Piglets’ acute responses to local anesthetic injection and surgical castration: Effects of the injection method and interval between injection and castration

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TYPE Original Research PUBLISHED 29 September 2022 DOI 10.3389/fvets.2022.1009858 Piglets’ acute responses to local OPEN ACCESS anesthetic injection and surgical castration: Effects of the EDITED BY T. Bas Rodenburg, Utrecht University, Netherlands REVIEWED BY Regula Bettschart, injection method and interval University of Zurich, Switzerland Nancy De Briyne, Federation of Veterinarians of between injection and castration Europe, Belgium *CORRESPONDENCE Mathilde Coutant1*, Jens Malmkvist1 , Marianne Kaiser1 , Mathilde Coutant mathilde.coutant@anis.au.dk Leslie Foldager1,2 and Mette S. Herskin1 SPECIALTY SECTION 1 Department of Animal Science, Aarhus University, Tjele, Denmark, 2 Bioinformatics Research This article was submitted to Centre, Aarhus University, Aarhus, Denmark Animal Behavior and Welfare, a section of the journal Frontiers in Veterinary Science Although applied in some countries, efficacy of local anesthetics based on RECEIVED 02 August 2022 ACCEPTED 02 September 2022 procaine to mitigate acute responses to piglet castration remains questioned. PUBLISHED 29 September 2022 This paper presents results from a factorial study examining the effects of CITATION two methods of injection of a procaine-based drug (intra-funicular, IF, vs. Coutant M, Malmkvist J, Kaiser M, Foldager L and Herskin MS (2022) intra-testicular, IT), and four intervals between drug injection and castration Piglets’ acute responses to local (2.5, 5, 10, and 30 min) on acute responses of 3–4 day old piglets. The anesthetic injection and surgical study involved 597 male piglets, and 13 treatments: surgical castration castration: Effects of the injection method and interval between injection without anesthesia (CC), local anesthesia followed by castration involving all and castration.

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of the injection method and interval between injection without anesthesia (CC), local anesthesia followed by castration involving all and castration. combinations of injection method and interval, and sham handling separated Front. Vet. Sci. 9:1009858. by the same four intervals (SH). Responses of piglets to drug injection, doi: 10.3389/fvets.2022.1009858 castration and sham handling were evaluated based on quantification of COPYRIGHT © 2022 Coutant, Malmkvist, Kaiser, intra-procedural vocalizations and leg movements, as well as saliva cortisol Foldager and Herskin. This is an concentration in samples taken before and after castration. No differences open-access article distributed under were found between IF and the simpler IT injection method. Intervals of 2.5 the terms of the Creative Commons Attribution License (CC BY). The use, or 30 min led to stronger piglet responses than the other intervals. Overall, distribution or reproduction in other treatments involving anesthesia led to significantly stronger responses than forums is permitted, provided the original author(s) and the copyright sham handling, during both injection and castration. All treatments, even sham owner(s) are credited and that the handling, led to a significant increase in saliva cortisol, with no differences original publication in this journal is between anesthesia treatments and controls. Based on these results, castration cited, in accordance with accepted academic practice. No use, distribution 5–10 min after intra-testicular injection of procaine seems to be preferable or reproduction is permitted which as compared to the other treatments tested. However, piglets still showed does not comply with these terms. measurable signs of pain and stress during both injection and castration,

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ents tested. However, piglets still showed does not comply with these terms. measurable signs of pain and stress during both injection and castration, while handling alone (including the use of a castration bench) triggered a noticeable stress response. In light of these findings, the overall benefit of the procedure in terms of piglet welfare remains arguable. KEYWORDS local anesthesia, castration, pig, pain, stress, acute responses Frontiers in Veterinary Science 01 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 Introduction as intra-funicular injections to a similar level as experienced veterinarians. Consequently, the possibility for diverging efficacy Surgical castration is a routine practice that involves millions of the procedure when performed in practice, as compared of pigs yearly in the EU and other global regions. The procedure, to studies led by veterinarians, means that predictions for the consisting of the removal of the male piglets’ testes via severing actual welfare impact of castration involving local anesthesia, as of the spermatic cords, is primarily performed to prevent commonly performed commercially, may be uncertain. A field boar taint, perceived by humans as an unpleasant odor, and trial setup was therefore implemented in the present study. potentially present in meat from entire males (1). Implemented While the efficacy of local anesthesia administered prior without pain mitigation, surgical castration leads to substantial to piglet castration has been mostly reported in studies using pain and stress in piglets, as measured for example by a higher lidocaine, procaine is the active ingredient most frequently prevalence of high frequency vocalizations (2, 3), increased used as anesthetic for pig castration in the EU (17).

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e active ingredient most frequently prevalence of high frequency vocalizations (2, 3), increased used as anesthetic for pig castration in the EU (17). Textbooks plasma cortisol concentrations (4–7), and in-pen behavioral reviewing lidocaine and procaine report differences in terms of alterations (5, 8, 9) compared to sham handled piglets. As onset of action, efficacy and potency (24–26). In addition, recent a result, pain mitigating strategies such as administration of studies suggested unsatisfactory pain mitigation of procaine non-steroidal anti-inflammatory drugs (NSAIDs) and/or local administered alone before surgical castration of 3–7 day old anesthetics prior to the procedure have been developed and piglets (12, 22, 27). implemented. Studies suggest that local anesthesia limits the Irrespectively of the drug used, knowledge on the welfare expression of high pitch vocalizations (2, 10, 11), reduces impact of castration with prior administration of local levels of leg movements interpreted as resistance (11–14), and anesthetics remains limited, as only few studies have reported decreases the plasma cortisol response (10, 15). In accordance piglets’ response to the injection itself. As reviewed by Kongsted with the ‘European Declaration on alternatives to surgical et al. (28), studies reporting effect of different methods of castration in pigs’, stating that from 2012, all piglets should injection are also scarce. In addition, the effect of specific receive prolonged pain relief (16), various European countries modalities of the anesthetic administration remain poorly have enforced the use of local anesthetics prior to piglet documented. For instance, while the method of injection is castration, and the practice is common, but not systematic, in often

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hetics prior to piglet documented. For instance, while the method of injection is castration, and the practice is common, but not systematic, in often described with regards to the anatomical location (intra- the European pig industry (17). funicular, intra-testicular), descriptions of the orientation of the In Denmark, the fourth largest producer of pigs in Europe needle, pressure applied on the testes, or distribution of the (18), systemic pain relief using NSAIDs has been mandatory injected liquid is often lacking (29). Potential effects of the since 2009, and the administration of local anesthetics became interval between injection of the anesthetic and castration have a code-of-practice in 2019, following an initiative from the been discussed with regards to the onset of action, but, to our Danish pig industry (19). Farmers and herdsmen are allowed knowledge, only one study compared the impact of interval on to administer the anesthetics themselves after completing a the acute response to castration for a specific drug (30). training led by a veterinarian, following the guidelines provided The aim of the present study was to compare the effect by the Danish Veterinary and Food Administration [DVFA, of two methods of injection of procaine as a local anesthetic (19)]. These include administration of the anesthetic using and four time intervals between injection and castration on an intra-funicular injection (injection into the spermatic cord, piglets’ responses to injection and castration in field trial testis, and scrotal skin), also referred to as a “three-step” conditions resembling commercial practice. Acute responses method, and a 5 to 10 min time interval between injection and were evaluated based on vocalizations and number of leg castration (19). The

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e responses method, and a 5 to 10 min time interval between injection and were evaluated based on vocalizations and number of leg castration (19). The procedure is performed using one of the movements, interpreted as resistance, during injection and two anesthetics drugs (both procaine-based) legalized for piglet castration, as well as saliva cortisol concentrations before and castration in Denmark. after surgical castration. Yet, most studies investigating the efficacy of local anesthesia to mitigate piglet pain have been carried out in laboratory- like conditions, involving procedures performed by skilled Materials and methods veterinarians [e.g., (10, 12)]. Thereby, the procedures likely considered subtle aspects of the injection techniques such as the Animals speed or the pressure applied on the testes, which potentially may affect the pain triggered as well as administration efficacy The experiment was carried out between July and October (20, 21). Other studies have been performed on-farm, but still 2020 and was conducted in a Danish conventional sow herd involved a trained veterinarian for the administration of local with approximately 1,300 sows giving birth to (Landrace × anesthetic and castration (22, 23). Thus, in the absence of a Yorkshire) × Duroc crossbred piglets. Sows were loose-housed comparative study, it is not known whether herdsmen, despite in farrowing pens measuring 3.1 × 2.8 m, set as a crate in the their mandatory training, perform complex procedures such first week post-farrowing, i.e., during the experiment. Frontiers in Veterinary Science 02 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 Trials were conducted 2 days per week, corresponding to the during vocalizing. To further ensure a proper fit in the bench, routine days of

