Proposed multidimensional pain outcome methodology to demonstrate analgesic drug efficacy and facilitate future drug approval for piglet castration

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Animal Health Research Proposed multidimensional pain outcome Reviews methodology to demonstrate analgesic cambridge.org/ahr drug efficacy and facilitate future drug approval for piglet castration Review Angela Baysinger1 , Sherrie R. Webb2, Jennifer Brown3, Johann F. Coetzee4, Cite this article: Baysinger A et al (2021). Sara Crawford5, Ashley DeDecker6, Locke A. Karriker7, Monique Pairis-Garcia8, Proposed multidimensional pain outcome methodology to demonstrate analgesic drug Mhairi A. Sutherland 9 and Abbie V. Viscardi4 efficacy and facilitate future drug approval for 1 piglet castration. Animal Health Research Merck Animal Health, 35500 W 91st Street, DeSoto, Kansas 66018, USA; 2American Association of Swine Reviews 22, 163–176. https://doi.org/10.1017/ Veterinarians, 830 26th Street, Perry, Iowa 50220, USA; 3Prairie Swine Centre, 2105 8th Street East, Saskatoon, S1466252321000141 Saskatchewan, S7H 5N9, Canada; 4Department of Anatomy and Physiology, College of Veterinary Medicine, Coles Hall, Kansas State University, Manhattan, Kansas 66506, USA; 5National Pork Board, 1776 NW 114th Street, Clive, Received: 19 April 2021 Iowa 50325, USA; 6Smithfield, 4134 US 117, Rose Hill, North Carolina 28458, USA; 7Department of Veterinary Revised: 30 August 2021 Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa Accepted: 30 September 2021 First published online: 3 December 2021 50011, USA; 8North Carolina State University, College of Veterinary Medicine, 1060 William Moore Drive, Raleigh, North Carolina 27607, USA and 9Beef + Lamb New Zealand, 154 Featherston St., Wellington, New Zealand Key words: Piglet; pain; castration; biomarkers; endpoints; Abstract mitigation Castration of male piglets in the United States is

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ington, New Zealand Key words: Piglet; pain; castration; biomarkers; endpoints; Abstract mitigation Castration of male piglets in the United States is conducted without analgesics because no Author for correspondence: Food and Drug Administration (FDA) approved products are labeled for pain control in Angela Baysinger, swine. The absence of approved products is primarily due to a wide variation in how pain E-mail: angela.baysinger@merck.com is measured in suckling piglets and the lack of validated pain-specific outcomes individually indistinct from other biological responses, such as general stress or inflammation responses with cortisol. Simply put, to measure pain mitigation, measurement of pain must be specific, quantifiable, and defined. Therefore, given the need for mitigating castration pain, a consortium of researchers, veterinarians, industry, and regulatory agencies was formed to identify potential animal-based outcomes and develop a methodology, based on the known scientific research, to measure pain and the efficacy of mitigation strategies. The out- come-based measures included physiological, neuroendocrine, behavioral, and production parameters. Ultimately, this consortium aims to provide a validated multimodal methodology to demonstrate analgesic drug efficacy for piglet castration. Measurable outcomes were selected based on published studies suggesting their validity, reliability, and sensitivity for the direct or indirect measurement of pain associated with surgical castration in piglets. Outcomes to be considered are observation of pain behaviors (i.e. ethogram defined behaviors and piglet grimace scale), gait parameters measured with a pressure mat, infrared thermography of skin temperature of the cranium and periphery of the eye, and blood

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ace scale), gait parameters measured with a pressure mat, infrared thermography of skin temperature of the cranium and periphery of the eye, and blood biomarkers. Other measures include body weight and mortality rate. This standardized measurement of the outcome variable’s primary goal is to facilitate consistency and rigor by developing a research methodology utilizing endpoints that are well-defined and reliably measure pain in piglets. The resulting methodology will facilitate and guide the evaluation of the effectiveness of comprehensive analgesic interventions for 3- to 5-day-old piglets following surgical castration. Introduction In the United States, surgical castration is commonly performed on commercial pig produc- tion farms within the first 3–5 days of life to prevent the accumulation of boar taint and agon- © The Author(s), 2021. Published by istic behaviors (Rault et al., 2011), and the procedure is typically performed without Cambridge University Press. This is an Open administration of an analgesic or anesthetic. Current evidence demonstrates that neonates Access article, distributed under the terms of experience pain and, if left untreated, can result in permanent neuroanatomic or behavioral the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), changes (Mellor and Gregory, 2003; Sneddon et al., 2014). Thus, pain management is essential which permits unrestricted re-use, distribution for young animals. and reproduction, provided the original article The castration of piglets is recognized as a significant welfare concern, and guidelines for is properly cited. the use of analgesia and or anesthesia have been developed and implemented in the EU and Canada (National Farm Animal Care Council, 2014; European Commission,

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of analgesia and or anesthesia have been developed and implemented in the EU and Canada (National Farm Animal Care Council, 2014; European Commission, 2017). The European Commission reported pain intervention methods via a survey conducted from June 2016 to October 2016. The use of anesthesia and/or analgesia for piglet castration found the mixed application of pain mitigation strategies focusing on the concerns of animal https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 164 Angela Baysinger et al. welfare, economic sustainability, practical application of the indirect measurement of pain associated with surgical castration method, environmental impact, and human health concerns. in piglets. Outcomes to be considered are observation of pain The application of pain mitigation for piglet castration in the behaviors (i.e. ethogram defined behaviors and piglet grimace EU ranges from gaseous or injectable anesthesia (CO2/O2, scale), gait parameters measured with a pressure mat, infrared ketamine, azaperone, isoflurane), local anesthesia (lidocaine), thermography (IRT) of skin temperature of the cranium and per- non-steroidal anti-inflammatory drugs (flunixin, meloxicam, iphery of the eye, and blood biomarkers. Other measures include metamizol), or various combinations for anesthetic/analgesic body weight and mortality rate. effect (European Commission, 2017). However, there was no The information herein supports the inclusion of multiple consensus on the best method for animal welfare with the endpoints to evaluate their validity and reliability for demonstrat- practicality of on-farm ease of use. ing control of pain in piglets undergoing surgical castration. For Conversely, US farmers and veterinarians are currently limited endpoint

