. Injection of Local Anaesthetics and Onset of Action The local anaesthetics

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e corresponding observations using 1.4 x MAC of halothane anaesthesia. 4.2. Injection of Local Anaesthetics and Onset of Action The local anaesthetics were injected into the testis and subscrotal tissue. The re- sults of the preliminary study demonstrated, that the application at these sites showed a widespread distribution of a mixture of contrast medium and lidocaine beneath the scrotal skin, in the testis, and in the spermatic cord [47]. However, radiolabelled lidocaine injected into the testis did not readily diffuse through the tunica vaginalis and into the cremaster muscle. Therefore, Ranheim and Haga [48] assumed incomplete block of the sensory innervation resulting in insufficient analgesia during castration. This is confirmed by the results of our study, since regardless of the type and dosage of the local anaesthetic used, a complete absence of changes in the observed pain indicators could not be achieved, even in the M0.5 group being the most effective group with the least deviations from the baseline values. When using local anaesthetics, the maximum amount that can be applied to a piglet must be considered to avoid toxicity [9]. A wide variety of doses and concentrations of local anaesthetics used in piglet castration have been described, but toxic side effects were rarely mentioned. Nevertheless, in the present study, the recommended drug associated maximum dosages given by VETIDATA, a web-based veterinary information system (www. vetidata.de (accessed on 11 February 2020), and an easy to handle volume of commercially available products (0.5 mL) were compared. No toxic side effects were observed, despite exceeding the maximum recommended dose by a multiple when using 0.5 mL of the local anaesthetics per injection site (2 mL per piglet). Due to the close

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xceeding the maximum recommended dose by a multiple when using 0.5 mL of the local anaesthetics per injection site (2 mL per piglet). Due to the close relationship between systemic toxicity and the plasma concentration of local anaesthetics [49], the maximum plasma level is decisive for the onset of side effects. Scott et al. [50] demonstrated that the maximum plasma concentration of lidocaine occurred between 10 and 20 min after injection comparing 4 injection sites (intercostal, subcutaneous vaginal, lumbar epidural, subcutaneous abdominal). During this time, however, the infiltrated testis has already been Animals 2022, 12, 2833 18 of 24 removed, so that complete absorption could not take place, which may help to protect the piglet from systemic side effects. Comparing the median onset of action of the local anaesthetics, no significant differ- ences except for mepivacaine in the low application volume (M0.3 ) could be detected, M03 and S03 had the longest median with 11 min. Interestingly, quite a few animals, which got saline 0.9%, did not show any aversive reactions to pinching of the scrotal skin within the 15 control minutes. Despite the standardized procedure, the lack of reaction may have been caused by the inhalation anaesthesia or reflects the individual variation of pain perception [51]. 4.3. Pain Assessment during Injection We compared pain induced reactions during intratesticular/subscrotal injection with intramuscular injection, because the latter is a routine route of drug administration in pigs. Therefore, the infliction of such pain is generally accepted also by the German Animal Welfare Act. The diameter of the canula for the i.m. injections was comparable to routinely used cannulas in the field. To reduce the pain caused by needle insertion into

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meter of the canula for the i.m. injections was comparable to routinely used cannulas in the field. To reduce the pain caused by needle insertion into the testis, a smaller needle was used, as recommended [52,53]. Pain associated reactions were only assessed in connection with injection of the local anaesthetic or saline solution. However, pain responses to the sole puncture of the skin and the testis might also be important in the assessment of animal welfare aspects in piglet castration. Regardless of the localisation and the content, every injection has caused nociception, but there were marked variations in the pain response. Volunteers recognized differences after intradermal and subcutaneous infiltration of five local anaesthetics, which were not related to the acidity, ionization, protein binding, sodium chloride concentration or osmolality, but a relation to lipid solubility was assumed [54]. Accordingly, the injection of mepivacaine was perceived as more painful than lidocaine [54]. The current results indicate more influence of the injected volume than lipid solubility, since no differences were found after injection of mepivacaine or lidocaine. In fact, after smaller intratesticular and subscrotal volumes fewer pigs showed nocifensive movements compared to the control group in which the pigs received 0.5 mL saline i.m. At the same time, RR as well as MAP rose above the 10% of baseline in those groups which received a volume of 0.5 mL per injection site, regardless of the agent. An increase of 10% from baseline was defined as a positive response to the invasive intervention according to Otto et al. [55] and Roizen et al. [56]. Additionally, although not statistically significant, the pain induced reactions after the larger volume of saline led to stronger