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22.1009858 Trials were conducted 2 days per week, corresponding to the during vocalizing. To further ensure a proper fit in the bench, routine days of castration at the farm. Earlier in the week, litters considering the variation in piglets’ body size, a soft material reaching 3 to 4 days of age on the weekday of experimentation (5-mm yoga matt; Figure in Supplementary Figure S1) could be (with day 0 defined as the day of birth of the last piglet in a placed in the bench. litter), and counting at least six males, were clinically assessed, In-between procedures, piglets were returned to the heated and male piglets selected. All selected piglets were clinically box with their littermates. Immediately after castration or healthy and free of overt anatomical malformations. Piglets last sham handling, piglets were individually subjected to weighting <0.9 or >2.3 kg on the day of selection were not complementary testing not reported in the present paper, and included in the study, due to a risk of improper fit in the brought back to the sow in the farrowing pen. On average castration bench during testing. Each of six male piglets selected 17 min after castration or last sham handling, a second saliva within a litter was randomly assigned to one of 13 treatment sample was taken in the farrowing unit. Later in the afternoon, groups (Table 1) according to a randomization plan balancing approximately 6 h after castration, a last saliva sample was taken treatments between litters, experimental days, and experimental for cortisol determination. weeks. On the day of castration, the health status of experimental In accordance with the clinical trial permit (described piglets and sows were re-assessed, and piglets were excluded below), piglets were closely monitored for

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ance with the clinical trial permit (described piglets and sows were re-assessed, and piglets were excluded below), piglets were closely monitored for drug-related side from the experiment if the inclusion criteria were not fulfilled effects up to 72 h after anesthetic injection and castration. (severe diarrhea, lameness, or sow rectal temperature higher Two experimenters were present in the testing area: an than 39◦ C). experimenter performing the procedures, and an experimenter All piglets were identified by a number written on their back starting and stopping the recordings. These two were not using a food-safe marker. Experimental piglets were allowed blinded to the experimental treatments. All other experimenters, to be cross-fostered in the first days of life, but could not be selecting the piglets, sampling them, recording the data and moved from their litter after selection. Cross-fostering of non- creating the datasets were blinded until the start of the experimental littermates was permitted up to the morning prior statistical analysis. to castration. The piglets were administered a suspension of 45 mg toltrazuril and 200 mg gleptoferron (ForcerisTM , 1.5 mL, Ceva Animal Health A/S, Libourne, France) on day 1 after Treatments farrowing. The experimental piglets were not ear tagged, tail docked, or teeth clipped before castration. In order to A total of 597 piglets were assigned to one of thirteen avoid confounding of the results on the efficacy of the local treatments (Table 1): Surgical castration without local anesthesia anesthetic, piglets were administered an NSAID (intramuscular involving a single stay in the bench (control-castrated; CC), injection of 1.5 mg meloxicam; Melovem, 0.3 mL, Dopharma, intra-funicular (IF) injection of 0.5 mL of local

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stay in the bench (control-castrated; CC), injection of 1.5 mg meloxicam; Melovem, 0.3 mL, Dopharma, intra-funicular (IF) injection of 0.5 mL of local anesthetic per The Netherlands) as analgesic after completion of the data testis and subsequent castration after 2.5 min (IF02), 5 min collection and within 24 h after castration. (IF05), 10 min (IF10) or 30 min (IF30), intra-testicular (IT) injection of 0.5 mL of local anesthetic per testicle and subsequent castration after 2.5 min (IT02), 5 min (IT05), 10 min (IT10) or Study design 30 min (IT30), sham handling (SH) with two stays in the bench without tissue damage inflicted (sham anesthesia and sham On the day of castration, experimental piglets were weighed. castration), separated by 2.5 min (SH02), 5 min (SH05), 10 min Saliva samples were taken approximately 35 to 40 min before (SH10) or 30 min (SH30). bringing the piglets to the testing area, a calm room outside the farrowing room. All experimental piglets plus one extra littermate selected at random were transported together in a plastic box (size: 71.5 × 53.0 × 39.5 cm) layered with straw, and Procedures placed underneath a heat lamp (averaging 20◦ C, ranging from 15 to 25◦ C at recipients) upon arrival in the testing area. Piglets All surgical and injection procedures were performed by were injected, castrated or sham handled one by one, respecting the same experimenter, an experienced farm staff from Aarhus a randomized testing order, and following a predefined schedule University trained in accordance with standards from the ensuring that the experimental intervals between procedures DVFA (19). To achieve a uniform injection technique among were respected. During all procedures, piglets were fixated while the experimental piglets, the experimenter

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hieve a uniform injection technique among were respected. During all procedures, piglets were fixated while the experimental piglets, the experimenter received additional lying on their back, in a commercially available castration training led by a veterinarian and practiced the different bench (Unitron A/S, Kolding, Denmark). For the experimental procedures on approximately 50 piglets before experimentation. purpose, the bench was modified to enable larger amplitudes of For all piglets, the precise duration of each procedure (injection, front leg movements, and more natural opening of the mouth castration, sham handling) was recorded to the nearest second. Frontiers in Veterinary Science 03 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 1 Description of the 13 treatment groups involved in the study. IF02 IF05 IF10 IF30 IT02 IT05 IT10 IT30 CC SH02 SH05 SH10 SH30 IM IF IF IF IF IT IT IT IT – SH SH SH SH TI 2.5 5 10 30 2.5 5 10 30 – 2.5 5 10 30 N 50 50 50 49 50 49 50 49 50 50 25 25 50 Apart from CC (control castrated), all treatments were a combination of a method of injection (IM): IF, intra-funicular injection; IT, intra-testicular injection; SH, sham handling; and time interval between injection and castration (TI, min): 02, 2.5 min; 05, 5 min; 10, 10 min; 30, 30 min. N, number of piglets tested per treatment. Anesthetic drug The anesthetic used in the study was a procaine hydrochloride 2% solution (Procamidor R Vet., 20 mg/mL, Richter Pharma AG, Wels, Austria). The product was administered using an automatic syringe (Prima Tech R ; 0.5 mL in 0.1 mL increments) with a 25G needle for the intra-funicular injection (0.5 × 16 mm, BD MicrolanceTM 3, BD, New Jersey, USA). For the intra-testicular injection, in order to get the shortest needle length

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icular injection (0.5 × 16 mm, BD MicrolanceTM 3, BD, New Jersey, USA). For the intra-testicular injection, in order to get the shortest needle length possible, a 26G needle (0.45 × 12 mm, Sterican R Insulin needle, B Braun Medical SA, Barcelona, Spain) was used, together with a 5 mm plastic stopper. Needles were changed between each piglet. Injection types Piglets were fixated in dorsal recumbency position in the castration bench, and testes were fixed carefully in the distal FIGURE 1 The two methods of injection of the local anesthetic. (A) The end of the scrotum. The scrotum area was not disinfected angle of insertion of the needle is shown for the intra-funicular prior to anesthetic injection nor castration. The right testis (IF) and (B) intra-testicular (IT) methods. was fixed caudally between the thumb and index finger of the experimenter, applying a steady but low pressure during the fixation. For the intra-funicular injection, the needle was Castration inserted at a 45-degree angle pointing in dorsal direction After fixation in the castration bench, a disposable and a 10-degree angle pointing in lateral direction from a scalpel (Scalpel no. 24, carbon steel sterile blade, Swann- caudocranial view (Figure 1). The needle was inserted in its full Morton, Sheffield, England) was used to perform an incision length (16 mm) through the center of the testis and aiming (approximately 1 cm) through the scrotal skin and spermatic for the spermatic cord. The anesthetic was administered by fasciae. The right testis was then gently pressed between the continuously dispensing the drug while withdrawing the needle, index and the thumb of the experimenter until fully outside of and releasing the testis, also referred to as “push and pull the scrotum. The testis was then

