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Assessment of the affective component of pain in dairy calves Ede, Thomas 2020

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  ASSESSMENT OF THE AFFECTIVE COMPONENT OF PAIN IN DAIRY CALVES by  Thomas Ede  M.Sc., Bordeaux Sciences Agro, 2015  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Applied Animal Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   November 2020  © Thomas Ede, 2020   ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  ASSESSMENT OF THE AFFECTIVE COMPONENT OF PAIN IN DAIRY CALVES   submitted by Thomas Ede in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Applied Animal Biology  Examining Committee: Dr. Daniel Weary, Professor, Applied Animal Biology, UBC Supervisor  Dr. Marina A. G. von Keyserlingk, Professor, Applied Animal Biology, UBC Supervisory Committee Member  Dr. Sumeet Gulati, Professor, Environmental and Resource Economics, UBC Supervisory Committee Member Dr. Murat Aydede, Professor, Philosophy, UBC University Examiner Dr. Anthony Farrell, Professor, Applied Animal Biology, UBC University Examiner        iii Abstract Whether they live in our homes, farms or laboratories, many animals are subjected to painful procedures. In humans, pain assessment is mostly done through verbal self report, but the assessment of pain in non-verbal humans and animals remains a challenging task. Pain can be divided in two main components: a reflex response, and an emotional experience. The focus of pain research has largely been centered around reflex responses when animals (and human neonates until recently) are the ones subjected to pain. The aim of this thesis was to address this gap by developing a method to assess the emotional experience (or ‘affective state’) that dairy calves go through during painful procedures. To do so, the focus of our novel approach was on how animals formed memories of procedures they were subjected to.  In the first chapter, I reviewed the literature on the assessment of emotion in dairy cattle. I highlighted that many measures reflect how aroused animals are rather than whether they are in a positive or negative state (valence), but that measures based on cognition were promising in evaluating valence. In the second chapter, I studied the pain caused by different injection methods by looking at how much milk calves were willing to give up to avoid injections. Although all methods were more aversive than not receiving an injection, intramuscular injections were more aversive than subcutaneous or intranasal. In the third, fourth and fifth chapter, I studied how calves remembered different methods of disbudding (a procedure that prevents horn growth) by looking at how much they would avoid the place where they experienced the procedure. I found that calves remember disbudding as negative and that learned aversion is reduced if calves are provided pain control both during and after the procedure.    iv In summary, calves showed not to be limited to a ‘knee-jerk’ response to pain. Rather, they formed impactful memories that affected their future behaviour, exhibiting complex emotional processing of pain.     v Lay Summary Dairy calves routinely undergo painful procedures. In this thesis, I developed and applied novel methods of assessing pain in calves. My methods focused on the calves’ memory of the experience; the results suggest that calves remember painful procedures as negative and will avoid the place where they experienced them. Refining procedures with less painful alternatives and use of appropriate pain control can reduce this learned aversion. The paradigms developed in this thesis are adaptable to the study of other emotions and for use in other species.    vi Preface A version of Chapter 1 has been published as Ede, Thomas, Benjamin Lecorps, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Symposium review: Scientific assessment of affective states in dairy cattle”. Journal of Dairy Science 102, no 11 (2019): 10677-94. https://doi.org/10.3168/jds.2019-16325. TE and BL wrote the initial manuscript. MvK and DW provided critical input and revisions. This project did not require ethic approval. A version of Chapter 2 has been published as Ede, Thomas, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Approach-aversion in calves following injections”. Scientific Reports 8, no 1 (2018): 9443. https://doi.org/10.1038/s41598-018-27669-7. TE designed, conducted and wrote an initial report of the study. MvK and DW provided critical input and revisions. The study was approved by the UBC Animal Care Committee (Application A16-0310). A version of Chapter 3 has been published as Ede, Thomas, Benjamin Lecorps, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Calf aversion to hot-iron disbudding”. Scientific Reports 9, no 1 (2019): 5344. https://doi.org/10.1038/s41598-019-41798-7. TE and BL designed and conducted the study, TE wrote an initial report. MvK, DW and BL provided critical input and revisions. The study was approved by the UBC Animal Care Committee (Application A16-0310). A version of Chapter 4 has been published as Ede, Thomas, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Assessing the affective component of pain, and the efficacy of pain control, using conditioned place aversion in calves”. Biology Letters 15, no 10 (2019): 20190642. https://doi.org/10.1098/rsbl.2019.0642. TE designed, conducted and wrote an initial    vii report of the study. MvK and DW provided critical input and revisions. The study was approved by the UBC Animal Care Committee (Application A16-0310). A version of Chapter 5 is in press for publication in the Journal of Dairy Science. TE designed, conducted and wrote an initial report of the study. MvK and DW provided critical input and revisions. The study was approved by the UBC Animal Care Committee (Application A16-0310).     viii Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Figures .................................................................................................................................x List of Abbreviations ................................................................................................................... xi Acknowledgements ..................................................................................................................... xii Chapter 1: Introduction ............................................................................................................... 1 1.1 General introduction ................................................................................................... 1 1.2 Spontaneous responses................................................................................................ 4 1.3 Learned responses ..................................................................................................... 17 1.4 Mood assessment ...................................................................................................... 23 1.5 Pharmacological interventions .................................................................................. 28 1.6 Discussion ................................................................................................................. 29 1.7 Conclusions ............................................................................................................... 31 1.8 Thesis objective ........................................................................................................ 31 Chapter 2: Approach-aversion in calves following injections ................................................ 32 2.1 Introduction ............................................................................................................... 32 2.2 Methods..................................................................................................................... 33 2.3 Results ....................................................................................................................... 36 2.4 Discussion ................................................................................................................. 38 Chapter 3: Calf aversion to hot-iron disbudding ..................................................................... 42 3.1 Introduction ............................................................................................................... 42 3.2 Methods..................................................................................................................... 44 3.3 Results ....................................................................................................................... 49 3.4 Discussion ................................................................................................................. 51 3.5 Conclusion ................................................................................................................ 54 Chapter 4: Assessing the affective component of pain, and the efficacy of pain control, using conditioned place aversion in calves................................................................................ 