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Roizen et al. [56]. Additionally, although not statistically significant, the pain induced reactions after the larger volume of saline led to stronger responses. Therefore, it is assumed that the pain induced reaction is caused in particular by the pressure in the tissue, as the volume of the testicles cannot expand due to the firm layers. In contrast, Coutant et al. [57] could not observe any differences in vocalisation and foreleg movements after intratesticular application of 0,3 mL or 0.5 mL procaine 2%. According to previous studies, procaine injection causes the greatest discomfort [13,19,58]. Tissue irritation due to the significantly lower pH of procaine (pH of 3.7) compared to lidocaine (pH of 5.0) and mepivacaine (pH of 5.5) cannot be ruled out as a cause. The sensation of nociception was confirmed by the changes in the LF/HF ratio of the HRV. Increase in LF and LF/HF ratio reflects sympathetic baroreflex activity [59,60], while HF reflects parasympathetic influences and corresponds to the HR variation depending on the respiratory cycle (respiratory sinus arrhythmia) [61]. The increase in the LF/HF ratio is accompanied by an increase in MAP, one of the most sensitive nociceptive indicators in pigs [25]. The simultaneously observed increases in time domain indices (SDHR, SDRR) were unexpected as with an increased heart rate accelerated by the sympathetic nerves, time between heartbeats (RR intervals) decreased and less time for variability occur, which means, HRV should actually decrease. Additionally, the heart rate did not change as expected. We could notice a small decrease in heart rate (<3.5% from baseline) as well as an increase in RR-intervals during the minute after start of injection in the L0.3 , M0.3 and S0.3 Animals 2022, 12, 2833 19 of 24 group.

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) as well as an increase in RR-intervals during the minute after start of injection in the L0.3 , M0.3 and S0.3 Animals 2022, 12, 2833 19 of 24 group. Clement et al. [62] reported sudden and relatively sustained falls in both arterial pressure (up to 56% from baseline) and heart rate (up to 30% from baseline) after noxious deep somatic and noxious visceral manipulations. Haga and Ranheim [63] also observed a decrease in the pulse rate during injection of lidocaine into the funiculus spermaticus in piglets, while Saller et al. [18] reported a decrease in blood pressure and heart rate in response to the castration. This paradoxical effect is the result of a vasovagal response, whereby bradycardia is caused by a sudden increase in vagal activity and hypotension results from a sudden reduction in sympathetic activity and relaxation of arterial resistance vessels [64]. As HRV reflects the activity of the sympathetic and parasympathetic nervous systems, changes in the recorded parameters in those three groups could be expected by increasing HRV due to the prolonged RR interval. However, SDHR and SDRR increased in all groups. Burton et al. [65] observed a temporary increase in the heart rate (7.0 ± 2.0 %) after inducing pain by intramuscular or subcutaneous injections of hypertonic saline which returned to baseline within 60 s after inducing pain reflecting only brief arousal responses, but causing an increase in the LF/HF ratio of HRV. Radeisen [31] also could relate changes in frequency domain indices to intraoperative pain associated nocifensive movements during castration of boars in the pubic region, especially during incision of the skin and the vaginal process and during the traction of the spermatic cord, whereby time domain HRV did not reflect those single pain

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incision of the skin and the vaginal process and during the traction of the spermatic cord, whereby time domain HRV did not reflect those single pain events. Using the LF/HF ratio Raue et al. [66] could detect nociceptive stimulation in cats in only 60 s post stimulation epoch measurements, while longer time periods revealed a readjustment to the basal values. Generally, short-term measures of HRV rapidly return to baseline after transient disturbances [28], therefore the recording time has a significant influence especially on time domain values [30] and a rapid recovery of the heart rate after the injection might be the reason for the rise in SDHR and SDRR. 4.4. Pain Assessment during Castration In group M0.5 (n = 17; 85.0%) the lowest number of animals showed nocifensive movements during castration followed by group L0.5 (n = 15; 75.0%). Even in each of the placebo groups there were two animals (10.0%) that showed no withdrawal movements during castration possibly as a result of isoflurane anaesthesia. Saller et al. [18] also noticed three out of nine piglets of a positive control group, which received sodium chloride (without pain relief) without limb movements during castration using a light isoflurane anaesthesia model. Autonomic responses to castration were most pronounced in the placebo groups as well as in the M0.3 group during castration. Due to the high dilution of mepivacaine (depending on bodyweight of the piglet 1:23–1:45), which was based on the toxicity limit for the horse, as no corresponding value was available for the pig, effective local anaesthesia could hardly be expected. An increase in RR and MAP of over 10% above the baseline value was noted, which was not seen in the other groups, so that a marked perception of pain must be assumed. The