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and the thumb of the experimenter until fully outside of and releasing the testis, also referred to as “push and pull the scrotum. The testis was then carefully lifted vertically, and technique” (19). After each injection, a drop of the anesthetic the spermatic cord cut a few millimeters below the testis using was left on the surface of the skin of the scrotum (Cutis scroti). the scalpel. The incision was repeated on the left testis. A new For the intra-testicular injection, the needle was inserted in the scalpel was used for each piglet. A video of the procedure is center of the right testis, in a dorsal direction at an angle of 90 available in Supplementary Video S1. degrees from a caudocranial view (Figure 1). A custom-made 5 mm plastic stopper was placed on each needle to ensure a Sham handling standardized needle length of 7 mm. The anesthetic was injected Piglets were fixated in the castration bench, as previously slowly into the testicle (over approximately 3 s) while gradually described, for a duration of approximately 25 s (corresponding loosening the grip around the testicle. The procedures were then to the average duration of the procedures of local anesthesia and repeated for the left testicle, and the piglet removed from the castration as assessed in a pilot study), during which they did castration bench and placed in a heated area with littermates not experience any tissue damage nor physical stimulation of the until castration. groin area. Frontiers in Veterinary Science 04 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 Ethical and other permits TABLE 2 Description of the vocal parameters analyzed for each piglet during injection of local anesthetic, castration, or sham handling, all performed while the piglet was in the castration bench.

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alyzed for each piglet during injection of local anesthetic, castration, or sham handling, all performed while the piglet was in the castration bench. The study was performed in compliance with the EU Directive 2010/63/EU for animal experiments, the Ministry of Parameter (unit) Description Food, Agriculture and Fisheries, and The Danish Veterinary and Food Administration under act 474 of 15. May 2014 and Call proportion Proportion of time spent vocalizing during the executive order 2028 of 14. December 2020. The experiment procedure, calculated as call duration /procedure was approved as a clinical trial by the Danish Medical Agency duration. (reference number 2020061784). All procedures were ethically Call per second (s−1 ) Number of calls per s of the procedure evaluated and approved by the Danish Animal Experiments Mean call duration (s) Average duration of a call during the procedure, Inspectorate (Approval number 2019-15-0201-00263). calculated as sum of call durations/number of calls. Mean energy (dB) Average energy, calculated as an average of the Data collection energy of each call during the procedure. Max energy (dB) Maximum value of energy recorded for all calls Vocalizations during the procedure. The vocal responses of the piglets were recorded during each Max power (dB) The maximum power recorded for all calls during procedure, using a microphone (Sennheiser E614, Sennheiser the procedure, relative to the specific recording Wennebostel, Germany) fixed 30 cm ahead of the piglet’s snout, set-up. at the level of the head of the piglet. The microphone was Aggregated entropy (kilobits) Aggregated disorder for the procedure obtained by connected to an amplifier (Audiobox USB R 96, PreSonus, analyzing the energy distribution within each call. Louisiana, USA)

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the procedure obtained by connected to an amplifier (Audiobox USB R 96, PreSonus, analyzing the energy distribution within each call. Louisiana, USA) connected to a computer, from which Higher entropy values correspond to greater recordings were manually started and stopped upon piglet’s disorder in the sound whereas a pure tone would placement and removal from the castration bench. Duration have zero entropy (31). of each procedure was recorded. All vocal files were analyzed Max entropy (kilobits) Highest value of disorder recorded for all calls in Raven Pro 1.6 bioacoustics analysis software (Cornell Lab during the procedure. of Ornithology, Ithaca, New York, USA) using the band limited energy detector function, as described in Coutant et al. (27). This function allowed each intra-procedural call observers, blinded to the experimental treatments, were trained to be automatically detected based on a pre-set of parameters to recognize and count these behaviors, and practiced recording (data in Supplementary Text S1), and characterized in terms on approximately 100 random video clips, using the Behavioral of number, duration, energy, and entropy. After running Observation Research Interactive Software [BORIS; (32)]. Each the automatic call detection, each procedural recording was video sequence was then analyzed, and the occurrence of manually checked to ensure that every call was properly each type of behavior was counted for each front leg in the selected, and to de-select surrounded noise or experimenter’s interval between closing and opening of the castration bench. voices wrongfully detected as a call. For all piglets, vocal Movements that were too sudden to be categorized despite characteristics of each procedure were then defined (Table 2) the low speed of

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piglets, vocal Movements that were too sudden to be categorized despite characteristics of each procedure were then defined (Table 2) the low speed of video analysis were not counted. Reversely, and analyzed. These procedures were performed by one person movements performed relatively slow (duration >1 s) were not (MC), blinded to the experimental treatments. An auditory considered as resistance and therefore not recorded. In addition, example of a piglet vocalizing during castration can be consulted duration of blocking in the bench, corresponding to a leg being in Supplementary Video S1. mechanically unable to move due to physical blocking, were also recorded. The two observers showed a high inter-observer Resistance movements reliability with an intraclass correlation coefficient (ICC) close Four distinct types of front leg movements, interpreted as to 96% (95% confidence interval: 90–99%; comparing 15 video resistance movements, were recorded during each procedure clips). An example of leg resistance movements performed by using a camera (GoPro HERO7 Black, GoPro, San Mateo, a piglet during castration can be consulted in the video in California, USA; 60 frames per sec, FPS) placed on a stand Supplementary Video S1. 30 cm to the right of the castration bench, approximately 50 cm above the bench. This distance allowed a full picture of the piglets’ front legs. Resistance movements were quantified using a Saliva cortisol concentrations novel method developed in Coutant et al. (27). Video clips were For baseline, one saliva sample per piglet was collected in observed at low speed (5 FPS) to detect four types of movements: the home pen on average 35 (±14; SD) min before the first flexion, extension, kick, and blow (Table 3; Figure 2). Two procedure. For changes in

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ments: the home pen on average 35 (±14; SD) min before the first flexion, extension, kick, and blow (Table 3; Figure 2). Two procedure. For changes in saliva cortisol in response to the Frontiers in Veterinary Science 05 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 3 Description of the leg resistance movements recorded during injection of local anesthetic, castration, or sham handling, all performed while the piglet was in the castration bench. Category Description Flexion Piglet vertically bends his front leg, provoking a flexion of the elbow of at least 90 degrees. Extension Piglet fully extends his front leg while lowering the head in the bench. May be accompanied by trembling of the leg and/or by a subtle lift of the piglet’s back. Kick Piglet front leg performs a sudden upwards movement, changing from a flexion to a tense upwards position. Blow Piglet suddenly draws back his front leg forwards or backwards for at least half a bench length, from a normal upright position to an extended position, with little or no flexion of the elbow. Leg blocked Piglet’s front leg is blocked in the bench cone, preventing movement. Each leg was scored separately. Figure 2 shows a visualization of the leg movements. FIGURE 2 Visual representation of the four leg resistance movements recorded during injection of local anesthetic, castration, or sham handling while in the castration bench. F, Flexion; K, Kick; E, Extension; B, Blow. The visualizations only show the leg procedures, one sample per piglet was collected on average 17 movements, see further description of the categories in Table 3. (±9) min after castration and again approximately 6 h after castration (5 h 47 min ±26 min). Saliva samples were performed using a novel method recently used in Coutant

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tration and again approximately 6 h after castration (5 h 47 min ±26 min). Saliva samples were performed using a novel method recently used in Coutant et al. (27); a cotton swab (Salivette R , Sarstedt, Aktiengesellschaft & Co., coefficient of variation was 3.7 and 5.6%, respectively, for low Numbrecht, Germany) was cut in pieces (approx. 2.0 × 0.5 cm), and high control, and the inter-assay variation was 7.2 and 9.8% soaked in concentrated apple juice (nectar from concentrated for low and high control, respectively. The minimal detectable juice, min 60%, Rynkeby Foods A/S, Ringe, Denmark) for 1 h, concentration was 45 pg/mL. The procedure outlined by the and dried in an electric oven at 60◦ C for 5 h. A pilot study manufacturer was followed. revealed an increase in saliva production with this method compared to the use of a non-pre-soaked piece of cotton. Similar results were obtained with soaking the cotton pieces in citric acid (fresh lemon juice), but after this method, saliva Statistical analysis sampling seemed more aversive for the piglets, and the method was therefore abandoned. During sampling, the cotton swab Three piglets were removed from the analysis: two piglets was fixed at the end of a straight pean clamp, and gently due to experimental issues during the procedures, and one introduced into the piglet’s mouth, while the piglet was held due to cryptorchidism (only one testis descended) discovered in the experimenter’s arms. The cotton was rotated gently in during injection of the local anesthetic. In addition, for the piglet’s mouth for 30 to 45 s, with insistence around the two piglets, insufficient amounts of saliva rendered cortisol salivary glands. This procedure was performed by one of four analysis impossible, and eight piglets were