55 4.1 Introduction ............................................................................................................... 55 4.2 Methods..................................................................................................................... 57 4.3 Results ....................................................................................................................... 62 4.4 Discussion ................................................................................................................. 63 4.5 Conclusion ................................................................................................................ 66 Chapter 5: Conditioned place aversion of caustic paste and hot-iron disbudding in dairy calves…………………………………………………………………………………………….67 5.1 Introduction ............................................................................................................... 67 5.2 Methods..................................................................................................................... 68 5.3 Results ....................................................................................................................... 72 5.4 Discussion ................................................................................................................. 74 5.5 Conclusion ................................................................................................................ 77 Chapter 6: General discussion ................................................................................................... 78 6.1 Thesis summary ........................................................................................................ 78    ix 6.2 Contributions............................................................................................................. 80 6.3 Limitations and future research areas ....................................................................... 81 6.4 General conclusion.................................................................................................... 89 References .....................................................................................................................................91     x List of Figures Figure 1-1. Theoretical framework of affect including two components: valence and arousal. .... 2 Figure 1-2. Example of operant motivation testing. ..................................................................... 23 Figure 2-1. Experimental apparatus. ............................................................................................. 35 Figure 2-2. Latencies of calves to approach a milk reward, depending upon size of the reward and treatment. ................................................................................................................................ 37 Figure 3-1. Experimental apparatus. ............................................................................................. 45 Figure 3-2. Time (√s) that calves spent in the different pens during test sessions relative to the treatment they received in that pen during treatment sessions. .................................................... 50 Figure 3-3. Pen in which calves lay down during test sessions relative to the treatment they received in that pen during treatment sessions. ............................................................................ 51 Figure 4-1. Experimental apparatus. ............................................................................................. 58 Figure 4-2. Time (√s) that calves spent in test pens (a) and the pen in which calves eventually lay down (b) during test sessions. ....................................................................................................... 63 Figure 5-1: Experimental apparatus. ............................................................................................. 69 Figure 5-2. Place aversion results comparing hot-iron disbudding to caustic paste. .................... 74     xi List of Abbreviations ACTH = Adrenocorticotropic hormone CI = Confidence interval HPA = Hypothalamic pituitary adrenal HR = Heart rate HRV = Heart rate variability IM = Intramuscular IN = Intranasal NSAID = Non-steroidal anti-inflammatory drug SC = Subcutaneous SD = Standard deviation SNS = Sympathetic nervous system       xii Acknowledgements I would like to thank Dan and Nina for their guidance over the last four years. Their dedication and trust were invaluable in building the confidence needed to make this thesis. Thank you for the countless stimulating discussions and challenging feedback. Thanks to Sumeet for his helpful feedback on the thesis. I am grateful I had the chance to be part of the Animal Welfare Program, an impressive group of professors and students who are as bright as they are supportive. Specifically, I would like to thank my first farm mentors: Heather, Joao and Becky who encouraged me to come back as a graduate student. Thanks to Ben, Eugenie, Tracy, Augusto, Allison and many other farm and ‘blue house’ students for all the fun times. Thank you Stephanie for the great moments spent exploring this wonderful country. I would like to show my gratitude to the staff of the Agassiz farm: Nelson, Mary-Ann, Brad, Barry, Bill, Ted, Lyndon, Madison and Alex for their help, advice and patience. Finally, thanks to my family and friends from France for their continuous support, I have missed you all during these years.        1 Chapter 1: Introduction A version of Chapter 1 has been published as Ede, Thomas, Benjamin Lecorps, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Symposium review: Scientific assessment of affective states in dairy cattle”. Journal of Dairy Science 102, no 11 (2019): 10677-94. https://doi.org/10.3168/jds.2019-16325. 1.1 General introduction A key component of animal welfare is the animal’s affective state; in other words, how it feels. Affective states can be negative like pain and fear, as well as positive like pleasure and happiness (Boissy et al., 2007; Webb et al., 2018). Affective states are subjective by nature, making them out of reach of direct assessment. As Panksepp (2005) puts it, ‘There is no mind scope, and may never be, that can directly monitor any psychological process’. The ‘hard problem’ (Chalmers, 1995) of studying the subjective experience of emotions is not limited to animals; it is also a critical and controversial topic in human literature. With humans, researchers can ask (most) patients how they feel, providing a type of gold standard for research on competent, verbal humans. Given that this approach is not applicable to dairy cows (or other animals), the challenge is to identify useful surrogate measures. As David Fraser puts it: ‘There is no simple English word to capture the class of concepts such as ‘pleasure’, ‘pain’, ‘suffering’, and ‘happiness’. They are sometimes called ‘feelings’, but that term seems too insubstantial for states like pain and suffering. They are sometimes called ‘emotions’, but emotions do not include states like hunger and thirst. Perhaps the most accurate, if rather technical, term is affective states, a term that refers to emotions and other feelings that are experienced as pleasant or unpleasant rather than hedonically neutral.’ (Fraser, 2008)         2 This quote serves to illustrate one component of affect: valence (i.e. whether something is experienced as positive or negative). Valence is of great relevance to animal welfare as minimizing negative affective states and maximizing positive ones is key to improving welfare (Fraser and Duncan, 1998). However, limiting affect to valence might be inadequate. Borrowing from human literature (Barrett, 1998; Bradley and Lang, 1994), Mendl et al. (2010) provided a framework that integrated valence and arousal (relating to the degree of tension or agitation of the affective state). For example, fearfulness and sadness are both negative states, but sadness is typically lower in arousal (Figure 1.1). As further illustration, when humans were asked to position their affective reactions to images, they rated a cemetery as negative-low arousal, an aimed gun as negative-high arousal, a roller-coaster as positive-high arousal and a cow as positive-low arousal (Bradley and Lang, 1994). In this review we use the words affect, emotion and feeling synonymously (as defined by Mendl et al., 2010) and avoid claims about consciousness or self-awareness.  Figure 1-1. Theoretical framework of affect including two components: valence and arousal.         3 Words in italics are examples of affective states in each quadrant, adapted from (Barrett, 1998; Mendl et al., 2010). Words in parentheses refer to the examples from (Bradley and Lang, 1994).  Although we adopted the two-dimensional arousal/valence framework for this review, we acknowledge that it might be incomplete; authors have mentioned a third dimension including the control (or agency) over the experience (Bradley and Lang, 1994; Warriner et al., 2013). Additionally, dimensions might be different from the continuous spectra assumed here. For example, some have argued that positive and negative valences are two distinct processes rather than varying along a single continuum (Fraser and Duncan, 1998; Shriver, 2014).  