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d MAP of over 10% above the baseline value was noted, which was not seen in the other groups, so that a marked perception of pain must be assumed. The groups P0.5 , L0.5 and M0.5 differed significantly from the placebo groups in differences of MAP, HR, and RR interval recorded during castration, while only in the L0.5 group the HR slightly dropped and consequently the RR intervals increase. Nevertheless, SDHR and SDRR increased according to the changes during the injection, but this could be related to the alternating influences of the parasympathetic and sympathetic nervous systems during the different steps of castration and the pain inflicted in the sequence of skin incision, exteriorization of the testicles and emasculation. Especially the influence of the parasympathetic nervous system leads to rapid changes in the range of milliseconds, while the effects of the sympathetic nervous system are rather slow (time scale of seconds) [67]. Besides the differences mentioned above, L0.5 and the Mepi0.5 differed significantly from the placebo groups in the differential values of the LF/HF ratio. This suggests that Animals 2022, 12, 2833 20 of 24 both substances are best at blocking sympathetic baroreflex activity, but only if it is given in the appropriate dosage. Compared to basal levels, the M0.5 group showed the least changes in HR variability parameters and therefore appears superior in efficacy to lidocaine and procaine. 4.5. Pain Assessment Post Castration In the immediate postoperative phase, the values of RR and MAP in group P0.3 , M0.3 , S0.3 , and S0.5 remained at a similar level or showed a further increase compared to the values recorded during castration, indicating ongoing nociception due to the lack of analgesia during and after castration. This observation

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ed to the values recorded during castration, indicating ongoing nociception due to the lack of analgesia during and after castration. This observation also illustrates, that despite the timely administration of the NSAID, acute postoperative pain is not effectively eliminated. SDHR, SDRR and LF/RF ratio returned to baseline levels from before castration in all groups, according to the statement of von Borell et al. [28], that short-term measures of HRV rapidly return to baseline after transient distress. Overall, during castration, the lowest deviations in autonomous responses from the baseline values were observed in the L0.5 and M0.5 groups, so that the most effective pain reduction is also assumed for the postoperative phase with these two agents. 4.6. Limitations Limitations of this study include the change in administered volume and dose between the 0.3 and 0.5 groups so that the individual effects of either dose or volume cannot be differentiated between treatments. Isoflurane was used to facilitate immobility of the piglets. As it influenced electrical brain activity it may have blunted the EEG parameters despite the very low concentration of isoflurane used. Moreover, arterial cannulisation was not possible in all piglets so that data for MAP were not available for all piglets. 5. Conclusions Contrary to our hypotheses, the EEG-Parameters NI, PTOT , MF, and SEF95 did not show any changes in connection with the painful procedures and are therefore not assessed as suitable for pain detection during injection or castration of piglets. In contrast, the concordance of changes in the LF/HF ratio with the further pain indicators collected demonstrates the applicability and utility of frequency-based HRV for the detection of pain responses during suckling piglet

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pain indicators collected demonstrates the applicability and utility of frequency-based HRV for the detection of pain responses during suckling piglet castration. In comparison to the intramuscular injection, more pronounced pain responses in piglets were observed with intratesticular application of the larger volume of 0.5 mL of local anaesthetics as well as saline than with the use of 0.3 mL, although regular statistical evidence is lacking. Pain-related responses during castration were reduced using the higher volume and thus the higher content of mepivacaine and lidocaine, while procaine appears unsuitable for local anaesthesia in piglet castration The agreement regarding pain assessment with a combination of several physiologic variables and behavioural observations leads to the conclusion that a completely pain-free castration is not feasible by means of intratesticular and subscrotal injection of procaine, lidocaine or mepivacaine in the applied concentration of 2%. Whether a higher concen- tration of mepivacaine and lidocaine, with a reduced volume, would lead to sufficient distribution in the tissues and complete analgesia during castration needs to be investigated in further studies. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani12202833/s1, Table S1: Mean and standard deviation of respiratory rate [breaths min−1 ], mean arterial blood pressure (MAP [mmHg]), and pulse rate [pulses min−1 ]) for groups P0.3 , P0.5 , L0.3 , L0.5 , M0.3 , M0.5 , S0.3 , S0.5 and IM for the timepoints baseline Injection (BL-INJ), injection (INJ), baseline castration (BL-CA), castration (CA) and post castration (POST-CA); Table S2:Heart rate variability. Mean and standard deviation of heart rate