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ufficient amounts of saliva rendered cortisol salivary glands. This procedure was performed by one of four analysis impossible, and eight piglets were excluded from the trained experimenters blinded to the experimental treatments. resistance movement analysis due to technical issues with The sample was then placed in an experimental tube (provided the recording of the videos (five for injection and three as part of the Salivette R ), labeled, and stored at −18◦ C for castration). Malfunctioning and technical issues of the until cortisol concentration determination at the departmental vocalization recording set-up resulted in 168 missing files for laboratory. Samples were defrosted and centrifuged for 6 min anesthesia and 182 missing files for castration, leaving valid at 1,000 × g. Concentrations of cortisol were determined vocalization data from 376 piglets for anesthesia and 412 piglets using a direct enzyme immunoassay without extraction and for castration. previously validated for saliva (Arbor Assays, Cat. K003-H1W, Vocalizations and resistance movements were analyzed Michigan, USA). With this method, the antiserum cross-reacts separately for local anesthesia and castration. During the with cortisol and some cortisol metabolites, and values have injection of the local anesthetic, time interval had not yet any to be interpreted as cortisol immunoreactivity. The intra-assay bearing, and thus for the first procedure only three treatments Frontiers in Veterinary Science 06 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 were relevant: injection by intra-funicular method (IF), injection Results by intra-testicular method (IT), and sham handling (SH). The vocalization parameters (Table 2) were log-transformed Vocalizations if necessary (to obtain

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intra-testicular method (IT), and sham handling (SH). The vocalization parameters (Table 2) were log-transformed Vocalizations if necessary (to obtain normality) and analyzed in a linear mixed effects model with treatment as the main explanatory variable, Vocal responses of piglets during administration of the weight (range: 0.94–2.68 kg), age (3 or 4 days), time of day local anesthetic were affected by the treatments. A significant when starting the procedure (decimal h, range: 8.21–14.66) and difference was observed in 6 out of 8 indicators (Table 4), i.e., duration of procedure (range: 21–117 s) as covariates, and litter for call proportion, call per second, mean energy, max energy, as a random effect. Rate of vocalizations and other variables max power, and aggregated entropy. Across all indicators, no where duration of the procedure was an integrated part of differences were found between the two methods of injection the calculated response, were analyzed by similar models, but of the local anesthetic, and both types of procedures resulted without duration of the procedure as covariate. in higher values of vocal parameters compared to sham The counts of each type of resistance movements were handled (i.e., not injected, not castrated) piglets. There was summed per piglet during each procedure (Table 3). Total no effect of treatments in terms of mean call duration and observation duration was defined as the sum of observation max entropy. time per leg, subtracting the duration of left leg and right leg At castration, all vocalization indicators showed significant occasionally being blocked while in the castration bench. The treatment differences (Table 5), except for max entropy. sum of piglets’ resistance movements was analyzed by a negative Generally, IF

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tion bench. The treatment differences (Table 5), except for max entropy. sum of piglets’ resistance movements was analyzed by a negative Generally, IF did not differ significantly from IT, except binomial mixed effects model including treatment as main in mean call duration, where IF resulted in longer calls explanatory variable, weight, age and time of day as covariates, than IT when pigs were castrated 2.5 min after anesthetic logarithm of total observation duration (range: 1–211 s) as offset, injection, while IT resulted in longer calls than IF when pigs and litter as a random effect. were castrated after 30 min. Piglets castrated 2.5 min after Changes in saliva cortisol in response to the procedures were IF injection showed mean call durations, maximum energy log transformed and analyzed in a mixed model with treatment, and maximum power levels not differing significantly from sampling point (early - late) and their interaction as fixed effects CC. Additionally, no differences were found between vocal of main interest, and weight, age, time of sampling (decimal h, responses of piglets castrated 30 min after IT injections and range: 7.82–20.50), and baseline cortisol concentration (range: the ones of CC in terms of call proportion, mean call 2,001–48,570 pg/mL) as covariates, with sample point as a duration and max energy. In five out of eight indicators repeated measure, and litter as random effect. (i.e., call proportion, mean call duration, mean energy, max For all outcomes, initial models were reduced by stepwise energy, and max power), no differences were found between removal of fixed effects at P > 0.10, starting with the interaction vocal responses of IT and IF vs. SH, when piglets were of highest order, however, never removing the main effect

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> 0.10, starting with the interaction vocal responses of IT and IF vs. SH, when piglets were of highest order, however, never removing the main effect castrated after 5 and 10 min. Vocal responses of piglets in for the variables of key interest. In linear mixed effects the treatments IF and IT were, though, greater than SH in models, Satterthwaite’s approximation of denominator degrees the case of castration after 2.5 and 30 min. IT and IF vocal of freedom was used. Deviations from assumption of normality responses were greater than SH, regardless of the interval and variance homogeneity were monitored visually by plotting between local anesthesia and castration, and did not differ residuals at each step. Covariates with significant effects were significantly from those of CC, in terms of call per second maintained in the final models, but effects not reported, at the and aggregated entropy. Piglet weight was associated with exception of weight. mean energy (F1,289= 7.1, P = 0.008), max energy (F1,281= In case the final model showed significant effect of the 4.6, P = 0.033), and max power parameters (F1,277= 7.5, treatments (P ≤ 0.05), pairwise comparisons between treatments P = 0.007), with greater values observed in heavier pigs. were performed with p-values adjusted for multiple comparisons Overall, piglets’ vocal responses to castration did not differ using the Tukey-Kramer method (marked by Padj ). For analyses between intra-testicular and intra-funicular injections. Stronger of castration, however, Tukey-Kramer was not applied as the responses, often comparable to those of piglets castrated without 13 treatment groups would result in 78 pairwise comparisons, anesthesia, were shown by piglets castrated 2.5 and 30 min many of which were not relevant for the

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ent groups would result in 78 pairwise comparisons, anesthesia, were shown by piglets castrated 2.5 and 30 min many of which were not relevant for the aims of the after anesthetic injection, while most vocal parameters did not present study. Instead, intervals were compared within injection differ from sham handled animals when piglets were castrated methods, and methods were compared within intervals, using 5 and 10 min after local anesthesia. For two indicators, i.e., unadjusted P-values reported as P. All calculations were call rate and aggregated entropy, the vocal parameters of performed using SAS 9.4 (SAS Institute Inc., Cary, North piglets castrated after injection of local anesthetic did not differ Carolina, USA). All data used for statistical analysis can significantly from those of piglets castrated without anesthesia, be consulted in the dataset in Supplementary Dataset S1. regardless of the time interval between local anesthesia Descriptive measures are presented as average ± standard error. and castration. Frontiers in Veterinary Science 07 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 4 Averages (± SE) of vocal parameters recorded during injection of the local anesthetic. IF IT SH F-test P Call proportion 0.63 ± 0.01a 0.64 ± 0.01a 0.47 ± 0.02b F2,314= 29.4 <0.001 −1 a a b Call per second (s ) 0.90 ± 0.02 0.88 ± 0.02 0.66 ± 0.03 F2,314= 39.3 <0.001 Mean call duration (s) 0.75 ± 0.03 0.76 ± 0.02 0.72 ± 0.03 F2,311= 1.6 0.201 Mean energy (dB) 84.80 ± 9.17a 85.13 ± 9.12a 46.41 ± 10.10b F2,310= 5.6 0.004 Max energy (dB) 319.98 ± 8.54a 318.51 ± 7.94a 288.21 ± 10.46b F2,311= 3.2 0.041 Max power (dB) −14.24 ± 0.74a −14.40 ± 0.67a −17.06 ± 0.89b F2,311= 4.3 0.015 Agg entropy (kilobits) 130.54 ± 3.27a 122.08 ± 2.85a