Affective states are one of the three commonly discussed components of animal welfare, the remaining two being natural living and physical health (Fraser et al., 1997). As described by Webb et al. (2018), natural living and physical health belong to the ‘assumed’ aspects of welfare (as we assume natural environments and good health to improve welfare) while affective states belong to the ‘apparent’ aspect (as this directly relates to the animal’s experience). This review will focus on affective states, because of their importance to welfare and because of the challenges in developing assessment methods for these states. Moreover, practical efforts to address welfare problems typically require that we know the specific affective state of concern (e.g. is the cow experiencing pain or fear, or both), if we are to develop methods of prevention or treatment. Thus, where possible, we also discuss if different measures can be useful in drawing inferences regarding specific affective states. The objective of this critical review is to assess the strengths and weaknesses of scientific methods used to assess affective states in animals. We discuss inferences regarding physiological and spontaneous behavioural responses, especially regarding arousal. We also discuss what       4 inferences regarding valence can be drawn from studies of learned behaviours. We have also considered how affective states contribute to mood (i.e. the sum of affective states; Boissy and Lee, 2014), how mood itself can be assessed, and finally how experimental treatments using drugs can be used to draw inferences regarding specific affective states. Where possible we use examples of research on dairy cattle, but also refer to work on other species to provide historical context and note the translatability of studies to dairy cattle and highlight approaches that have not yet been applied to cattle. Our examples with dairy cattle focus on cows and calves, as little relevant research has been published addressing this issue in other types of dairy cattle (e.g. dairy bulls, etc.). 1.2 Spontaneous responses  Physiological responses Stress has been the focus of much research on farm and laboratory animal welfare (Broom, 1991, 1988; Jensen and Toates, 1997; Veissier and Boissy, 2007), leading to an interest in measures related to the activation of the hypothalamic pituitary adrenal (HPA) axis and sympathetic nervous system (SNS) in response to stressful events. The term ‘stress’ can sometimes imply a negative experience. A broader definition of stress – that we adopt in this review – includes reactions to all challenges to homeostasis (Mellor et al., 2000; Rushen, 1986a). In the following section, we will argue that responses related to activation of the HPA and SNS are of most value as non-specific indicators of arousal. Stress responses generally show some proportionality to challenge intensity (Mellor et al., 2000), but the non-specific nature of these responses means that thorough knowledge of the subjects’ circumstances and experiences preceding the assessment (such as housing, interactions with pen mates, handling…) is required to inform inferences regarding valence, let alone the specific emotion of interest.        5 Stress responses have been studied in dairy cattle on a wide array of scales, from hormones like adrenaline (Hopster et al., 2002) and cortisol (Stock et al., 2013), to organ systems including circulatory (Kovács et al., 2015) and respiratory (Rizk et al., 2012) responses, and whole animal measures such as activity (Webb et al., 2017). Reviewing each of these responses is beyond the scope of this review. Instead, we focus on just four responses (cortisol, heart rate variability, eye/nose temperature and eye white), based on their prevalence in the cattle literature and our personal experience.  1.2.1.1 Cortisol Cortisol and other measures related to activation of the HPA axis have often been used in studies of affect in cattle, including studies on painful procedures (Adcock and Tucker, 2018). For example, in one review on cattle dehorning (Stock et al., 2013), the word ‘cortisol’ was mentioned 275 times (in comparison, ‘adrenaline’ and ‘ACTH’ were mentioned 4 and 3 times, respectively). Plasma cortisol as an index is most commonly described, but sampling from milk (Verkerk et al., 1998), saliva (González et al., 2010), hair (Burnett et al., 2014), urine (Redbo, 1993) and feces (Palme et al., 2000) have been mentioned. Elevated cortisol levels have been observed in calves following dehorning (Stilwell et al., 2010), castration (Fisher et al., 1996) and branding (Schwartzkopf-Genswein et al., 1997a). Changes in plasma cortisol concentration are sensitive to pain mitigation strategies; for example, the use of a local anesthetic (lidocaine) on the cornual nerve virtually eliminates the cortisol peak response observed in the 2 h following horn amputation in calves (Stafford and Mellor, 2005). Moreover, the cortisol increase usually observed as local anesthesia wanes is reduced by the use of post-operative analgesics (Stafford and Mellor, 2011). Similar responses have been observed after castration procedures (Stafford and Mellor, 2005).        6 However, the association between cortisol and pain is imperfect. For example, Coetzee et al. (2008) found no differences in cortisol between calves that had been castrated (without anesthesia or analgesia) and uncastrated calves. This example reveals a vulnerability of cortisol measures to ceiling effects; in this case it is likely that other events associated with the surgery (restraint, blood sampling, etc.) were sufficiently stressful that animals showed maximal cortisol responses, leaving no room to detect an effect of pain. Other work has shown that cortisol responses can be brief (with differences detectable for only a few hours after the procedure) even when behavioural differences indicative of pain persist for weeks (Thüer et al., 2007). Thus, cortisol measures may be most relevant for the acute phase after a painful event but insensitive to longer-term effects. In addition, individuals typically display a decrease in cortisol responses with repeated exposure to a stressful event (i.e. the cortisol habituation response; Andrade et al., 2001; Solano et al., 2004; Wüst et al., 2005; Wong et al., 2010). Finally, it is important to note that the delay between a painful event and a change in cortisol levels makes this measure insensitive during the first few minutes after a procedure (Mellor and Stafford, 2000). Cortisol is not specific to negative events: although not studied in cattle, elevated levels of corticosteroids have been observed in positive situations such as sexual stimulation in stallions and rats (Colborn et al., 1991; Szechtman et al., 1974), voluntary exercise in mice and rats (Stranahan et al., 2008) and exposure to an enriched environment in chickens (Solomon, 1997). Cortisol responses are also dependent upon learning and coping capacities (Rushen, 1986a), meaning that higher levels could be reflective of a more novel and unpredictable environment, but not necessarily a negative one.  In summary, measures linked to the activation of the HPA axis reflect an increased demand of resources from the organism. In some cases (such as in the absence of ceiling effects       7 or outside the response duration), such measures may relate to arousal. The non-specific nature of this response means contextual factors must be considered, including the animal’s previous experiences, before drawing inferences regarding valence. Inferences regarding what specific affective state the animal is experiencing will generally be impossible without the use of drug treatments as discussed later in this paper. See Rushen (1986a) and Mellor et al. (2000) for a more detailed discussion of the strengths and limitations of using evidence of HPA axis activation to draw inferences about pain and other affective states. 1.2.1.2 Heart rate variability Heart rate (HR) was used in early studies of stress in humans (Lazarus et al., 1963) and animals, including work on cows examining the effects of heat stress, dehorning and electric shocks published in the 1950’s (Bianca, 1958; Graf and Petersen, 1953). These intuitively negative events were associated with a rise in HR, but so was any increase in physical activity (Graf and Petersen, 1953). More recently, heart rate variability (HRV) has been suggested as a more sensitive measure to characterize cardiac variations associated with stress (Montebugnoli et al., 2004). HRV is based on time interval irregularities between heart beats and is thought to reflect the balance between the two divisions of the autonomic nervous system: sympathetic and parasympathetic (Berntson et al., 1997; Bootsma et al., 1994; Camm et al., 1996). Some authors consider a balance towards the sympathetic system (thought to result in a low HRV) to correspond to the ‘fight or flight’ response; whereas, a balance towards the parasympathetic system (thought to be associated with a high HRV) to be the ‘rest and digest’ phase (Müller et al., 2017). The idea that HRV reflects autonomic control is based on animal and human studies blocking and amplifying either or both branches of the autonomic system and observing the       8 resulting cardiovascular variability (Parati et al., 2006). However, no consensus has been reached on the suitability of such measures and their use remains debated (Billman, 2013, 2011; Parati et al., 2006). The trend in human medical studies to use HRV to study diseases and mental states (Gorman and Sloan, 2000; Kristal-Boneh et al., 1995; Langewitz and Rüddel, 1989) may have increased the popularity of this measure in farm animal research (Kovács et al., 2014; von Borell et al., 2007).  