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ase of use. ing control of pain in piglets undergoing surgical castration. For Conversely, US farmers and veterinarians are currently limited endpoint measures not included for evaluation, the consortium’s in addressing this challenge due to the lack of analgesic or anes- decision was based on a lack of validated processes or practicality thetic drugs in the United States approved explicitly with an indi- of standardizing the on-farm application to justify use within the cation for the control of pain in swine. The lack of on-farm proposed methodology [i.e. vocalization and nociceptive with- analgesic use may be due to a limited ability to make solid recom- drawal response (Sheil and Polkinghorne, 2020)]. These end- mendations for effective pain management strategies by veterinar- points may ultimately be used in studies to demonstrate ians, the added cost, time, and effort involved with training substantial evidence of effectiveness, one component in the US caretakers and implementing pain management protocols Food and Drug Administration’s approval process of a pain miti- on-farm, in addition to a lack of US Food and Drug gation drug. This paper aims to describe a multidimensional Administration (FDA)-approved analgesics labeled with an indi- methodology to directly or indirectly assess behavioral, physio- cation for the control of pain for swine (Rault et al., 2011; logical, and neuroendocrine changes in piglets associated with Tuyttens et al., 2011; O’Connor et al., 2014). The US Food and pain resulting from surgical castration. This methodology will Drug Administration (FDA) has oversight of approval and safety use multiple outcome variables to, in summation, demonstrate of all products used in animals, including those animals used for analgesic efficacy in

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y use multiple outcome variables to, in summation, demonstrate of all products used in animals, including those animals used for analgesic efficacy in the post-surgically castrated piglet, satisfying human consumption. Pharmaceutical companies must use meth- the FDA efficacy requirement of a product. ods to assess animal responses that are well-defined and reliable to demonstrate products’ efficacy and safety when seeking FDA new Pain definition drug approval or label amendments. Veterinarians can prescribe FDA-approved products for extra-label purposes under the Pain is defined as an unpleasant sensory and emotional experi- Animal Medicinal Use Clarification Act (AMDUCA). However, ence associated with actual or potential tissue damage (IASP they must have reliable data to demonstrate the efficacy and safety Subcommittee on Taxonomy, 1979). The emotional component of food products derived from animals treated with a drug of pain is an affective state that cannot be directly measured. approved for use in other species. The sensory component of the pain response involves nocicep- A literature review reveals a lack of consistent data related to tion, including the detection, transduction, and transmission of the efficacy of pain mitigation products primarily due to the noxious stimuli by the peripheral and central nervous systems. lack of uniform testing methodology and protocols (O’Connor Collectively the sensory component of the pain response produces et al., 2016). This, in turn, makes evaluating the efficacy of pain behavioral, physiological, and neuroendocrine responses. mitigation interventions complex and has prevented consensus on best practices for pain relief (Bateson, 1991). Lack of consistent Evidence of pain associated with castration protocols creates

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revented consensus on best practices for pain relief (Bateson, 1991). Lack of consistent Evidence of pain associated with castration protocols creates difficulty for pharmaceutical companies to sub- mit new product approvals or label claims related to pain, veter- Human beings and other vertebrate mammals, such as pigs, have inarians to confidently prescribe products for extra-label use, similar neuroanatomical structures associated with pain researchers to reliably assess pain and potential mitigation strat- perception [e.g. nociceptors, a pathway connecting nociceptors egies, and pig farmers to make future business decisions regarding to the brain, and brain structures to process pain analogous to animal welfare. the human cerebral cortex (Bateson, 1991)] and the capacity for Given the need for mitigating castration pain, a consortium of animals to experience pain is well-described. It was long believed researchers, veterinarians, industry, and regulatory agencies was that neonates were incapable of experiencing pain or did so less formed to identify potential animal-based outcomes and develop intensely than adults because of their immature nervous system a methodology based on the known scientific research, to measure and lack of specific behavioral signs (Bateson, 1991). For many pain and the efficacy of mitigation strategies. The consortium’s years, this concept was translated to veterinary medicine and live- goal is to improve pig welfare on-farm by effectively controlling stock production practices as producers and veterinarians provided pain associated with on-farm surgical procedures, such as castra- little to no analgesics or anesthetics to animals if painful proce- tion, in a manner that is safe for the animal and the consumer and dures were conducted at a

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no analgesics or anesthetics to animals if painful proce- tion, in a manner that is safe for the animal and the consumer and dures were conducted at a young age. Evidence now suggests neo- is compliant with US regulation. This evaluation’s primary goal is nates may have a heightened pain experience, and untreated pain to facilitate consistency and rigor by developing a research meth- could result in permanent changes to pain sensitivity and neuroa- odology utilizing validated endpoints that are well-defined and natomic or behavioral abnormalities, making pharmaceutical pain reliably measure pain in piglets. The resulting methodology, management even more critical for young animals undergoing a with validated outcomes, will facilitate and guide the evaluation surgical procedure (Mellor and Gregory, 2003; Sneddon et al., of the effectiveness of comprehensive analgesic interventions for 2014). 3- to 5-day-old piglets following surgical castration. Surgical castration of piglets causes acute pain, as evidenced by Measurable outcomes were selected based on previous studies behavior and physiologic changes. Piglets display several pain suggesting their validity, reliability, and sensitivity for the direct or behaviors post-operatively in response to the surgical castration https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press Animal Health Research Reviews 165 procedure, including an increase in stiffness, trembling, scratching • Differences between SHAM and CAST can be interpreted the rump, tail wagging, awake inactive or restless behaviors, and as the effects of pain or tissue damage rather than environ- spending more time isolated from littermates (Viscardi and mental or other non-pain factors. Turner, 2018a). Piglets also produce

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rather than environ- spending more time isolated from littermates (Viscardi and mental or other non-pain factors. Turner, 2018a). Piglets also produce distinct high-frequency voca- • Differences between SHAM and SHAM + TRT can deter- lizations associated with the castration procedure (Weary et al., mine if the intervention causes a difference in the tested 1998; Leidig et al., 2009) and spend more time in contact with variable in the absence of pain and tissue damage (e.g. seda- the sow, which has been suggested to produce analgesic-like effects tive effects). by promoting endorphin release in neonates (Field and Goldson, (b) To validate the measurable outcomes in this protocol, the 1984; Blass, 1994). Behavioral alterations associated with castration authors would use buprenorphine (0.04 mg kg−1) (Viscardi persist beyond 24 h, with some abnormal behaviors present 4 days and Turner, 2018a), administered intramuscularly, before sur- later (Hay et al., 2003; Llamas Moya et al., 2008). gical castration as a ‘gold standard’ intervention. While this Surgical castration also causes a physiologic response. Piglets opioid drug is not approved for use in swine or other food show an increase in heart and respiration rate with higher animals, buprenorphine was chosen as the gold standard blood cortisol, lactate, and adrenocorticotropin hormone concen- because of its potency as an analgesic drug and ability to trations after castration (White et al., 1995; Prunier et al., 2006; bind to opioid receptors in the brain, spinal cord, and periph- Kluivers-Poodt et al., 2012). Peripheral vasoconstriction caused ery to suppress pain signal transmission (Chahl, 1996). by activation of the sympathetic nervous system results in a Furthermore, buprenorphine has proven to be effective