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b F2,311= 3.2 0.041 Max power (dB) −14.24 ± 0.74a −14.40 ± 0.67a −17.06 ± 0.89b F2,311= 4.3 0.015 Agg entropy (kilobits) 130.54 ± 3.27a 122.08 ± 2.85a 108.82 ± 4.40b F2,330= 33.8 <0.001 Max entropy (kilobits) 5.94 ± 0.01 5.91 ± 0.01 5.93 ± 0.02 F2,312= 1.1 0.340 a,b Different letters within a row indicate significant differences between treatments, P adj ≤ 0.05; IF, intra-funicular injection (n = 138); IT, intra-testicular injection (n = 135); SH, sham anesthesia (n = 103). Fn,d denotes an F-test on n, numerator and d, denominator degrees of freedom for the effect of treatment. Resistance movements F12,503= 0.8, P = 0.673; Table in Supplementary Table S1). A weight effect was observed (F1,485= 12.2, P < 0.001), with Piglets’ leg movements during injection of the local higher values of baseline cortisol in lighter piglets. anesthetic differed significantly among treatments, with greater The interaction between treatments and timing of sampling levels of movements observed in IF and IT than in SH. The leg was not significant (F12,1044= 1.56, P = 0.097). After removing movements observed during intra-testicular and intra-funicular the interaction from the model, the main effect of treatment was injections did not differ significantly, although piglets injected also not significant, although a tendency was observed (F12,1112 by the intra-funicular method displayed an average of 17.6% = 1.67, P = 0.067). However, the average cortisol concentrations more leg movements than piglets injected by the intra-testicular following the procedures were significantly affected by the method. At castration, the number of resistance movements sample point (F1,1056 = 708.3, P < 0.001), with higher differed significantly among treatments (Table 6). Regardless of

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number of resistance movements sample point (F1,1056 = 708.3, P < 0.001), with higher differed significantly among treatments (Table 6). Regardless of concentrations observed 17 min after castration (17,378 ± 8,216 the time interval between injection and castration, IF did not pg/mL) than 6 h after castration (9,479 ± 5,672 pg/mL). In this lead to a significantly different response from IT, and both model, an effect of weight was also observed (F1,456= 6.2, P = methods resulted in more resistance movements than SH. Piglets 0.013), with higher cortisol concentration in lighter piglets. castrated at various intervals after IF injection did not differ Considering the significant difference between cortisol significantly in their response, but piglets castrated after 30 min concentrations measured at 17 min and 6 h post-procedure, showed a response that did not differ significantly from the two separate analyses were performed. At 17 min, treatment one of CC. Similarly, piglets castrated after IT injection did not groups differed in cortisol concentration (Table 7). Across IF differ significantly in their response, regardless of the interval. In and IT, no difference was found among intervals between addition, levels of resistance movements of piglets castrated after injection and castration, but both injection methods led to, 2.5 or 30 min did not differ significantly from the ones of CC. or tended to lead to, a greater response than shown by sham All SH piglets responded significantly less than castrated piglets, piglets at 10 and 30 min. Within the IF treatments, piglets regardless of interval between stays in the castration bench. showed a greater cortisol response when castrated 30 min after Overall, the level of resistance movements during castration administration

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tion bench. showed a greater cortisol response when castrated 30 min after Overall, the level of resistance movements during castration administration of the local anesthetic compared to 2.5 and did not differ between injection methods nor interval between 5 min. Piglets anesthetised by the IT method did not differ procedures. Responses of piglets castrated 2.5 or 30 min after significantly, irrespective of the time interval between injection injection of anesthetic led to responses that did not differ of the local anesthetic and castration. Regardless of the method from those of piglets castrated without anesthesia. In addition, of injection of the local anesthetic, piglets castrated after 10 anesthetised piglets, regardless of the method, showed more and 30 min showed a greater cortisol response than CC. Within resistance movements than sham handled piglets. SH, no differences were observed for intervals 2.5, 5, and 30 min, but piglets offered a 10 min interval between stays in the castration bench had a lower cortisol concentration Saliva cortisol concentrations compared to intervals of 2.5 min and 5 min. The latter intervals were also significantly different from CC. Cortisol responses As expected, baseline cortisol concentrations from decreased with piglet weight (F1,356 = 9.49, P = 0.002). Overall, samples obtained 35–40 min before the procedures did not cortisol responses 17 min after castration did not differ between differ significantly among treatments (approx. 9,700 pg/mL, injection methods, but greater responses were observed in Frontiers in Veterinary Science 08 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 5 Averages (±SE) of vocal parameters recorded during castration. IF IT SH F-test P Call proportion 2.5 min 0.61 ± 0.03b,1 0.59 ±

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ts.2022.1009858 TABLE 5 Averages (±SE) of vocal parameters recorded during castration. IF IT SH F-test P Call proportion 2.5 min 0.61 ± 0.03b,1 0.59 ± 0.03b,1 0.43 ± 0.04b,2 F12,370 = 6.8 <0.001 b b b 5 min 0.59 ± 0.03 0.60 ± 0.03 0.51 ± 0.07 10 min 0.55 ± 0.03b,1 0.58 ± 0.02b,1/2 0.44 ± 0.05b,2 30 min 0.60 ± 0.03b,1 0.66 ± 0.03ab,1 0.43 ± 0.04b,2 a a CC 0.72 ± 0.02 0.72 ± 0.02 0.72 ± 0.02a Call per second 2.5 min 0.87 ± 0.051 0.87 ± 0.051 0.68 ± 0.05b,2 F12,363 = 6.7 <0.001 5 min 0.91 ± 0.041 0.89 ± 0.051 0.63 ± 0.07b,2 10 min 0.95 ± 0.041 0.94 ± 0.031 0.69 ± 0.07b,2 1 1 30 min 0.94 ± 0.04 0.87 ± 0.04 0.64 ± 0.05b,2 CC 0.92 ± 0.04 0.92 ± 0.04 0.92 ± 0.04a Mean call duration (s) 2.5 min 0.75 ± 0.05ab,1 0.70 ± 0.05abc,2 0.63 ± 0.04b,2 F12,364 = 2.4 0.006 5 min 0.70 ± 0.05bc 0.70 ± 0.05b 0.81 ± 0.11b 10 min 0.60 ± 0.04c 0.62 ± 0.03c 0.67 ± 0.08b bc,2 ab,1 30 min 0.68 ± 0.04 0.81 ± 0.05 0.68 ± 0.05b,2 a a CC 0.82 ± 0.04 0.82 ± 0.04 0.82 ± 0.04a b,1 b,1 Mean energy (dB) 2.5 min 34.58 ± 15.75 52.37 ± 16.6 0.15 ± 15.52b,2 F12,358 = 4.8 <0.001 5 min 25.88 ± 16.43b 29.59 ± 18.05b 42.88 ± 29.40b 10 min 19.28 ± 19.23b 12.93 ± 11.93b −2.34 ± 21.16b b,1 b,1 30 min 50.27 ± 18.81 59.10 ± 18.68 6.74 ± 15.40b,2 a a CC 131.20 ± 16.32 131.20 ± 16.32 131.20 ± 16.32a ab,1 b,1 Max energy (dB) 2.5 min 299.20 ± 16.71 302.53 ± 15.36 244.89 ± 18.90b,2 F12,359 = 3.5 <0.001 bc b 5 min 273.55 ± 18.78 285.58 ± 19.65 271.39 ± 24.91b 10 min 265.70 ± 20.45c 268.42 ± 17.46b 250.20 ± 26.68b 30 min 292.07 ± 18.28bc,1 302.51 ± 14.16ab,1 243.05 ± 18.59b,2 CC 349.24 ± 11.32a 349.24 ± 11.32a 349.24 ± 11.32a ab,1 b,1/2 Max power (dB) 2.5 min −15.22 ± 1.52 −15.20 ± 1.34 −20.20 ± 1.79b,2 F12,359 = 3.2 0.002 c b 5 min