Decreased HRV has been detected in dairy calves during social isolation, dummy teat removal and hot-iron disbudding (Clapp et al., 2015; Stewart et al., 2009). Cows receiving transrectal palpitation by students also showed decreased HRV (Giese et al., 2018). Decreased HRV has been observed immediately after dehorning without local anesthetics (Stewart et al., 2009) indicating the potential to use HRV to investigate immediate responses. However, inconsistencies have been noted between studies and depending on the HRV measure chosen (Stewart et al., 2009). The practical difficulties of long-term HRV data collection on cows have limited our knowledge of longer term effects of procedures on HRV (Kovács et al., 2014). One study showed a decrease in HRV up to 48 h after dehorning (Clapp et al., 2015), but in this study the calves were exposed to multiple stressors, making it difficult to draw specific inferences. A number of results undermine the idea that decreased HRV is reliably associated with pain or other negative affective states. For example, marked parasympathetic dominance has been found in calves following castration without anesthesia (Stewart et al., 2010) and in lame cows (Kovács et al., 2015). Since castrated and lame animals are expected to be less active (to avoid painful stimulation), this raises the concern that HRV (like HR) may be more related to physical activity rather than emotional state. Indeed, HRV parameters shift towards sympathetic       9 dominance simply when healthy animals stand up (Hagen et al., 2005), suggesting that activity differences can confound interpretation of HRV.  The study of HRV in dairy cows (and farm animals in general) has focused on negative events including disease, veterinary procedures, stressful handling, weaning and isolation (Kovács et al., 2014). To our knowledge, no study has investigated HRV in cows during a positive event, making it difficult to judge whether HRV responses are specific to negative affect. In humans, higher HRV is associated with calmness, cheerfulness and subjective well-being (Geisler et al., 2010), as well as with disgust and sadness (Kreibig, 2010). Lower HRV has been associated with daily worry (Brosschot et al., 2007) and with happiness (Kreibig, 2010), and the activation of the sympathetic nervous system has also been associated with sexual arousal (Meston, 2000).  Finally, major concerns have been raised regarding the influence of HR on HRV. Monfredi et al. (2014) and Kazmi et al. (2016) showed a non-linear inverse relationship between HR and HRV (i.e. variability in HR decreases as HR increases), implying that HRV might simply be an indirect measure of HR (Monfredi et al., 2014). The latter authors indicated that it is possible to control for the effect of HR on HRV with a non-linear correction. Unfortunately, none of the cattle studies previously mentioned used this correction. In summary, the results reviewed above suggest that HRV may be seen as a non-specific indicator of arousal, with little basis for inferences regarding affective valence let alone any specific state. 1.2.1.3 Eye/Nose temperature Emotions have been shown to induce physiological responses that include a shift in peripheral and internal temperatures. For instance, stressors, through the activation of the SNS, redirect peripheral blood to internal organs (i.e. the central nervous system and the muscles) via       10 vasoconstriction of the peripheral arteries; the associated increase in deep body temperature is sometimes referred to as ‘stress-induced hyperthermia’ (Zethof et al., 1994). This physiological mechanism is thought to support the fight or flight response by directing energy to internal organs. Infrared thermography offers a non-invasive way to measure subtle changes in peripheral body temperatures associated with the experience of different emotions. Research in humans has focused on changes in facial temperature, including specific regions of interest such as the mouth, cheeks, forehead and eyes (Clay-Warner and Robinson, 2015), providing some basis to predict changes in peripheral body temperatures that can be used to asses emotional states.  In animals, studies have focused on the eyes in various species such as dogs (Travain et al., 2016, 2015), sheep (Stubsjøen et al., 2009), horses (Dai et al., 2015) and cattle (Stewart et al., 2008) and on other areas such as the comb in laying hens (Moe et al., 2012), and the tail in rodents (Lecorps et al., 2016; Vianna and Carrive, 2005).  In dairy cattle, the eye temperature was found to quickly decrease (within the first minutes) after a painful procedure and then increase over baseline values for at least 15 to 20 min after the procedure (Stewart et al., 2008, 2010). These authors were able to detect when the effect of a lidocaine nerve block waned around 2.5 h after hot-iron disbudding (Stewart et al., 2009). Thus, eye temperature changes might be a useful indicator of post-operative pain.  For such examples it is difficult to disentangle the effects of fear and pain. In one study on castration, eye temperature decreased and then increased relative to baseline values in animals that were not given a nerve block, but animals given a nerve block showed an increase but no decrease in eye temperature (Stewart et al., 2010). These results could suggest that the initial drop in eye temperature, followed by an increase, is indicative of pain, while an increase only may be indicative of fear. Two recent studies found an increase in eye temperature when calves       11 were handled and transported (Lecorps et al., 2018) or claw trimmed (Gomez et al., 2018), and other studies have reported increased temperature associated with fear-related situations in dogs (Travain et al., 2015), horses (Dai et al., 2015), mice (Lecorps et al., 2016) and humans (Levine et al., 2001). The only cattle study that explored changes in eye temperature in positive situations found a slight increase during feeding (Gomez et al., 2018). Nasal temperature was found to decrease in both positive and negative situations (Proctor and Carder, 2016, 2015a). These authors used positive (receiving better feed than expected) and negative food-related contrasts (receiving unpalatable feed); further work is required to assess responses under more aversive situations. A study on laying hens also found decreased comb temperatures both during fear conditioning and consumption of a reward (Moe et al., 2012).  Collectively these results suggest that measures of peripheral temperature may provide a non-specific indication of arousal. Many factors can affect temperature recordings, including camera settings, distance and angle between the camera and the region of interest, and environmental conditions such as ambient temperature, sunlight exposure, wind and humidity (Church et al., 2014; Clay-Warner and Robinson, 2015), so acquisition and interpretation of these measures requires care.  1.2.1.4 Eye white In humans, increased visible eye white (i.e. the percentage of eye white over the total area of the visible eye) has been used to assess emotional states including fear (Feng et al., 2009; Fox and Damjanovic, 2006; Whalen et al., 2004), but eye white has received little study in non-human animals. Work to date has focused primarily on sheep and cattle (Lambert (Proctor) and Carder, 2017; Proctor and Carder, 2015b; Reefmann et al., 2009). Increased eye white has been       12 noted after cattle were exposed to the sudden opening of an umbrella (Sandem et al., 2004) and when heifers were milked for the first time without prior habituation to the milking parlor (Kutzer et al., 2015). Sandem and Braastad (2005) found that eye white also increased when cows were separated from their calves and that the magnitude of the response was correlated with other responses (including sniffing the ground where the calf had been, looking toward where the calf was taken, vocalizing and pacing). In another example, hungry cows (7 h without feed) showed an increase in eye white when allowed to see their feed (through a plexiglass barrier) without being able to access it (Sandem et al., 2002). These cows also showed head shaking, tongue rolling and vocal responses, all of which were not observed in the cows that had full access to the feed. Conversely, eye white was observed to decrease once dams were reunited with their calves (Sandem and Braastad, 2005), when given access to feed after a period of restriction (Sandem et al., 2002) and during grooming (Proctor and Carder, 2015b). These results seem to indicate that increased eye white reflects negative affect and decreased eye white reflects positive affect, but other results undermine this idea. For example, eye white was observed to increase in cattle as much during undisturbed feeding as during restraint and claw trimming (Gómez et al., 2018). Increased eye white occurs in response to anticipation of feeding, thought to be a positive, high arousal situation (Sandem et al., 2006). Making use of ‘feed’ types thought to be positive (concentrate), neutral (standard ration), and negative (inedible woodchips), Lambert and Carder (2017) reported that both positive and negative stimuli resulted in increased eye white compared to the neutral feed. To our knowledge, this is the only study including stimuli varying in valence but focused on a similar event (feed provision); thus, likely to be similar in arousal.  