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signal transmission (Chahl, 1996). by activation of the sympathetic nervous system results in a Furthermore, buprenorphine has proven to be effective at decrease in cutaneous temperature, which, using an IRT camera, reducing pain and lameness in piglets (Hermansen et al., has been observed in piglets after castration (Bates et al., 2014). 1986; Meijer et al., 2015) and alleviating behavioral pain indi- Surgical castration can also result in a decreased growth rate lead- cators and facial grimacing with no sedative effect for more ing to production losses (McGlone et al., 1993; Kielly et al., 1999; than 24 h in 5-day-old piglets following castration (Viscardi Malavasi et al., 2006). Published studies that have examined anal- and Turner, 2018a). gesia and local anesthetic use in piglets undergoing surgical cas- (c) The research protocol would use a 2 × 2 factorial design with tration are summarized in Table 1. piglet (within litter) as the experimental unit. The treatment will be applied to the individual male piglet, and each treat- ment will be represented within a litter at least once. If Development of a research protocol more than four male piglets in the litter meet the enrollment The information presented herein serves as a support for develop- criteria, a treatment will be assigned to each additional piglet ing a research protocol with the primary objective of determining using a treatment randomization list. Using piglet as the the validity and reliability of endpoints for use in demonstrating experimental unit and blocking/controlling allocation to efficacy for FDA product approval. The endpoints could be treatment based on the litter (controlling for sow effect) used to evaluate a drug’s efficacy for controlling pain associated helps control inter-litter

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eatment based on the litter (controlling for sow effect) used to evaluate a drug’s efficacy for controlling pain associated helps control inter-litter variability (e.g. litter size, sow milk with castration of 3-to 5-day-old piglets. The 3-to-5 day-old piglet yield, and piglet sex ratio) (Festing, 2006; Lazic and is targeted due to the standard age at which piglets are castrated in Essioux, 2013). Many previous studies (Table 1) evaluating commercial production within the United States being approxi- pain reduction for castrated piglets have used piglets as the mately 3 days of age, and foundational research in pain mitigation experimental unit. As the experimental unit, the piglet pro- has targeted the 3-day-old piglet (Sutherland et al., 2010; vides more data for, and higher confidence in the power ana- Sutherland et al., 2012). Through a review of the literature lysis calculations used to determine the research protocol’s describing pain expression measures in piglets, multiple outcome sample size. measures were identified as the best candidates for further valid- ation and inclusion in future efficacy trials. This paper’s multidi- A potential criticism for using piglet as the experimental unit mensional outcomes include observation and scoring of pain rather than the litter is that having all four treatments represented exhibition or behavior, physiological biomarkers, and automated within a litter could have a confounding influence on piglet physical measurements. behavior and activity. Emotional contagion has been observed in groups of pigs when exposed to positive or negative treatments (Reimert et al., 2017; Yun et al., 2019). For this concern to be Protocol study design addressed, using litter as the experimental unit in the research In the development

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7; Yun et al., 2019). For this concern to be Protocol study design addressed, using litter as the experimental unit in the research In the development of a standardized protocol to determine anal- protocol, all uncastrated male and female littermates would gesic efficacy, the following protocol was outlined to provide con- need to be removed from the farrowing box so that only treated sistency in trial design and standardize the measurement of males remained, creating an unnatural environment. Having outcomes: uncastrated female littermates present in the environment is important when validating outcomes and evaluating intervention (a) Four primary treatment groups will be used in the research efficacy, as these animals will always be present in typical com- protocol: (1) sham castration (SHAM), (2) sham castration mercial production. with the intervention (SHAM + TRT), (3) surgical castration with placebo control (CAST), (4) and surgical castration with Measurable outcomes intervention (CAST + TRT). In the absence of other con- founding factors, four treatment groups will help determine Validity, reliability, and sensitivity are vital characteristics that the following (Weary et al., 2006; Ison et al., 2016): should be considered when choosing measurable outcomes for • Differences between CAST and CAST + TRT treatments practical pain assessment (Ison et al., 2016). Each measurable out- can be interpreted as the efficacy of the intervention to come identified for use in the proposed protocol has been cate- mitigate pain. gorized as either primary or ancillary. Primary outcomes were https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 166 Angela Baysinger et al. Table 1. Results of studies that have examined analgesia and local

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21000141 Published online by Cambridge University Press 166 Angela Baysinger et al. Table 1. Results of studies that have examined analgesia and local anesthetic use to alleviate pain in surgically castrated piglets Reference Piglet age Analgesia Local anesthesia Measured outcome Drug efficacya McGlone and 2 week – 2% lidocaine hydrochloride Nursing behavior Yes (2 week Hellman (1988) and intratesticular pigs); No (7 7 week week pigs) Maintenance behavior No (eating, drinking, standing, lying) McGlone et al. 8 week 22 mg kg−1 aspirin oral or – Pain behavior No (1993) 0.11 mg kg−1 butorphanol IV White et al. (1995) 1–24 d – 2% lidocaine hydrochloride Vocalization Yes intratesticular Heart rate Yes Horn et al. (1999) 10–14 d – 2% lidocaine hydrochloride Vocalization No intratesticular Resistant movements Yes Haga and Ranheim 22 d – 2% lidocaine hydrochloride Mean arterial pressure Yes (2005) intratesticular and intrafunicular Pulse rate Yes EEG Yes Zankl et al. (2007) 4–6 d – Procaine hydrochloride 2% Cortisol No lidocaine hydrochloride intratesticular Leidig et al. (2009) 3–4 d – 10 mg procaine intratesticular Pain behavior Yes −1 Keita et al. (2010) <1 week 0.4 mg kg meloxicam IM – Pain behavior Yes Cortisol Yes Sutherland et al. 3d – Cetacaine or Tri-solfen topical Vocalizations No (2010) Cortisol No Leukocyte count No Lying behavior No −1 Hansson et al. 3–4 d 1.0 mg meloxicam IM 10 mg mL lidocaine +5 μg Pain behavior Yes (M only) (2011) mL−1 epinephrine intratesticular Vocalization Yes (L only) Ear temperature Yes (L only and M + L) Schmidt et al. 5–7 d 0.4 mg kg−1 Meloxicam IM – Nursing behavior Yes (2012) Active behavior No Sutherland et al. 3d 2.2 mg kg−1 flunixin meglumine IM – Cortisol No (2012) C-Reactive Protein