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± 11.32a 349.24 ± 11.32a 349.24 ± 11.32a ab,1 b,1/2 Max power (dB) 2.5 min −15.22 ± 1.52 −15.20 ± 1.34 −20.20 ± 1.79b,2 F12,359 = 3.2 0.002 c b 5 min −18.42 ± 1.73 −17.44 ± 1.73 −18.65 ± 2.02b 10 min −17.87 ± 1.64bc −19.36 ± 1.59b −20.52 ± 2.20b 30 min −15.92 ± 1.54bc,1 −15.92 ± 1.15b,1 −20.39 ± 1.54b,2 CC −11.83 ± 1.04a −11.83 ± 1.04a −11.83 ± 1.04a 1 1 Agg. entropy (kilobits) 2.5 min 136.12 ± 7.23 136.52 ± 10.25 110.08 ± 9.03b,2 F12,363 = 6.3 0.001 1 1 5 min 144.72 ± 12.55 138.54 ± 7.39 100.73 ± 11.52b,2 10 min 149.04 ± 7.251 141.56 ± 6.841 103.88 ± 9.52b,2 30 min 149.11 ± 7.481 133.58 ± 5.921 108.61 ± 9.11b,2 CC 148.54 ± 6.23 148.54 ± 6.23 148.54 ± 6.23a Max entropy (kilobits) 2.5 min 5.97 ± 0.03 5.96 ± 0.03 5.93 ± 0.02 F12,361 = 1.7 0.067 5 min 6.00 ± 0.03 6.00 ± 0.03 5.97 ± 0.03 10 min 5.99 ± 0.04 5.99 ± 0.04 5.98 ± 0.04 30 min 5.96 ± 0.03 5.98 ± 0.03 6.00 ± 0.02 CC 5.89 ± 0.03 5.89 ± 0.03 5.89 ± 0.03 1,2 Different numbers within a row indicate significant differences between injection methods within intervals for the given parameter. a−c Different letters within a column indicate significant differences between interval within injection method fort the given parameter; IF, intra-funicular injection and castration after 2.5 min (n = 35), 5 min (n = 33), 10 min (n = 33) or 30 min (n = 36); IT, intra-testicular injection after 2.5 min (n = 30), 5 min (n = 35), 10 min (n = 36) or 30 min (n = 35); SH, sham handling at an interval of 2.5 min (n = 35), 5 min (n = 16), 10 min (n = 19) or 30 min (n = 35); CC, castration without pain mitigation (control-castrated, n = 35). Fn,d denotes an F-test on n, numerator and d, denominator degrees of freedom for the effect of treatment. Frontiers in

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on (control-castrated, n = 35). Fn,d denotes an F-test on n, numerator and d, denominator degrees of freedom for the effect of treatment. Frontiers in Veterinary Science 09 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 6 Averages (±SE) of leg resistance movements recorded during castration. IF IT SH χ2 -test P Anaest. 28.44 ± 1.26a 23.85 ± 1.02a 14.32 ± 1.08b X22 = 172.4 <0.001 b,1 ab,1 b,2 Cast. 2.5 min 24.62 ± 2.13 30.26 ± 2.71 13.96 ± 1.77 <0.001 5 min 24.23 ± 2.58b,1 26.49 ± 2.63b,1 11.08 ± 2.58b,2 10 min 30.22 ± 3.85b,1 26.96 ± 2.65b,1 16.08 ± 3.34b,2 X22 = 132.4 ab,1 ab,1 b,2 30 min 29.51 ± 2.41 30.96 ± 3.00 15.69 ± 2.01 CC 41.06 ± 2.72a 41.06 ± 2.72a 41.06 ± 2.72a At anesthesia (Anaest.), a,b different letters within a row indicate significant differences between treatments, Padj ≤ 0.05; IF, intra-funicular injection (n = 195); IT, intra-testicular injection (n = 196); SH, sham anesthesia (n = 148). At castration (Cast.), 1.2 different numbers within a row indicate significant differences between methods within intervals, P ≤ 0.05. a−c Different letters within a column indicate significant differences between intervals within methods, P ≤ 0.05. IF, intra-funicular injection and castration after 2.5 min (n = 50), 5 min (n = 48), 10 min (n = 50) or 30 min (n = 49); IT, intra-testicular injection after 2.5 min (n = 50), 5 min (n = 49), 10 min (n = 49) or 30 min (n = 49); SH, sham handling at an interval of 2.5 min (n = 50), 5 min (n = 25), 10 min (n = 25) or 30 min (n = 48); CC, castration without pain mitigation (control-castrated, n = 49). χn2 denotes a chi-squared test on n degrees of freedom for the effect of treatment. piglets castrated 10 or 30 min after injection, compared to vocal parameters developed in the

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est on n degrees of freedom for the effect of treatment. piglets castrated 10 or 30 min after injection, compared to vocal parameters developed in the study, and are in line with piglets castrated after 2.5 or 5 min, and to piglets castrated previous results (11, 14, 23, 27). Thus, these methods seem to without anesthesia. be able to detect subtle differences in piglets’ acute responses to At 6 h post-procedure, piglets’ cortisol responses did not early-life interventions. differ significantly among treatments (F12,523= 0.7, P=0.721, Saliva-sampling of 3–4 day old piglets is difficult as they Table in Supplementary Table S1). At this point in time, produce relatively low amounts of saliva, and display less cortisol concentrations were not associated with piglet weight spontaneous chewing on cotton swabs during sampling than (F1,301= 0.2, P = 0.681). older piglets being experienced with solid feed intake. The present technique, developed in Coutant et al. (27), showed successful as it allowed gathering of enough saliva to perform Discussion the assays. In addition, samples taken on average 17 min after castration showed significantly higher cortisol concentrations We aimed in this study to assess piglets’ acute responses than baseline samples taken before, and post-procedural samples to two methods of injection of a local anesthetic, and surgical taken hours after the intervention, indicating that these samples castration following four different time intervals after the did record a robust response to the procedure. injection. Acute responses were evaluated based on piglet The present findings on saliva cortisol are, however, not in vocalizations, leg movements interpreted as resistance, line with previous studies showing a lowered cortisol response and saliva

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are, however, not in vocalizations, leg movements interpreted as resistance, line with previous studies showing a lowered cortisol response and saliva cortisol concentrations in samples obtained at to castration in piglets administered a local anesthetic prior two time points after castration (at approximately 17 min to castration compared to piglets castrated without anesthesia and approximately 6 h). Results showed no significant (10, 15, 33). Importantly, previous studies reporting these results difference between the two injection methods. Greater acute analyzed plasma cortisol, and used lidocaine as the anesthetic, responses were observed during castration performed 2.5 potentially in combination with an analgesic. To the best of and 30 min compared to 5 or 10 min after the injections. our knowledge, only one other study reported saliva cortisol Saliva cortisol concentrations in samples obtained from responses to castration following injection of procaine (27). This piglets castrated after injection with the local anesthetic study, using a similar design to the present one, showed no did not differ significantly from those of piglets castrated difference in cortisol response between piglets castrated with and without any anesthesia or sham handled. Below, these findings without anesthesia. A study on the development of the circadian are discussed in terms of methodology and in relation to pattern of saliva cortisol secretion in neonatal piglets reported animal welfare. a relatively high variation in cortisol concentrations from saliva Our results showed that, overall, administration of the sampled in piglets up to 3 days of age, with a stable circadian procaine-based local anesthetic reduced the acute responses pattern only observed from 10 days of age in males