Some authors have speculated that increased ‘eye openness’ reflects increased       13 attention to the environment (Sandem and Braastad, 2005). Increased attentiveness might be expected when animals are anxious but might also be expected when animals are anticipating positive outcomes, such as the opportunity to explore a novel environment or engage in social play. Thus eye white appears to be another non-specific indicator of affective arousal. One advantage of eye white measures is the fast response time. For example, changes in eye white following transition between stroking and not stroking took less than 30 s (Proctor and Carder, 2015b). Similarly, increases in eye white due to food deprivation were apparent after 1 min (Sandem et al., 2002). One study found that eye white in cows remained higher than baseline for 5.5 h following separation with their calf (Sandem and Braastad, 2005), but otherwise there is little evidence that changes in eye white reflect longer term events. Based on the evidence reviewed above, we suggest that measures related to the activation of the HPA axis and the sympathetic and parasympathetic nervous systems mainly allow inferences about affective arousal, with less basis for inferences regarding whether affect is positive or negative, let alone the specific affective state experienced.   Behavioural responses 1.2.2.1 Escape, wound directed and evoked behaviours Pain is often assessed using behaviours expressed by animals during and following a painful condition but not expressed by control animals (Millman, 2013; Weary et al., 2006). Escape behaviours provide one obvious example. For instance, calves disbudded with a hot-iron without local anesthesia rear and push (Stafford and Mellor, 2011; 2005), calves surgically castrated struggle and kick (Fell et al., 1986) and steers branded exert more force on a head gate and squeeze chute compared to steers receiving a sham treatment (Schwartzkopf-Genswein et al., 1997b).        14 Although these behaviours are associated with nociceptive processes (i.e. the sensory detection of physical damage) they may be less useful for drawing inferences regarding the affective component of pain (Sneddon, 2017; Weary et al., 2017); just as our immediate withdrawal reflex from heat is not evidence of a response relating the affective component of pain. Wound directed behaviours do allow inferences about the where of the affective experience, even if details about the what are less clear. For example, in the hours following hot-iron disbudding, calves show increased attention towards the injured area, including an increased frequency of ear flicks and head shakes (McMeekan et al., 1999; Sylvester et al., 2004; Winder et al., 2018). However, some variation in results might be explained by changes in behaviours less likely to be useful, such as changes in activity. For example, castration in lambs was associated with both decreased and increased physical activity depending on whether the surgical or rubber ring method was used (Mellor et al., 2000). More specific inferences about the where of pain may be possible in such cases if the specific anatomical structures involved in the movement are known. For example, an increased frequency of abdominal hunching (and decreased frequency of abdominal stretching) is expected in cases of injuries to abdominal muscles or inflammation of the visceral organs (Stojkov et al., 2015). In addition to these spontaneous responses, evoked behaviours (e.g. withdrawal in response to tissue stimulation) can also be used to make inferences about the where of pain. To draw stronger inferences about pain requires further interventions, for example, the use of appropriate analgesic treatments. Even in such cases, inferences regarding the affective component of pain will be limited, as some responses may simply reflect a nociceptive component of low intensity pain (as pointed out by       15 Stafford et Mellord (2005), interest in the scrotal area following castration may indicate ‘extreme pain or minor irritation, or anything in between’). 1.2.2.2 Vocalisations As described by Watts and Stookey (2000), cattle vocalisations can be seen as a subjective commentary by an individual on its own internal state. The challenge then lies in understanding that commentary. Vocalisations have been studied in contexts expected to be negative for cattle. For example, cows have been reported to vocalise when separated from their calves, and this response increases when cow and calf have been allowed to bond for several days (Flower and Weary, 2001; Stěhulová et al., 2008). Cattle also vocalize when food quantity is reduced (Schütz et al., 2013), as well as during and after painful procedures (Caray et al., 2015; Watts and Stookey, 1999).  However, the relationship between vocalisations and affect is far from straightforward. For example, increased calling is observed during estrus (Schön et al., 2007), where any link to affect is unclear. Increased vocalisations have also been observed in cows transitioned from tie-stalls to free-stalls (Pavlenko et al., 2018) and in calves during social play (Wagner et al., 2013), suggesting that a vocal response is not specific to negative events. Moreover, vocal responses are highly variable among individuals even in response to clearly negative events. For example, only 10% to 20% of cattle vocalize in response to castration (Stilwell et al., 2008), electric prodding (Grandin, 2001) and electro-ejaculation (Amaral et al., 2017). Of course, not all vocalizations have the same biological function, and more specific inferences about the nature of the affective experience may be possible by classifying vocalisations (e.g. by their spectral components; Green et al., 2018). For example, high-      16 frequency vocalisations have been observed in cattle during social isolation (Rushen et al., 1999), calf-dam separation (Johnsen et al., 2018; Padilla de la Torre et al., 2015), feed restriction (Thomas et al., 2001) and hot-iron branding (Watts and Stookey, 1999). Conversely, cows produce low-frequency calls when in contact with their calves (Padilla de la Torre et al., 2015) or when lying down in a cubicle while ruminating (Meen et al., 2015). These results are consistent with the idea that high-frequency calls relate to negative affect and low-frequency calls relate to more positive states, but other studies have reported no difference in maximum frequency of vocalisations produced during social interaction, sexual behaviour and behaviours such as fleeing (Meen et al., 2015). This lack of difference might be due to similar arousal levels (Green et al., 2018).  Although vocalizations vary widely among species (Green et al., 2018), some insights may be drawn from studies on different species. For example, in a study assessing affect in goats, the number of calls seemed to reflect arousal whereas variability in pitch seemed more related to valence (Briefer et al., 2015). More variable frequencies of ‘rumbles’ were also observed in elephants subjected to a negative social context (Soltis et al., 2011). Research on this topic remains sparse in dairy cattle and more studies are required to draw strong conclusions regarding arousal or valence in cattle. 1.2.2.3 Facial expressions Efforts to use facial expressions as proxies for affective states and animal welfare have been made (Descovich et al., 2017). Langford et al. (2010) were among the first to use facial expressions and to explore the existence of a ‘pain face’ in mice, which has been replicated in other species (Holden et al., 2014; MacRae et al., 2018; McLennan, 2018). Interestingly, orbital       17 tightening is one of the key facial action unit found in the human ‘pain face’ (Prkachin, 1992) and has been found in many species in response to pain, but has not yet been explored in cattle.  Work to date on dairy cattle has focused mostly on ear postures. Some authors suggested that ear posture could be used as valence indicator in cattle. For instance, one study found different ear postures expressed in response to a positive contrast (receiving a better food than expected) and a negative contrast situation (receiving unpalatable woodchips instead of food; Lambert and Carder, 2019). Both situations triggered arousal as shown by increased heart rates but different ear postures (positive: backward ear posture; negative: forward ear posture). However, backward ear postures have been found both when cows were in pain (Gleerup et al., 2015) and when stroked, which has been suggested to induce positive affective states of low arousal (Proctor and Carder, 2015b; Schmied et al., 2008). Thus, more work on of ear postures is required before strong inferences regarding affective valence can be drawn.  