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cam IM – Nursing behavior Yes (2012) Active behavior No Sutherland et al. 3d 2.2 mg kg−1 flunixin meglumine IM – Cortisol No (2012) C-Reactive Protein No Substance P No Vocalization No Pain behavior No Activity No −1 Kluivers-Poodt 2–5 d 0.4 mg kg meloxicam IM 2% lidocaine intratesticular Pain behavior Yes (M only and et al. (2013) M + L) Bates et al. (2014) 5d meloxicam transmammary – Cortisol Yes Peripheral vasoconstriction Yes Viscardi et al. 5d 0.4 mg kg−1 meloxicam IM EMLA topical cream Pain behavior No (2017) (lidocaine 2.5%, prilocaine 2.5%) Activity level No Facial grimacing No (Continued ) https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press Animal Health Research Reviews 167 Table 1. (Continued.) Reference Piglet age Analgesia Local anesthesia Measured outcome Drug efficacya Viscardi and Turner 5d 0.04 mg kg−1 buprenorphine IM – Pain behavior Yes (2018a) Activity level Yes Facial grimacing Yes Vocalization No −1 Viscardi and Turner 5d 0.4 mg kg meloxicam IM or 1.0 mg – Pain behavior No (2018b) kg−1 meloxicam IM or 6.0 mg kg−1 ketoprofen IM Activity level No Facial grimacing No Vocalization No Burkemper et al. 3–5 d 1.0 mg kg−1 meloxicam oral – Pain behavior No (2019) Yun et al. (2019) 5d 0.4 mg kg−1 meloxicam IM or 0.5 ml 2% lidocaine Pain behavior No isoflurane (1.5%) with meloxicam IM intratesticular IV = intravenous; EEG = electroencephalogram; IM = intramuscular; M = meloxicam; L = lidocaine; CRP = C-reactive protein. aDrug efficacy is defined as successful (yes) if treatment administration minimized outcome measures or significantly minimized deviations to behavioral and physiological indicators of pain (Dzikamunhenga et al., 2014). defined as measures directly related to clinical

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minimized deviations to behavioral and physiological indicators of pain (Dzikamunhenga et al., 2014). defined as measures directly related to clinical signs of pain as wagging (Hay et al., 2003), all of which indicate pain. Also, nurs- recorded in the published literature and are repeatable when the ing (McGlone et al., 1993) and social behaviors decrease post- proper methodology is used. Ancillary outcomes were defined procedure, and greater duration and intensity of fighting with as newer methodologies in the published literature directly related pen mates can also be observed (Hay et al., 2003; Llamas Moya to clinical signs of pain or indirectly related to clinical signs of et al., 2008; Leidig et al., 2009; Sutherland et al., 2010; pain and support the primary outcomes. A combination of mul- Sutherland et al., 2012). Lastly, a reduction in activity levels (i.e. tiple outcome variables may provide a robust evaluation of a increased inactivity) is a commonly noted behavioral response castrated piglet’s pain profile and the tested intervention efficacy. post castration (McGlone et al., 1993; Hay et al., 2003; Llamas A brief justification is provided for each measurable outcome Moya et al., 2008). for the proposed protocol. Additionally, evidence for when and Among studies that have recorded piglet activity and pain how each outcome should be measured is provided. A recom- behavior following castration, current behavior sampling method- mended sample size is also included for each outcome based on ologies have not been validated, and there is considerable vari- previous literature. With the diversity in the methodology used ation in the methodologies used (O’Connor et al., 2014). For in the existing literature, performing a complete power analysis example, when

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ology used ation in the methodologies used (O’Connor et al., 2014). For in the existing literature, performing a complete power analysis example, when assessing activity and maintenance behaviors in proved difficult. castrated piglets, McGlone et al. (1993) used continuous live observations for 6 h following castration and found reduced suck- ling and standing and increased lying time in castrated piglets Outcomes variables compared to non-castrated littermates. In comparison, Hay Pain behavior and activity tracking et al. (2003) also used live observation but utilized scan sampling Diagnosis of pain in animals is a complicated process due to at 10-minute intervals and found that castrated piglets had unique individual experiences with pain (Gaynor and Muir, reduced activity at the udder and spent more time inactive (i.e. 2009) and differences in pain tolerance and reaction between lying, sitting, or standing). Similarly, Llamas Moya et al. (2008) breeds, sex, age, pain duration, procedure type, and stimulus recorded piglet behavior for 3 h in the afternoon on the day of severity (Matthew, 2000). From a scientific standpoint, a behav- castration using 3-minute scan samples and found that castrated ioral assessment may be the most practical endpoint to assess pigs spent less time walking than sham-handled piglets. These pain in livestock production systems as it is the most direct assess- few examples highlight the wide variation in behavioral method- ment of an animal’s welfare. However, given that the sensory ologies used to assess castration pain. Be that as it may, all these component of pain is also associated with a neuroendocrine studies were able to quantify deviations in piglet behavior and physiological response in the animal, measuring neuroendo-

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iated with a neuroendocrine studies were able to quantify deviations in piglet behavior and physiological response in the animal, measuring neuroendo- post-procedure. crine and physiological outcomes is essential to confirm and sup- To address limitations associated with behavioral methodolo- port behavioral endpoints and outcomes. A multimodal approach gies used in castration studies, validating the accuracy of different is the proposed best practice because these outcomes’ totality behavioral sampling methodologies is needed. Also, developing a reveals an overall assessment of pain. piglet behavioral pain scale using behaviors that can be reliably Piglets castrated without anesthetics or analgesics demonstrate measured by multiple people and are sensitive to detecting pain several behavioral changes indicative of pain, as demonstrated by would be valuable. Given the lack of validated behavior method- increased pain-specific behaviors post-procedure and deviations ologies for pigs, the authors recommend the following method- in maintenance behavior and piglet activity. ology for all behavioral observations, regardless of how behavior Piglets castrated without pain control demonstrate increased data are collected (i.e. manual or automated methods). Piglets trembling, rump scratching, prostration, and tail jamming/ should be individually identified and filmed in their home pens https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 168 Angela Baysinger et al. continually over a 24-hour period for three days. Identification of response was reduced when calves were given an injection of the individual piglets would be via a unique identifier consisting local anesthetic into the testes and scrotum 7 minutes before cas- of a number and a

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he individual piglets would be via a unique identifier consisting local anesthetic into the testes and scrotum 7 minutes before cas- of a number and a letter, with the letter randomly assigned and tration. Also, SHAM procedures only caused a minor change in represents the piglet’s treatment. The number identifier is placed eye temperature compared to the painful procedures (Stewart on both back legs and the letter on the piglet’s forehead and back et al., 2010). These results support that IRT can be used as an using a black permanent marker. The markings must be refreshed objective measure of the animal’s response to a painful stimulus. twice daily during study procedures. The video should be cap- In pigs, IRT has been used to detect disease (Cook et al., 2015), tured digitally, and behavioral software programming utilized to stress (Schaefer et al., 2002; Magnani et al., 2011; Sutherland capture data. et al., 2015), and pain (Bates et al., 2014). Specific to pain, Automated technologies for measuring piglet activity at castra- Bates et al. (2014) found that pigs given analgesia before castra- tion have not been used to date but have the potential to provide tion had greater cranial skin temperatures, as measured using more reliable data and a significant reduction in labor IRT, than castrated pigs that did not receive pain relief. (Nasirahmadi et al., 2017). For example, accelerometers (ped- A change in blood flow during stress can be detected using ometers), radio-frequency identification, and visual tracking sys- IRT on specific body regions such as the eye in cattle (Stewart tems have been used to assess activity in older pigs and other et al., 2010) and horses (Bartolomé et al., 2013), the comb in livestock species (Currah et al., 2009; Kashiha et al.,