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3 days of age, with a stable circadian procaine-based local anesthetic reduced the acute responses pattern only observed from 10 days of age in males (34). With of piglets to castration, as measured by the number of a relatively large individual variation in cortisol response, even foreleg movements interpreted as resistance and vocalization within a treatment, it cannot be excluded that the variation in characteristics (including number, duration, and intensity of saliva cortisol concentrations in piglets as young as 3 to 4 days calls), as compared to piglets castrated without anesthesia. of age may have reduced the possibility to detect differences These results are based on quantitative recording of resistance in acute responses, despite our relatively large sample size movements and automatic detection and characterization of (calculated based on previous plasma cortisol results). Further Frontiers in Veterinary Science 10 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 TABLE 7 Averages (±SE) of saliva cortisol concentrations (pg/mL) sampled at 17 min post-castration. IF IT SH F-test P 2.5 min 16,958 ± 1,100bc 17,518 ± 1,277ab 18,589 ± 1,255a F12,518= 2.5 0.003 bc ab ab 5 min 16,367 ± 1,196 16,291 ± 816 18,742 ± 1,750 10 min 19,132 ± 1,289ab,1 19,605 ± 1,482a,1 13,687 ± 1,289c,2 30 min 19,713 ± 1,325a,1 17,207 ± 936a,1/2 15,842 ± 1,053ab,2 c b CC 15,057 ± 858 15,057 ± 858 15,057 ± 858b 1,2 Different numbers within a row indicate significant differences between methods within intervals. a,b Different letters within a column indicate significant differences between intervals within methods, P ≤ 0.05. IF, intra-funicular injection and castration after 2.5 min (n = 50), 5 min (n = 48), 10 min (n = 50) or 30 min (n = 49); IT, intra-testicular

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≤ 0.05. IF, intra-funicular injection and castration after 2.5 min (n = 50), 5 min (n = 48), 10 min (n = 50) or 30 min (n = 49); IT, intra-testicular injection after 2.5 min (n = 50), 5 min (n = 48), 10 min (n = 49) or 30 min (n = 49); SH, sham handling at an interval of 2.5 min (n = 50), 5 min (n = 25), 10 min (n = 25) or 30 min (n = 49); CC, castration without pain mitigation (control-castrated, n = 49). Fn,d denotes an F-test on n numerator and d denominator degrees of freedom for the effect of treatment. studies investigating the plasma cortisol response of piglets authors concluded that herdsmen were able to effectively inject subjected to treatments similar to our study could therefore be a local anesthetic intra-testicularly, resulting in comparable relevant, although the general impact of the sampling method efficacy of anesthesia as reported in other studies involving should be carefully evaluated. We suggest that this way of trained veterinarians (13). Yet, this assessment was concluded saliva sampling is generally preferable as relatively non-invasive with no data or consideration of the piglets’ response to the compared to venepuncture. injection of the local anesthetic itself. Our study was not In relation to the interpretation of the cortisol results, designed to assess herdsmen’s ability to administer the local concern has been raised for years regarding the usefulness of anesthetic and subsequently perform castration, but rather to cortisol and other physiological indicators to inform about the study the acute response of the piglets, with approximation to affective component of pain, as these indicators may be more on-farm practice. A few adaptations to the castration routine related to the level of arousal induced by the procedure (35, 36). of

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cators may be more on-farm practice. A few adaptations to the castration routine related to the level of arousal induced by the procedure (35, 36). of the commercial farm had to be implemented for the sake Our results may therefore also reflect piglets’ stress response of data recording and standardization though. Piglets were following the combined procedures rather than the ability of brought outside of the farrowing room to be injected with the anesthetic to relieve acute pain only. If a longer duration the local anesthetic and castrated while in a castration bench, of the procedure (including injection of anesthetic, castration, whereas many farmers perform castration in the farrowing and the time interval in-between) is correlated with higher room, placing the piglets upside down or onto the herdsman’s stress responses, this suggestion could explain why, in our study, lap during both procedures. Although the impact of castration higher cortisol responses were observed in piglets castrated 10 or performed while the piglet is held upside down has recently 30 min after administration of the local anesthetic, compared to been reported (38), comparison of the potential implications of 2.5 or 5 min after, and compared to piglets non-anesthetized. It different restraining techniques for piglets’ responses to injection is, however, surprising that a similar pattern was not observed in of local anesthetics has not yet been investigated. We cannot sham handled piglets, where a 10 min interval between stays in exclude that fixation in the castration bench led to a different the castration bench led to a lower cortisol response than 2.5 or stress response compared to handling on the lap, however, we 5 min intervals. It is possible that a longer interval between stays

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response than 2.5 or stress response compared to handling on the lap, however, we 5 min intervals. It is possible that a longer interval between stays would expect that the placement of piglets in a calm, heated area in the bench resulted in a different shape of the curve of cortisol with littermates between injection and castration contributed response, the peak of which is not known. We also hypothesize to limiting the stress response compared to the typical on- that the lack of sensitivity of cortisol may have resulted in farm practice of placement in a cartwheel in the farrowing ceiling effects following handling alone, as already suggested by room. Similarly, application of the procedures in a calm previous authors (4, 11), especially in neonatal piglets, whose environment may limit the emotional social contagion of stress HPA-axis may be highly responsive (33). This suggestion is responses (among littermates, and with the sow) potentially supported, in our study, by comparable cortisol levels recorded happening when several litters are processed simultaneously in sham handled and castrated piglets. As a consequence of these in the farrowing room (39). Yet, further studies are required known concerns, the present study examined acute responses to to investigate these suggestions. Throughout the study, the injection of anesthetic and castration across several indicators same trained herdsman, hired for this specific role, performed including cortisol, vocalizations and resistance movements, the procedures on all experimental piglets. This was chosen thereby taking a multi-modal approach as suggested by Sheil and to strengthen accuracy, lower variation and thus increase the Polkinghorne, and Baysinger et al. (36, 37). power to detect differences when comparing

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to strengthen accuracy, lower variation and thus increase the Polkinghorne, and Baysinger et al. (36, 37). power to detect differences when comparing experimental To the best of our knowledge, only one other study treatments. Whether the results can translate directly into a farm has investigated the efficacy of a local anesthetic as pain setting is however not known. Further studies could investigate mitigating when administered in practice by farmers. The the efficacy of the administration of a local anesthetic when Frontiers in Veterinary Science 11 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 performed by farm employees, as part of their daily routines in is, however, poorly supported by our data as weight effects were the farrowing room. inconsistent and not recurrent across indicators. Yet, future Our study did not show a significant difference in acute studies could focus on the effects of anesthetic injection and responses of piglets exposed to the intra-testicular vs. intra- subsequent castration applied in piglets across a wider weight funicular injection methods. This result is in line with a previous range, e.g., across the legal age range of 2 to 7 days for surgical study performed in piglets under general anesthesia, and also castration after local anesthesia. comparing the two methods (40). It can be remarked, though, The effect of interval between injection of local anesthetic that the study by Haga & Ranheim (40) included a lidocaine- and castration is poorly investigated in the literature, and seem based drug, and involved piglets of 22 days of age, thus to only have been included in one study by Courboulay et al. significantly older than in the present study. Similarly, a recent (30). The study compared piglets’ response to castration

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study by Courboulay et al. significantly older than in the present study. Similarly, a recent (30). The study compared piglets’ response to castration 15 or study found no difference in vocalization and leg movements 30 min following administration of procaine, and found reduced in piglets administered 0.5 mL of procaine by an intra-funicular plasma cortisol response in piglets castrated 30 min compared injection, vs. 0.3 mL of the drug administered by intra-testicular to 15 min after injection (30). The interpretation of this result injection (23). The latter study involved the administration of in terms of the efficacy of the anesthetic can be questioned, different volumes of anesthetic for the two injection methods, though, as cortisol responses may not be accurate indicators evaluating the combined effects of the intra-testicular pressure of nociceptive pain, as already discussed. The authors of the resulting from the volume of liquid injected (20), the anesthetic study mentioned that previous studies have suggested an onset efficacy obtained from the dose of anesthetic administered, and of 20 min for procaine, and that most work evaluating the the pain response from the area of injection (21). Although efficacy of procaine used a 10 to 30 min interval between drug the present results did not reach statistical significance, our administration and surgery (30). Interestingly, guidelines given data showed close to 20% more resistance movements during to Danish farmers, and based on recommendations from the injection of the anesthetic in piglets subjected to an intra- manufacturers of Procamidor R Vet., recommend an interval of funicular injection vs. intra-testicular injection. The intra- 5 to 10 min. This timing is in line with our results, showing the funicular