1.3 Learned responses Researchers have used responses that are shaped by the test situation and the animal’s awareness of the contingencies to ask animals three types of question: this or that, yes or no, and how much?   This or that? By allowing animals to choose, preference tests provide an intuitively compelling measure of how animals rank the options provided to them. For example, Telezhenko et al. (2007) provided group-housed cows the choice between rubber and concrete standing surfaces and found that most chose to stand on the rubber surface; it seems likely that this preference was driven by increased comfort associated with standing on the softer rubber. Interestingly, the preference for rubber standing surfaces was less pronounced among lame cows, likely because these animals       18 were less well able to compete for access to the preferred rubber mats; for studies looking at lame cows standing and bedding preferences while alone, see Jensen et al. (2015) and Bak et al. (2016).  The social environment is one of several potential confounders in such studies; other factors such as age of the animal, time of the day and novelty of the procedure can all affect preferences (Fraser and Nicol, 2011).  In another study, researchers assessed preferences for three different lying surfaces: sand, straw and rubber mats (Manninen et al., 2002). Cows preferred straw in winter but there was no preference during summer, illustrating that preferences can also vary over time and with environmental conditions. Indeed, for some types of preference test there is an expectation that they vary with environmental conditions. For example, as researchers predicted, cows preferred a water sprinkler more during warmer weather (Chen et al., 2013). Preference tests have been used to tackle many topics: which plants cows prefer to graze (Dwyer, 1961; Horadagoda et al., 2009; Rutter et al., 2004), which flavors they like best (Nombekela et al., 1994), how much do they favor variety in their food (Meagher et al., 2017), and how to improve milking systems (Hopster et al., 1998; Prescott et al., 1998), free stalls (Tucker et al., 2004) and water troughs (Teixeira et al., 2006). Despite the popularity of preference tests, they have only limited value in drawing inferences related to affect. The results provide a rank assessment of valence (option A is better than B) but both options could be good or bad, and the results often provide little insight as to importance of the preferred choice to the animal.       19  Yes or no?  By definition, rewards can be considered to be what an animal approaches and punishers as what an animal seeks to avoid (Brush, 1971; Rashotte, 1979). Approach and avoidance responses can thus provide insight into the affective valence associated with options provided to animals. For example, cattle that were shouted at or shocked with an electric prod at the end of a race took more time and required more force from the handler to be walked down this race compared to cows receiving a neutral control treatment (a person standing still), suggesting (as expected) that the animals consider being shouted at or shocked as unpleasant (Pajor et al., 2000). Similarly, cows kept a greater flight distance from a handler who had previously slapped and yelled at them compared to a neutral handler (Hötzel et al., 2005). Conversely, cows that were stroked by their handlers during milking displayed less avoidance than control cows when approached during feeding (Windschnurer et al., 2009). Approach and avoidance responses thus seem to be able to reflect the affect state associated with positive and negative events. Some approach / aversion findings share similar limitations to preference tests: for example, less avoidance can be interpreted only as ‘better’ and not necessarily as ‘good’. However, if cows not only display less avoidance, but also actively seek out their handlers (Bertenshaw and Rowlinson, 2008; Schmied et al., 2008), this provides stronger evidence that the interaction is accompanied by positive affect. Bringing cows into the hoof-trimming chute has been reported to require more forceful interactions (such as hitting the cows or twisting their tails) over time (Lindahl et al., 2016), indicating 1) that hoof trimming is aversive, and 2) that the animals learn to recognize cues associated with this event. This type of learned aversion can lead to a negative spiral, as increased baulking associated with the trimming leads to rough handling by staff, making the       20 procedure even more aversive for the animal. One way to avoid this spiral is to use counter conditioning, by training cows to associate the experience with an added reward. The most obvious example of this on dairy farms is providing a food reward to cows for entering the milking stall; even a small reward can elicit a marked increase in the cow’s motivation to enter (Ceballos and Weary, 2002). This approach leads to the questions of how much reward is required to counterbalance various negative events? We turn to this issue in the following section.   How much? Animals can be asked how much they are willing to work to access something they are motivated to acquire, or how much they are willing to give up to avoid something aversive, by using experimental approaches that create economic contingencies (Jensen and Pedersen, 2008; Kirkden and Pajor, 2006).  Active motivation tests (assessing willingness to ‘work’) can be illustrated through one of the most fundamental motivational relationships: hunger and food. As we would expect, cows that have been food-restricted for longer periods are more motivated to access food (e.g. by walking greater distances; Schütz et al., 2006). In another study, motivation to access fresh food was assessed following milking with the use of a push gate which was increasingly weighted (von Keyserlingk et al., 2017). All cows were willing to push around 20 kg to enter the area with the food, but as weight was added to the gate, fewer cows were willing to push it open to access the food. In the same experiment, the cows’ motivation to access pasture was evaluated. The weight cows were willing to push for fresh food was similar to that for pasture access (an average maximum weight of approximately 35 kg), suggesting that fresh food and pasture access were judged as similarly positive. Using the same methodology, cows showed motivation to       21 access fresh feed and a mechanical brush (McConnachie et al., 2018), and were willing to push a much greater weight to access either of these resources than to enter an empty pen that contained neither feed nor brush. Although motivation to access fresh feed and the brush were not examined in identical ways, cows were motivated for both. Future studies are needed to compare the motivations.  An advantage of the weighted gate is that the task is relatively intuitive for the animal, but one important limitation is that at higher weights cows might not push open the gate simply because they are physically unable to do so, and not because they lack the motivation. This type of limitation may create a ceiling effect, limiting the ability to distinguish among highly motivated options. Other types of operant tasks may be more suitable in such cases, such as panel presses. For example, Holm et al. (2002) used this approach to show that calves work more for full social contact with a peer compared to head contact only. Webb et al. (2014) also used a panel-press design, this time integrating preference and motivational testing. Calves were trained to work for different types of forage from two simultaneously available feeders. Calves had to pay varying costs (i.e. number of presses) to access the different feeds and the strength of preference was inferred by deviations from equality (i.e. not paying the same cost for both rewards). Although motivation to work is used as evidence that the resource is viewed positively, work itself can also be a positive experience, perhaps especially in under-stimulating environments. This phenomena, known as contrafreeloading, is demonstrated when animals work for a resource that is also freely available (Osborne, 1977; Van Os et al., 2018). Contrafreeloading could result in overestimating animal motivation to access the resource and illustrates the importance of including appropriate controls in these designs.        