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ctivity in older pigs and other et al., 2010) and horses (Bartolomé et al., 2013), the comb in livestock species (Currah et al., 2009; Kashiha et al., 2013; poultry (Moe et al., 2012), and head, snout, vulva, and teats in Kulikov et al., 2014). Compared to most tracking devices, the pigs (Bates et al., 2014; Sutherland et al., 2015). Bates et al. small piglet size presents a challenge, but problems associated (2014) measured changes in skin surface temperature using IRT with these technologies are decreasing with continuing improve- on the top of the cranium, ears, and snout in pigs castrated ments in their size, accuracy, and affordability. Therefore, while with and without analgesia and found that cranial skin tempera- automated systems may help identify effective pain control mea- ture was the most reliable anatomical location for assessing pain sures, they are not currently recommended for efficacy evaluation in piglets in response to castration due to significant temperature of pain mitigation strategies at castration as they require further variability in other locations. Furthermore, Sutherland et al. validation for use in piglets. (2015) investigated the potential for IRT as a non-invasive meas- Piglet behavior and activity observations will be collected using ure of stress in pigs and compared whether the eye or the snout continuous behavior sampling on Day 0 (day before castration, or was a more sensitive region to measure stress. Temperature baseline), Day 1 (0–24 h post-treatment administration), and Day changes suggested that the eye may be a more reliable area to 2 (24–48 h post-treatment administration). Behaviors to be mea- assess stress than the snout in pigs. The literature shows that sured are defined in the ethogram in Table 2. Based on

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stration). Behaviors to be mea- assess stress than the snout in pigs. The literature shows that sured are defined in the ethogram in Table 2. Based on previous IRT can be used to measure animal pain; however, further valid- work outlined in Table 1, the authors recommend the sample size ation of this technique’s methodology is needed, including the for behavioral observations is 10–20 piglets/treatment. Before trial most reliable anatomical site for assessing pain. initiation, observers must be trained to ensure behavioral data are Based on data from previous studies (Stewart et al., 2010; Bates recorded consistently. To ensure inter-observer reliability, a 2-h et al., 2014), assuming a 5% significance level, and 80% power, the subset of continuous video is selected at random, observed, and authors recommend a sample size of 20 piglets/treatment based compared until 90% accuracy is achieved (Ross et al., 2019). on an expected difference in cranial skin temperature of one The ethogram defined in Table 2 should be used for all obser- degree Celsius and a standard error of the mean of 0.1 degrees vational data collected. All behaviors (Table 2) should be collected Celsius at 12 h after castration, assuming data are analyzed continuously by two trained observers utilizing software (e.g. using 2-sample t-tests. Observer XT program) or any data collection, coding, and ana- lysis tool that is easily validated with a time and date stamp. Cortisol Videos are randomized and assigned to observers who are blind Cortisol is a biomarker commonly used to measure stress and to treatment and time points. Inter-observer reliability is assessed pain in animals. Several painful husbandry procedures (e.g. cas- before data collection and at three-time points during the behav- tration,

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s assessed pain in animals. Several painful husbandry procedures (e.g. cas- before data collection and at three-time points during the behav- tration, tail docking, and dehorning) have been shown to cause ior scoring period. Throughout the trial, all inter-observer reli- an increase in cortisol concentrations in several species (e.g. ability tests should produce an R-value above 0.9, indicating sheep and cattle) (Dinniss et al., 1997; Kent et al., 1998; excellent agreement between scorers and no significant drift McMeekan et al., 1998; Sutherland et al., 1999, 2002; Stafford throughout the scoring period (Park et al., 2020). Analysis should et al., 2002) including surgical castration and tail docking in be completed in a repeated measures linear mixed model with the pigs (Prunier et al., 2005; Carroll et al., 2006; Sutherland et al., Poisson distribution. 2012). Numerous studies have shown that surgical castration causes a significant and marked increase in pigs’ cortisol concen- Infrared thermography trations (Prunier et al., 2005; Carroll et al., 2006; Sutherland et al., IRT is a non-invasive method of detecting the amount of infrared 2010, 2012, 2017). However, handling alone only causes a slight energy (heat) an object radiates and can be used to measure skin but non-significant increase in cortisol (Prunier et al., 2005), sug- temperature changes associated with activation of the sympathetic gesting that the increase in cortisol in response to castration is nervous system. When an animal is stressed or in pain, the sym- predominantly due to the pain of the procedure and not the stress pathetic nervous system becomes activated, which causes vaso- of handling. Moreover, in pigs given analgesia (e.g. lidocaine or a constriction and a shift in blood flow from the

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omes activated, which causes vaso- of handling. Moreover, in pigs given analgesia (e.g. lidocaine or a constriction and a shift in blood flow from the skin to the non-steroidal anti-inflammatory drug), the cortisol response to organs. The blood flow change results in a loss of heat in the surgical castration was reduced (Kluivers-Poodt et al., 2012; body’s periphery and decreased skin temperature (Stewart et al., Bates et al., 2014). 2005; Bates et al., 2014). Cortisol is commonly measured in plasma, serum, or saliva. Stewart et al. (2010) found that eye temperature measured After surgical castration in pigs, plasma cortisol concentrations using IRT significantly increased in response to castration. This peak between 15 and 60 min and return to baseline levels between https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press Animal Health Research Reviews 169 Table 2. Behavioral ethogram for piglets adapted from Hay et al. (2003) Category Behavior Definition Non-specific Udder massage or Nose in contact with the udder or teat in the mouth. Vigorous and rhythmic up and down head behaviors suckling movements or suckling movements. Looking for teat Attempts to find a teat by walking and pushing other piglets while most of the others are suckling. Nosing The snout is close to or in contact with a substrate or a pen-mate. Snout movements may be observed. Belly-nosing Repeated up and down massage movements with the snout onto another piglet or the sow (except the udder). Chewing or licking Rubbing the tongue over or nibbling at littermates (ears, tail or foot, etc.), substrates, floor, or pen walls. Playing Head shaking, springing (sudden jumping or leaping), running with vertical and horizontal bouncy movements. It can involve partners