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interval of funicular injection vs. intra-testicular injection. The intra- 5 to 10 min. This timing is in line with our results, showing the funicular injection is quite complex, requiring a deeper, more best efficacy after 5 to 10 min compared to the other intervals precise, injection into the testicle and toward the spermatic examined. This gap between the interval commonly used in cord. Precision of the injection is considered important for the literature and the interval recommended in practice could the efficacy of the anesthetic (41), while the speed of the drug be explained by potential discrepancies between pre-existing injection and the intra-testicular pressure may affect pain from knowledge on the low diffusing ability and onset of action of the injection (21). If one of the injection methods is to be applied, procaine (26), and the formula and characteristics of specific we therefore see an advantage of the use of the intra-testicular procaine-based products available commercially. Considering injection, as it appears to be easier and faster to perform the relatively low rate of diffusion of procaine (24), it cannot than the currently used intra-funicular injection type (23), with be excluded that an interval inferior to 5 min does not result comparable efficacy at castration. Yet, penetration of the testicle in sufficient neuronal blocking. In addition, a study using by the needle, and increased intra-testicular pressure resulting lidocaine injected intra-testicularly showed a 90% reduction in from the intra-testicular injection are considered painful (41). concentration of the product in the testes between 3 and 40 min In our study, injection of the local anesthetic, regardless of the post-injection, with a very low concentration in the spermatic method

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3 and 40 min In our study, injection of the local anesthetic, regardless of the post-injection, with a very low concentration in the spermatic method of injection, led to a rate of resistance movements close cord at 40 min, suggesting that a relatively long interval between to doubled compared to handling without injection. In addition, injection and castration would reduce the anesthetic efficacy the duration of the procedure was affected by piglet weight (41). Even with a potential lower rate of diffusion, a similar effect (results not shown), with longer procedures observed in lighter may be seen for procaine as of 30 min post-injection. An interval piglets, potentially because fixation of the testes and insertion of 5 to 10 min between the administration of procaine and of the needle at a correct angle may have been more difficult surgical castration therefore seems preferable as compared to 2.5 in small piglets. Weight and age effects were also observed in or 30 min. In practice, this interval also presents the advantage of the vocal responses of the piglets to the administration of the reducing the overall duration of handling, as compared to the 20 anesthetic and to castration, and in the saliva cortisol responses or 30 min used the literature, limiting the time spent away from as well. These effects may be related to a different fit in the the sow and home pen, and resulting stress and heat loss. castration bench for smaller piglets, and/or to weight-related Overall, our results showed that injection of procaine as characteristics, such as a smaller thoracic cage, potentially a local anesthetic allows some mitigation of piglets’ acute affecting the performance of vocalizations. It is, however, also responses to castration, but leads to significant responses

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igation of piglets’ acute affecting the performance of vocalizations. It is, however, also responses to castration, but leads to significant responses during possible that piglets’ experience of anesthesia and castration the injection of the anesthetic. Piglets in the present study did vary based on their weight, as the volume of anesthetic injected not receive any analgesic [as is mandatory in Denmark (19)] was fixed, and the dose of anesthetic administered per kg until after data collection was over, as our aim was to assess therefore decreased with an increase in weight. This possibility the efficacy of the local anesthetic, and because the comparison Frontiers in Veterinary Science 12 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 of the different intervals would have been confounded by the Funding use of an analgesic given at a specific time before castration. In practice, however, analgesics are administered only few minutes This study was commissioned and funded by the Danish before castration or at the same time, and are therefore likely Veterinary and Food Administration (DVFA) as part of the only efficient in mitigating post-castration pain and not the acute agreement with Aarhus University on research-based policy response to castration, as examined in the current study (42). support, as well as by Aarhus University. The funders had no role In the future, the use of local anesthetics prior to piglet in study design, data collection and analysis, decision to publish, castration in Europe may be further improved by the approval or preparation of the manuscript. of other local anesthetics than procaine, such as lidocaine, which may have an increased efficacy at castration compared to procaine (23). Yet, in both cases, the procedure requires

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procaine, such as lidocaine, which may have an increased efficacy at castration compared to procaine (23). Yet, in both cases, the procedure requires needle Acknowledgments injection and extra handling, which, in our study, resulted in clear stress responses, even in piglets not exposed to any tissue The authors thank the Danish pig farmer Niels Aage Arve damage but kept in a castration bench. and his staff for providing piglets and facilities for the study In conclusion, while our study provided further insights and for their flexibility. Technicians from Aarhus University into the administration of a procaine-based local anesthetic involved in the project are acknowledged for their hard work and prior to piglet castration, and has provided results allowing a dedication. We also thank Karoline V. Dohrmann for her help refinement of the procedure, we cannot oversee that castration in the data analysis, Hanne Kongsted, Aarhus University, for her with local anesthesia remains a welfare concern. This concern qualified advice in the design of the study, and Torben Larsen, has been raised previously [e.g., (11, 12, 27)], and is in line Aarhus University, for helping developing the saliva sampling with the ‘European Declaration on alternatives to surgical method and conducting the cortisol assays. castration in pigs’, a document produced after consultation of various stakeholders, judging castration with anesthesia as Conflict of interest an unsuitable alternative (16). Thus, further research into the practical application of alternatives to surgical castration or The authors declare that the research was conducted in alternatives to the use of local anesthesia is still recommended. the absence of any commercial or financial relationships that could be construed as a

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lternatives to the use of local anesthesia is still recommended. the absence of any commercial or financial relationships that could be construed as a potential conflict Data availability statement of interest. The reviewer ND declared a past collaboration with one of The original contributions presented in the study are the authors MH to the handling Editor. included in the article/Supplementary material, further inquiries can be directed to the corresponding author. Publisher’s note Ethics statement All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated The animal study was reviewed and approved by the Danish organizations, or those of the publisher, the editors and the Animal Experiments Inspectorate. Written informed consent reviewers. Any product that may be evaluated in this article, or was obtained from the owners for the participation of their claim that may be made by its manufacturer, is not guaranteed animals in this study. or endorsed by the publisher. Author contributions Supplementary material The study was designed by the project group involving The Supplementary Material for this article can be MH, JM, MC, and MK with advice from LF. MK developed found online at: https://www.frontiersin.org/articles/10.3389/ the anesthesia injections and trained the herdsman. MC was in fvets.2022.1009858/full#supplementary-material charge of the data collection, data processing, and data analysis SUPPLEMENTARY VIDEO S1 with supervision from JM, LF, and MH. LF performed the Video of the intra-testicular injection procedure. sample size calculations. MC drafted the manuscript. All authors SUPPLEMENTARY DATASET S1 contributed to the article and approved the submitted version. Dataset used for

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tions. MC drafted the manuscript. All authors SUPPLEMENTARY DATASET S1 contributed to the article and approved the submitted version. Dataset used for statistical analysis. Frontiers in Veterinary Science 13 frontiersin.org Coutant et al. 10.3389/fvets.2022.1009858 SUPPLEMENTARY FIGURE S1 SUPPLEMENTARY TEXT S1 The adapted castration bench used during injection, castration and Description of the parameters used for calibration of the band limited sham handling. Picture of the bench (1a), with microphone for energy automatic detection of calls in the Raven Pro software. vocalisation recording (1b), camera for recording of the resistance movements (1c), and soft layer matt ensuring the fitting of each piglet SUPPLEMENTARY TABLE S1 during the procedures (2). Saliva cortisol concentrations recorded for each sampling time. References 1. Bonneau M, Le Denmat M, Vaudelet JC, Veloso Nunes JR, Mortensen AB, 17. De Briyne N, Berg C, Blaha T, Temple D. Pig castration: will the Mortensen HP. Contributions of fat androstenone and skatole to boar taint EU manage to ban pig castration by 2018? Porc Heal Manag. (2016) 2:1– : 1. sensory attributes of fat and pork meat. Livest Prod Sci. (1992) 32:63– 11. doi: 10.1186/s40813-016-0046-x 80. doi: 10.1016/S0301-6226(12)80012-1 18. NYT fra Danmarks Statistik. Unchanged Pig Population Of 12.7 Million Pigs. 2. Marx G, Horn T, Thielebein J, Knubel B, Von Borell E. Analysis (2020). Available online at: https://www.dst.dk/Site/Dst/Udgivelser/nyt/GetPdf. of pain-related vocalization in young pigs. J Sound Vib. (2003) 266:687– aspx?cid=30377 (accessed June 10, 2022). 98. doi: 10.1016/S0022-460X(03)00594-7 19. Danish Agriculture and Food Council. Retningslinjer for kurser i 3. Taylor AA, Weary DM. Vocal responses of piglets to castration:

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