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baseline castration (BL-CA), castration (CA) and post castration (POST-CA); Table S2:Heart rate variability. Mean and standard deviation of heart rate [beats min− 1 ], RR-Interval [ms], and median, minimum and maximum of Standard deviation of heartrate (SD HR Animals 2022, 12, 2833 21 of 24 [beat min− 1 ]), Standard deviation of RR-Interval (SD RR [ms]) and Low Frequency High Frequency ratio (LF/HF-ratio) for groups P0.3 , P0.5 , L0.3 , L0.5 , M0.3 , M0.5 , S0.3 , S0.5 and IM for the timeperiods baseline Injection (BL-INJe ), injection (INJe ), baseline castration (BL-CAe ), castration (CAe ) and post castration (POST-CAe ). Author Contributions: F.J.S. and S.W. carried out the clinical studies and drafted the manuscript; A.B. conducted the statistical analysis; J.T. and K.-H.W. (deceased) participated in the design of the study, S.B.R.K. and A.v.A. conceptualized, designed, and supervised the study and edited the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Ministry of Food, Agriculture, and Consumer Protection, Lower Saxony, Germany (ML), Project No. 206/204.1-04032-165. This Open Access publication was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—491094227 “Open Access Publication Funding” and the University of Veterinary Medicine Hannover, Foundation. Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki. An application for permission of this project was submitted to the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES), Hannover, Germany in accordance with §8 (1) of the German Animal Health and Welfare Act and approved under the permit number

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and Food Safety (LAVES), Hannover, Germany in accordance with §8 (1) of the German Animal Health and Welfare Act and approved under the permit number 33.9-42502-04-19/3218. Data Availability Statement: The data presented in this study are available in Supplementary Materials. Additional data are available on request from the corresponding authors. Acknowledgments: The authors thank Martin Beyerbach for providing help with statistical analysis. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. References 1. European Commission. European Declaration of alternatives to surgical castration of pigs. Available online: https://food.ec. europa.eu/system/files/2016--10/aw_prac_farm_pigs_cast-alt_declaration_en.pdf (accessed on 23 September 2020). 2. Federal Ministry of Food and Agriculture. Debate on piglet castration. Available online: https://www.bmel.de/EN/topics/ animals/animal-welfare/debate-piglet-castration.html (accessed on 20 December 2020). 3. Hodgson, D.S. An inhaler device using liquid injection of isoflurane for short term anesthesia in piglets. Vet. Anaesth. Analg. 2006, 33, 207–213. [CrossRef] 4. Rüdebusch, J.; Kästner, S.; Waldmann, K.H.; Wendt, M.; Von Altrock, A. Investigation into the optimization of automated isoflurane anesthesia for the performance of safe, painless castration of male suckling pigs. Berl Munch Tierarztl Wochenschr 2022, 135, 1–13. [CrossRef] 5. Schwennen, C.; Kolbaum, N.; Waldmann, K.H.; Holtig, D. Evaluation of the anaesthetic depth during piglet castration under an automated isoflurane-anaesthesia at farm level. Berl Munch

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[CrossRef] [PubMed] 19. Skade, L.; Kristensen, C.S.; Nielsen, M.B.F.; Diness, L.H. Effect of two methods and two anaesthetics for local anaesthesia of piglets during castration. Acta Vet. Scand. 2021, 63, 1. [CrossRef] 20. Hancock, T.M.; Caulkett, N.A.; Pajor, E.A.; Grenwich, L. An investigation of the effects of intratesticular alfaxalone and lidocaine during castration in piglets. Vet. Anaesth Analg. 2018, 45, 858–864. [CrossRef] 21. Waldmann, V.; Otto, K.H.; Bollwahn, W. Piglet castration–pain sensation and pain elimination. Dtsch. Tierarztl. Wochenschr. 1994, 101, 105–109. [PubMed] 22. Abendschon, N.; Senf, S.; Deffner, P.; Miller, R.; Grott, A.; Werner, J.; Saller, A.M.; Reiser, J.; Weiss, C.; Zablotski, Y.; et al. Local Anesthesia in Piglets Undergoing Castration-A Comparative Study to Investigate the Analgesic Effects of Four Local Anesthetics Based on Defensive Behavior and Side Effects. Animals 2020, 10, 1752. [CrossRef] [PubMed] 23. Ison, S.H.; Clutton, R.E.; Di Giminiani, P.; Rutherford, K.M. A Review of Pain Assessment in Pigs. Front. Vet. Sci. 2016, 3, 108. [CrossRef] 24. White, R.G.; DeShazer, J.A.; Tressler, C.J.; Borcher, G.M.; Davey, S.; Waninge, A.; Parkhurst, A.M.; Milanuk, M.J.; Clemens, E.T. Vocalization and physiological response of pigs during castration with or without a local anesthetic. J. Anim. Sci. 1995, 73, 381–386. [CrossRef] 25. Haga, H.A.; Tevik, A.; Moerch, H. Electroencephalographic and cardiovascular indicators of nociception during isoflurane anaesthesia in pigs. Vet. Anaesth. Analg. 2001, 28, 126–131. [CrossRef] 26. Sheil, M.; Polkinghorne, A. Optimal Methods of Documenting Analgesic Efficacy in Neonatal Piglets Undergoing Castration. Animals 2020, 10, 1450. [CrossRef] [PubMed] 27. Byrd, C.J.; Johnson, J.S.; Radcliffe,

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