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pen walls. Playing Head shaking, springing (sudden jumping or leaping), running with vertical and horizontal bouncy movements. It can involve partners (gentle nudging or pushing, mounting, chasing). Aggression Forceful fighting, pushing with the head, or violently biting littermates. Walking Slowly moving forward with one leg at a time. Running Trot or gallop without a sudden change in direction or speed. Awake inactive No special activity but awake. Lying, sitting, or standing. Sleeping Lying down, eyes closed. Pain behavior Prostrate Awake, sitting or standing motionless, with the head down, lower than shoulder level. Huddled up Lying with at least three legs tucked under the body. Stiffness Lying with extended and tensed legs. Trembling Shivering as with cold. The animal may be lying, sitting, or standing. Spasms Quick and involuntary contractions of the muscles under the skin Scratching Scratching the rump by rubbing it against the floor or the pen walls. Tail wagging Tail’s movements from side to side or up and down. Postures Lateral lying Motionless; body weight supported by side. Shoulder in contact with the floor. Ventral lying Motionless; body weight supported by belly. Sternum in contact with the floor. Sitting Motionless; body weight supported by hind-quarters and front legs. Standing Motionless; body weight supported by the four legs. Kneeling Motionless; body weight supported by front carpal joints and hind legs. Location Udder Close to (<10 cm) or in contact with the udder. Sow’s back Close to (<10 cm) or in contact with sow’s back. Heat mat On the heat mat. Social cohesion Isolated Aside from other piglets, alone or with one pen-mate at the most. A distance of at least 40 cm (about the width of two piglets) separates the animal from the closest

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r piglets, alone or with one pen-mate at the most. A distance of at least 40 cm (about the width of two piglets) separates the animal from the closest group of littermates. Desynchronized Activity different from that of most (at least 75%) littermates (e.g. sleeps while most other littermates suckle). Other Other The pig’s behavior cannot be determined, or the animal was not seen. 120- and 180-minutes post-procedure (Prunier et al., 2005; California) (Kluivers-Poodt et al., 2012; Bates et al., 2014) and Sutherland et al., 2012, 2017). Therefore, to evaluate the efficacy EIA kits such as Assay Designs (Ann Arbor, Michigan) (Carroll of different pain mitigation strategies to reduce the pain caused et al., 2006; Sutherland et al., 2010, 2012) and IMMULITE/ by surgical castration, cortisol concentrations should be measured IMMULITE 1000 Cortisol (Global Siemens Healthcare, immediately before the procedure to assess baseline levels, Erlanger, Germany) (Sutherland et al., 2008). between 15 and 60 min post castration to assess changes in Changes in cortisol should be included as a biomarker for pain peak cortisol levels and then again at 120 min to confirm that in pigs as it has reliably been shown to increase in response to levels have returned to baseline. Cortisol concentrations in pigs pain and be reduced or abolished in response to different pain can be measured using validated in-house radioimmunoassay mitigation strategies after controlling for confounding factors (RIA) or enzyme immunoassay (EIA) techniques. However, in such as handling, restraint, and tissue trauma (Sheil and the recent literature, cortisol concentrations in pigs have been Polkinghorne, 2020). Besides, cortisol can be reliably measured more commonly measured using commercially available RIA in

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ntrations in pigs have been Polkinghorne, 2020). Besides, cortisol can be reliably measured more commonly measured using commercially available RIA in suckling pigs’ serum or plasma using commercially available kits such as Coat-a-Count (Siemens Medical Solutions RIA or EIA kits. However, when commercial diagnostic kits Diagnostics [formally Diagnostic Products Corp], Los Angeles, developed and validated for the diagnosis of endocrine disorders https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 170 Angela Baysinger et al. such as hyperadrenocorticism are repurposed as analytical meth- output can be converted to PDF, allowing the outcomes to be ods to measure the concentrations of biomarkers in healthy ani- reconstructed after the study is completed. mals undergoing painful procedures, researchers must ensure Using data collected from an unpublished pilot study, assum- that the diagnostic kit validation data provided by the manufac- ing a 5% significance level and 80% power, the authors recom- turer is reliable and accurate. Furthermore, it is a good scientific mend using a sample size of 30 piglets/treatment based on an practice to validate the diagnostic kit’s performance in the facility expected per cent change in front stride length from baseline mea- conducting the sample analysis. surements of 30%, a standard error of the mean of 10% assuming Using data collected from previous studies (Sutherland et al., the data are analyzed by ANOVA. 2010, 2012, 2017; Bonastre et al., 2016), assuming a 5% signifi- cance level and 80% power, the authors recommend using a sam- Blood biomarkers ple size of 20 piglets/treatment based on an expected difference in serum cortisol concentrations of 20 ng mL−1 and a standard error Several

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iomarkers ple size of 20 piglets/treatment based on an expected difference in serum cortisol concentrations of 20 ng mL−1 and a standard error Several circulating biomarkers targeting the indirect assessment of of the mean of 5 ng mL−1 at 60 min after surgical castration, pain and stress have been measured in piglets at the time of cas- assuming data are analyzed using 2-sample t-tests. tration. Specifically, these include markers of the neuroendocrine response (e.g. corticotropin, β-endorphins, epinephrine, norepin- ephrine, and substance P), inflammatory response (e.g. haptoglo- Ancillary outcomes bin, c-reactive protein, serum amyloid A, and prostaglandin E2), Stride length, contact pressure, contact area, and stance phase and adrenocortical response (e.g. cortisol) (Weary et al., 2006; duration Dzikamunhenga et al., 2014). Also, immune response assessments Objective gait parameters, measured using a commercially avail- have been made using hematological endpoints such as neutrophil able floor mat-based pressure/force measurement system to lymphocyte ratio (N:L) derived from a complete blood count. (MatScan, Tekscan, Inc, South Boston, Massachusetts), were These biomarkers have been correlated with stress because cortisol used in conjunction with lameness scores as primary endpoints release is typically rapid and difficult to quantify, but the asso- in a pivotal study that supported FDA approval of the first anal- ciated stress leukogram lasts longer and could be less time- gesic drug labeled for use in cattle in the United States (Banamine sensitive. Also, the production of unconjugated pterins (neopterin Transdermal, Merck Inc, Madison, New Jersey) (US Food and and biopterin) have been associated with stressful situations such Drug Administration, 2017).

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ansdermal, Merck Inc, Madison, New Jersey) (US Food and and biopterin) have been associated with stressful situations such Drug Administration, 2017). Pressure mat analysis is recognized as piglet castration (Marsálek et al., 2011; Maršálek et al., 2015). within a validated foot rot model as a reliable pain assessment Blood biomarkers are indirect measures of pain and inflamma- endpoint in cattle, thus satisfying the FDA Guidance Document tion. Furthermore, many of these outcomes have low specificity 123 requirements (US Food and Drug Administration, 2006). and can be altered by other factors such as handling stress. Pressure mat technology has been used to record and analyze nat- These outcomes are susceptible to confounding by other aspects urally occurring or experimentally induced changes in gait in cat- of the experiment, specifically blood sampling and handling. tle and swine due to lameness (Kotschwar et al., 2009; Schulz Most all the analytical methods have not been validated to et al., 2011; Karriker et al., 2013; Coetzee et al., 2014; Good Laboratory Practice or Good Clinical Practice specifications. Pairis-Garcia et al., 2015) and surgical castration (Nasirahmadi Attempts at validating these outcomes in piglets have been con- et al., 2017; Kleinhenz et al., 2018). Taken together, these data founded by challenges with extracting the analytes from plasma support the assessment of stride length, contact pressure, contact and serum. Additional challenges surround the sample collec- area, and stance phase duration using the pressure mat in the pro- tion’s optimal timing to ensure that the outcomes are correlated posed efficacy study. One of the proposed study’s key outcomes with the painful event. An initial screening of blood biomarkers would be to