22 Another way to avoid this bias is to adopt a reversed approach and determine how much animals are willing to sacrifice. In a recent study, we trained dairy calves to approach a 1 L milk reward after 12 h of feed restriction. As expected, all calves were quick to approach the reward. These calves were then randomly assigned to 1 of 4 injection methods that they received upon reaching the milk (intramuscular, subcutaneous, intranasal and a control group that was not injected). Initially, injected calves did not differ from the control group in latency to approach the reward. However, as the volume of milk rewarded decreased, injected groups took longer to reach the reward, especially when calves were injected intramuscularly (Ede et al., 2018). By giving up a resource that was highly valued, calves demonstrated the price they were willing to pay to avoid injections, most likely in relation to the pain they associated with these injections. Another example of this ‘giving up something valued’ approach is an older study on stray voltage (Whittlestone et al., 1975). Cows were initially presented with two panels they could press, both with an equal probability of delivering a food reward (crushed barley). Then, one panel was switched off (i.e. it would not deliver rewards anymore) until cows directed around 90% of their presses to the other, still active panel. Once this threshold was reached, presses to the active panel were associated with electric shocks of increasing current until the cows switched their presses back to the unrewarded (and unelectrified) panel (Figure 1.2). This design was used to pinpoint what electric shock was sufficiently negative that the food reward was abandoned.        23  Figure 1-2. Example of operant motivation testing. Results adapted from Whittlestone et al. (1975). Cows abandoned their interactions with a panel delivering food rewards when it was associated with electric shocks of increasing current. In this example, the threshold current at which the cow switched to the non-electrified panel was 5.0 mA.  A potential limitation of learning paradigms is the development of ‘learned helplessness’ resulting in animals abandoning their attempts to avoid the negative event due to a perceived lack of control (Maier and Seligman, 1976); care is required to avoid confounding this phenomena with the event being perceived as neutral.  In summary, addressing the questions this or that, yes or no, and how much provides insight into what animals judge as positive or negative, and in the latter case, how important obtaining or avoiding the stimulus is to the cow.  1.4 Mood assessment Moods are considered as persistent emotional states that result from the accumulation of positive and negative emotional experiences (Boissy and Lee, 2014). Mood states are increasingly viewed as important for animal welfare (Paul et al., 2005). Studies have attempted       24 to assess changes in mood associated with housing conditions (Zidar et al., 2018), painful procedures (Neave et al., 2013) and chronic stress (Destrez et al., 2013) in farm animals. The majority of these studies have relied on cognitive approaches to assess these states.  Cognitive biases  Cognitive bias refers to how emotions can affect cognitive processes such as attention, memory and judgment (Paul et al., 2005). The literature provides a basis for making predictions about how affect, especially valence, induces positive or negative biases. We will explore how these methodologies have been used or could be used in cattle. 1.4.1.1 Attention Attention bias refers to ‘the differential allocation of attention towards one stimulus compared to others’ (Crump et al., 2018). Many studies have shown that humans and animals direct their attention to cues depending upon their affective state. Negative emotions trigger increased attention to negative information and potential threats, while positive emotions increase attention to positive information (Crump et al., 2018; Mathews and MacLeod, 1994). Different experimental designs have been used in animals (reviewed in Crump et al., 2018), but most aim at comparing the attention given to different cues, usually of positive or negative value, presented either separately or simultaneously. In this type of paradigm, measures of attention bias include what stimulus is looked at and for how long. One design used in farm animals, focuses on attention to a threat (i.e. a dog) by cattle and sheep approaching a food reward. Both species were more attentive and slower to eat the reward when an anxiogenic drug was administrated compared to controls (Lee et al., 2016, 2018), providing support for an affect-basis to these responses. In addition, Monk et al. (2018) suggest that attention bias tests can differentiate pharmacologically induced anxiety from depression, as depressed sheep increased       25 attention towards the threat whereas anxious sheep increased their attention towards a positive stimulus (i.e. a picture of a conspecific). Attention biases do not require extensive training and can thus be applied relatively easily, especially compared to other cognitive bias tasks (Crump et al., 2018). However, some studies have failed to detect mood-induced differences. For example, a study on European starlings found no effect of the birds’ emotional state (anxiety induced by threatening calls) on level of attention, perhaps because the negative stimulus was not sufficiently threatening (Brilot et al., 2009). Other studies have observed results that go in the opposite direction than expected. For example, Bethell et al. (2012) measured gaze in stressed vs. non-stressed macaques and found that the stressed monkeys gazed for a shorter duration at the threatening stimulus. The stressed monkeys might have displayed avoidance because they perceived the negative stimulus as more threatening. Work on humans has also found increased and decreased attention to different threatening cues (Cisler and Koster, 2010).  Attention bias tasks share features with some paradigms used for personality testing in animals (i.e. startle, novel object and unfamiliar human tests; Forkman et al., 2007). These tests rely on novelty, so responses are expected to change with repeated testing. How reliable attention bias tasks are over repeated testing is unexplored. Future research should use different designs (i.e. single vs. dual stimuli presentation and threat-directed attention) in different emotional-eliciting situations in dairy cattle. Previous research has reported that anxious cattle pay more attention to an immediate threat (i.e. a dog; Lee et al., 2018), but new research is needed to generalize this response to other negative affects.       26 1.4.1.2 Memory Emotions and moods can affect memory retrieval and storage, phenomena generally referred to as mood-congruent biases in memory (Paul et al., 2005). Positive affect is expected to improve storage and recall of positive memories and negative affect to improve storage and recall of negative memories (Burman and Mendl, 2018). Few studies have used mood-congruent memory biases to assess affective states in any animals (for a recent example on rats see Burman and Mendl, 2018), and to our knowledge no study has done so in farm animals.  1.4.1.3 Judgment Most studies using cognitive bias have focused on how decisions are affected by emotional state (Mendl et al., 2009). Animals are initially trained to discriminate between a cue associated with a positive outcome and another cue associated with a negative or a less positive outcome. Once trained, animals are subjected to ambiguous cues intermediate relative to the training cues. Animals show stimulus generalization (i.e. show intermediate responses to the intermediate test cues), but in some cases the responses are more positive (or negative) than expected, suggesting that the animals are expecting a positive (or negative) outcome. A positive bias can be interpreted as optimistic and a negative bias as pessimistic. An abundance of studies has shown a pessimistic response bias when animals were exposed to treatments expected to induce negative affect. For instance, work on dairy calves found a negative judgment bias in the hours following hot-iron disbudding (Daros et al., 2014; Neave et al., 2013), and separation from the dam (Daros et al., 2014). Work on other farm animals has explored the effects of different types of housing (pigs; Douglas et al., 2012), prenatal stress (lambs; Coulon et al., 2015) and chronic stress (sheep; Destrez et al., 2012). No study to date has examined judgment biases in adult dairy cattle.       27 Judgment bias tests allow for inferences regarding affective valence (Paul et al., 2005), but have several limitations (Roelofs et al., 2016). The most obvious of these is the need to train animals to discriminate between the training cues. An important premise of judgment bias tests is that test cues are ambiguous. However, with repeated testing animals may learn to recognize these cues. For example, Barker et al. (2018) found decreased responsiveness to (unrewarded) ambiguous cues following repeated presentations. Different strategies have been used to mitigate this effect, including partial reinforcement of training cues (Neave et al., 2013) and reducing the number of ambiguous cues presented during testing (Hintze et al., 2018); these methods are likely to slow learning but not entirely inhibit it. Another limitation is that changes in response rate may reflect changes in how much animals value the reward rather than changes in their expectation of a reward; for example, feed motivation can vary with mood, as described in the next section. Finally, future studies should explore whether judgment bias tests are sensitive to moods of different intensity. An interesting approach would be to compare animals that receive different post-operative pain control medications after disbudding. We predict that animals with more efficient pain-control would show lower judgment bias compared to control animals (receiving only a sedative and a local block). If true, this evidence would suggest that judgment biases can be used to draw inferences about valence and intensity of the mood change.  