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correlated posed efficacy study. One of the proposed study’s key outcomes with the painful event. An initial screening of blood biomarkers would be to compare the pressure mat outcomes with behavioral relative to the painful event may help identify outcomes that war- and physiological outcomes. rant further investigation and validation. The pressure mat will be calibrated daily using the expected Blood biomarker assessment is currently predicated using body weight of the piglets, and each time the computer software immunoassays or automated hematological methods validated is engaged using a known mass to ensure the accuracy of the mea- for human medicine. The collection protocol (timing and amount surements at each time point. The pressure mat, measuring 6–8 of blood needed) will depend on the study’s biomarkers. After feet in length, must be set up on a flat surface where piglets can collection, the analyte stability remains a significant challenge; be directed to walk at a steady pace across the mat so that the dis- therefore, samples must be stored on ice and, in some cases, liquid tance between multiple footfalls, pressure, and stance can be mea- nitrogen. In the case of neuropeptides such as substance P, add- sured. Footfalls are recorded when the foot strikes the loaded or itional steps must be taken to ensure that serine proteases do ‘contact’ sensing elements inside a measurement box. Research not degrade after collection (Mosher et al., 2014). One method grade software (HUGEMAT Research 5.83, Tekscan, Inc., South involves the addition of benzamidine hydrochloride, a protease Boston, Massachusetts) is used to determine the contact pressure, inhibitor, at 1 mM mL−1 of whole blood collected in EDTA. contact area, stance phase duration, and stride

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) is used to determine the contact pressure, inhibitor, at 1 mM mL−1 of whole blood collected in EDTA. contact area, stance phase duration, and stride length. The walk- Hematological outcomes are also measured using blood collected ing pig’s video is captured digitally and synchronized to ensure in EDTA, while acute phase proteins, such as haptoglobin, are consistent gait between and within piglets for each time point. collected in serum tubes. Previous studies investigating blood bio- Readings are taken before castration and at 6, 12, 24, and 48 h markers have generally enrolled between 10 and 20 piglets/treat- after castration. The per cent change from baseline for all mea- ment (Rault et al., 2011; Sutherland et al., 2012; sures will be calculated and analyzed statistically using a Dzikamunhenga et al., 2014). Initially, all of the biomarkers men- mixed-effects model. Before trial initiation, observers must be tioned above would be measured to determine usefulness as an trained to ensure stride length data is recorded consistently. The indirect outcome variable in the multimodal protocol to measure observers will achieve 80% inter-observer reliability and be analgesic efficacy. Upon identification of the biomarker(s) that blinded to treatment to control for observer bias. The analysis’s consistently correlates with the primary outcome measures of https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press Animal Health Research Reviews 171 Fig. 1. Piglet grimace scale developed by Viscardi et al. (2017). pain, one or more biomarkers would be utilized in the multi- pain. They have been developed for non-verbal humans as well modal protocol. as many animals, including mice, rats, rabbits, horses, sheep, lambs, cattle, and piglets

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been developed for non-verbal humans as well modal protocol. as many animals, including mice, rats, rabbits, horses, sheep, lambs, cattle, and piglets (Langford et al., 2010; Herr et al., Piglet grimace scale 2011; Sotocinal et al., 2011; Keating et al., 2012; Costa et al., Facial grimace scales are a novel, non-invasive tool for pain 2014; Gleerup et al., 2015; Di Giminiani et al., 2016; Guesgen assessment using quantifiable changes to facial features to detect et al., 2016; McLennan et al., 2016; Häger et al., 2017). A Piglet https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 172 Angela Baysinger et al. Grimace Scale (PGS) developed by Viscardi et al. (2017) 25%). The study found that among heavier pigs, castrates had described changes to three facial action units in response to piglet lower ADG at weaning compared to non-castrated pigs. surgical castration and tail docking pain (Fig. 1). A facial grimace Furthermore, low- and medium-weight piglets that were castrated in piglets is characterized by narrowing the orbital area (eyes had a higher likelihood of pre-weaning mortality than their non- squeezing shut), ears pulled back against the head, and a promin- castrated littermates (Morales et al., 2017). ent bump or bulge on the snout resulting from cheek tightening. While body weight changes are not a direct measure of pain, According to the PGS, the maximum grimace score is 5, and it initial body weights should be considered for enrollment pur- has corresponded well to displayed pain behaviors (e.g. an poses. Control animals should be non-castrated male littermates increase in pain behavior corresponded to higher facial grimacing rather than females. Piglets weighing 1.5 kg or less should be in piglets) (Viscardi and Turner,

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pain behavior corresponded to higher facial grimacing rather than females. Piglets weighing 1.5 kg or less should be in piglets) (Viscardi and Turner, 2018a, b). excluded. Although an increase in piglet facial grimacing has been corre- lated to a decrease in activity level and corresponds to an increase Mortality rate in pain behavior (Viscardi et al., 2017; Viscardi and Turner, The mortality rate is not an indicator of pain, and intervention 2018a, b), the PGS has not been validated as a pain assessment efficacy trials are not typically designed to detect statistical differ- tool. This proposed study methodology will determine if facial ences between treatments. However, when supplemented with a grimacing can be correlated to pain behavior or other non- complete necropsy of the pigs that die, the mortality rate can be invasive outcome measures (e.g. IRT). The PGS has only been used as a non-specific indicator of negative impacts on health, used to retrospectively assess pain and analgesia efficacy by scor- toxicity when chemical interventions are applied, and secondary ing still-images of piglet facial expressions extracted from video complications that might influence the adoption of an interven- recordings. Individuals used to score facial expressions were tion. As some unrelated baseline, mortality is likely to occur in undergraduate, graduate, or veterinary students. To improve its most populations, and occasional euthanasia of animals is war- practicality for on-farm use, the PGS should be validated for real- ranted for unrelated reasons. Mortality is a non-specific endpoint, time detection of pain and producers and swine veterinarians’ and a stepwise evaluation process should be implemented to use ability to use the PGS to identify pain in piglets accurately.

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and swine veterinarians’ and a stepwise evaluation process should be implemented to use ability to use the PGS to identify pain in piglets accurately. study resources effectively. Previous work using grimace scales have found 1–3 facial First, potential physiological impacts that can progress to death images captured per animal per time point was sufficient to assess and are specific to the intervention being compared should be pain and analgesic efficacy (Sotocinal et al., 2011; Costa et al., identified a priori at the start of the study. Any specific necropsy 2014; Miller et al., 2015; Miller and Leach, 2015a, b; Miller lesions and post-mortem diagnostic testing to confirm or refute et al., 2016; Viscardi and Turner, 2018a). These studies also the intervention’s involvement in the mortality must be recorded. used 2–5 individuals to score facial expressions. Researchers aver- For example, if a drug intervention has the potential for harmful aged the individuals’ resulting scores before analysis and con- toxicity, the appropriate tissue, diagnostic test, and the testing ducted inter-observer reliability tests to ensure scoring laboratory should be identified and included in the study protocol consistency. as a standard component of the necropsy evaluation. The second step is to record the pig’s identity, time and date Body weight observed, and whether the death was the outcome of euthanasia Changes in body weight can be used as an indirect measure of the or occurred naturally. In cases where euthanasia was the cause pain experienced by piglets at castration. Multiple studies in a of death, the reason for euthanasia should be recorded. range of species demonstrate that when animals experience The third step is to perform a complete external exam and nec-

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uthanasia should be recorded. range of species demonstrate that when animals experience The third step is to perform a complete external exam and nec- pain, feed consumption decreases, resulting in a reduction in ropsy procedure, as outlined by Torrison (2012). The observation body weight and average daily gain (ADG) (Malavasi et al., 2006). of gross lesions or the confirmation of absence should be recorded Although there is little evidence of the long-term impact of for all the main body systems. The use of a checklist (Fig. 2) by castration on body weight gain in piglets, reductions in ADG trained observers is necessary to maintain consistency of evalua- due to castration have been found in the days following the pro- tions and data collection. cedure. Kielly et al. (1999) found that pigs castrated at 3 days of Suppose gross lesions indicate a specific organ system’s age gained less weight than weight-matched controls over the involvement, but a cause of death cannot be determined by the 3 days following castration, while those castrated at 14 days of necropsy procedure alone. In that case, the fourth step is to age showed no difference in ADG compared to controls, suggest- employ the appropriate tissue collection as outlined by Torrison ing that delayed castration may benefit piglets. In contrast, Hay (2012), followed by submission to a qualified diagnostic labora- et al. (2003) compared the body weights of piglets castrated at tory with appropriate history and context. All correspondence 5 days of age and sham handled controls twice per day for 4 with the diagnostic laboratory and test results should be copied days after treatment and found no differences in ADG. into the study record. Most studies have measured body weights at weaning and found no effect of

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days after treatment and found no differences in ADG. into the study record. Most studies have measured body weights at weaning and found no effect of castration with or without pain control on Discussion body weight (Kielly et al., 1999; Cassar et al., 2014; Burkemper et al., 2019). Contrary to this, McGlone et al. (1993) found that It is well documented that piglets experience pain associated with piglets castrated at one day of age had lower weaning weights castration, and efforts to mitigate this pain should be explored. than those castrated at 14 days, while female pigs were intermedi- Previous studies on pain mitigation at castration have typically ate. Because female pigs were used as controls rather than sham- included similar techniques and outcomes, but wide variation handled males, this result is difficult to interpret. A more recent in experimental design and data collection approaches give non- study by Morales et al. (2017) compared ADG in over 3000 comparable results and hinders a comprehensive interpretation castrated versus intact males and categorized piglets based on ini- of the science (Sheil and Polkinghorne, 2020). This paper tial body weight as low (lowest 25%), medium, or high (highest describes a methodology to assess behavioral, physiological, and https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press Animal Health Research Reviews 173 Fig. 2. Checklist of relevant mortality information, necropsy observations, including organ systems to be observed and lesions to be considered. neuroendocrine changes associated with pain in piglets resulting (observation of pain behaviors, activity tracking, IRT, and cortisol from surgical castration. The methodology is being further devel- concentrations) and ancillary

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f pain behaviors, activity tracking, IRT, and cortisol from surgical castration. The methodology is being further devel- concentrations) and ancillary outcomes (stride length, blood bio- oped into a research protocol template to facilitate and guide the markers [including neuroendocrine, inflammatory, immuno- validity and reliability of endpoints to evaluate drugs’ effectiveness logical, and stress response markers], piglet grimace scale, body to control post-operative pain in 3- to 5-day-old piglets following weight, and mortality rate) being either directly related to clinical surgical castration. signs of pain or indirectly related to clinical signs of pain and For the experimental design and the purposes of endpoint val- lending support to the direct outcomes will be measured, vali- idation, the authors recommend four primary treatments, includ- dated and analyzed. ing (1) sham castration, (2) sham castration with the ‘gold standard’ intervention, (3) surgical castration with a placebo con- Conclusion trol, and (4) surgical castration with the ‘gold standard’ interven- tion. Piglet (within litter) is the experimental unit with all The experimental design and measurable outcomes selected from treatments represented within each litter at least once. the validation study are intended to promote a consistent Outcomes being directly related to clinical signs of pain approach to determining more effective therapies for pain https://doi.org/10.1017/S1466252321000141 Published online by Cambridge University Press 174 Angela Baysinger et al. mitigation. This paper supports the inclusion of specific outcomes Di Giminiani P, Brierley VLMH, Scollo A, Gottardo F, Malcolm EM, in the validation study and summarizes the need for further val- Edwards SA and Leach MC

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Giminiani P, Brierley VLMH, Scollo A, Gottardo F, Malcolm EM, in the validation study and summarizes the need for further val- Edwards SA and Leach MC (2016) The assessment of facial expressions idation of emerging outcomes. The development of similar proto- in piglets undergoing tail docking and castration: toward the development of the piglet grimace scale. Frontiers in Veterinary Science 3, 100. cols for determining the validity and reliability of endpoints to Dinniss AS, Mellor DJ, Stafford KJ, Bruce RA and Ward RN (1997) Acute evaluate the efficacy of pain mitigation therapies targeted to cortisol responses of lambs to castration using a rubber ring and/or a cas- other painful procedures or conditions in swine, such as tail dock- tration clamp with or without local anaesthetic. New Zealand Veterinary ing or lameness, should be considered. Journal 45, 114–121. Dzikamunhenga RS, Anthony R, Coetzee JF, Gould S, Johnson A, Karriker Author contributions. Conceptualization, SRW and AD; methodology, L, McKean J, Millman ST, Niekamp SR and O’Connor AM (2014) Pain AKB, SRW, JB, JFC, SC, AD, LAK, M.P-G., MAS, AVV; writing-original management in the neonatal piglet during routine management procedures. draft preparation, AKB and SRW; writing- review and editing, AKB SRW Part 1: a systematic review of randomized and non-randomized intervention and M.P-G. All authors have read and agreed to the published version of studies. Animal Health Research Reviews 15, 14–38. the manuscript. European Commision (2017) Pig castration : methods of anaesthesia and Financial support. No monetary funding was sourced for the development analgesia for all pigs and other alternatives for pigs used in traditional pro- of this manuscript. ducts. [Online] Available at:

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