Anhedonia Anhedonia, defined as a reduced ability to pursue and enjoy pleasurable activities (Treadway and Zald, 2011), is a sign of depression in humans. Propensity to forgo pleasurable activities has been explored in laboratory rodents, mainly assessed using the consumption of a sweet solution (Rizvi et al., 2016). Surprisingly little work has focused on anhedonia in farm animals. One exception is a study by Figueroa et al. (2015), showing that restraint and social       28 stress decreased consumption of a sweet solution in pigs. No research to date has used this approach in cattle but a recent study showed that dairy calves reduce their interest in the positive cue (i.e. milk) in a judgment bias test 6 h after hot-iron disbudding (Lecorps et al., 2019), a result consistent with the existing models of anhedonia. Other work on cattle has measured changes in other behaviours assumed to be pleasurable. For example, play behaviour has been observed to decrease after hot-iron disbudding (Mintline et al., 2013; Rushen and de Passillé, 2012) and weaning (Rushen and de Passillé, 2012).Thus, measures of anhedonia seem to show some promise for assessing affective valence in dairy cattle.  1.5 Pharmacological interventions Drugs with known effects on emotional states in humans can be used to study affective states of animals. In cattle, the inclusion of drugs has mostly focused on sedatives (e.g. xylazine), anesthetics (e.g. lidocaine) and analgesics (e.g. meloxicam, ketoprofen) in the study of painful procedures (Adcock and Tucker, 2018). These studies typically rely upon physiological and acute behavioural responses discussed above, and thus may be of limited value for drawing inferences about the felt component of affective states.  More sophisticated approaches allow for stronger inferences. For example, Van Reenen et al. (2009) reported that calves treated with the anxiolytic drug brotizolam spent more time near, and in contact with, a novel object compared to calves treated with saline, suggesting that interaction with a novel object is related to fear in calves. Another interesting approach are self-selection protocols, for example lame chickens were found to prefer eating feed spiked with an analgesic (carprofen) rather than un-spiked feed; whereas, healthy chickens showed no preference (Danbury et al., 2000). We note that other authors have failed to replicate these results (Siegel et al., 2011). Following the same principle, mice housed in unpredictable conditions       29 displayed a preference for water spiked with an anti-anxiety drug (midazolam); whereas, mice housed in a predictable environment did not (Sherwin and Olsson, 2004). The strength of this approach is that it addresses the question of what (pain in the case of selection of analgesics, fear in the case of anxiolytics) and yes/no/how much (by assessing preference and sometimes motivation). Unfortunately, experiments demonstrating self-selection of drugs have not yet been reported in cattle. Given the strength of inferences allowed by this approach, it seems odd that so few studies have been published. We speculate that the dearth of studies is due to the difficulty in training animals to associate ingestion of a drug (typically delivered in feed/water) with the delayed effects of the drug, a delay likely accentuated due to the dilution of pharmaceutical agents in cattle’s large digestive track. Yet, self-selection paradigms still show potential; work on cattle and other ruminants suggests that they are able to vary their dietary choices to self-medicate, for example, by increasing roughage intake after sub-acute ruminal acidosis induction (Keunen et al., 2002; but see also Paton et al., 2006).   Another experimental approach (known as ‘drug naming’) relies upon animals comparing their current affective state with that induced by a drug. For example, rats were trained to press specific levers depending on whether they were treated with saline or an anxiogenic drug (pentylenetetrazole). Following exposure to a cat (also assumed to be anxiogenic), rats displayed the response associated with pentylenetetrazole (Gauvin and Holloway, 1991). To our knowledge, such a paradigm has not yet been explored in cattle.  1.6 Discussion  Spontaneous physiological and behavioural responses can be helpful in the study of affect, but most measures are more reflective of arousal. This means that these measures may be useful in showing the intensity of an affective state (especially during acute events), but less       30 valuable when investigating if something is experienced positively or negatively. In scenarios where the valence of an event is intuitive (e.g. dehorning without pain control), arousal responses might be sufficient, but researchers need to be aware of any ceiling effects and consider that high arousal states are unlikely to be maintained over long periods of time due to metabolic limits.  Preference tests, approach/aversion and motivational tests allow for stronger inferences regarding affective valence, but these tests also have limitations. First, many of the tests rely on comparative judgments, which do not allow inferences on absolute valence (i.e. positive or negative), only the relative value (i.e. better or worse). Furthermore, any test requiring animals to perform a task (which includes mood assessments such as cognitive biases and anhedonia tests) may be difficult in bored, lethargic, ill or postpartum individuals. More generally, tests involving activity should consider that animals’ ability to move might be compromised, especially after surgical procedures or drug use, potentially confounding results. On a more fundamental note, motivational paradigms provide a clear estimate of what animals want but this might not be identical to what animals like (Gygax, 2017; Pool et al., 2016). Pharmacological interventions, such as self-selection and drug-naming paradigms, allow for inferences regarding specific affective states (e.g. anxiety versus pain), rather than just valence. Again however, there are limitations. For instance, some drugs are initially aversive and then become appetitive (Tzschentke, 1998). Other drugs may have muscle relaxing or memory altering properties (Hall et al., 2001), making it difficult to draw inferences without the use of well-considered controls. Moreover, most drugs have not been validated in cattle and assumptions regarding efficacy across species require caution, especially when interpreting null results.       31 Paradigms based on learned responses rely on the animal’s capacity to learn a connection between their behaviour and an outcome, and thus also on the researcher’s ability to allow the animal to learn. Training typically requires some knowledge of the animal (e.g. regarding vision, smell, hearing, social structure, locomotion, etc.) and researchers need to reflect upon the suitability of training methods when faced with null results. Among the methods presented, it can be difficult to decide which to choose when attempting to assess affect. We suggest that researchers refrain from including many measures in a single experiment as this practice increases the risk of ‘false positives’, weakening the reliability of conclusions. Rather, we encourage researchers to limit their focus to just one or a few measures with clearly stated predictions.  1.7 Conclusions Within the valence/arousal framework, spontaneous behavioural and physiological responses more likely reflect arousal than valence. Paradigms relying on learned responses such as preference tests, approach/aversion, motivation and mood assessments appear to be more valuable when assessing valence. The use of drugs with known effects on emotional states in humans can allow for inferences regarding specific affective states in animals.  1.8 Thesis objective The aim of this PhD will be to explore the affective states of dairy calves exposed to common painful practices. The focus will be on the study of valence of the calves’ subjective experience rather than its arousal levels. Thus, learned responses will be utilized, with and without the use of pharmacological interventions. More specifically, paradigms relying on place aversion will be used. Two pain models will be investigated: first, the acute pain associated with needle injections. Second, the longer-term pain resulting from hot-iron disbudding.       32 Chapter 2: Approach-aversion in calves following injections A version of Chapter 2 has been published as Ede, Thomas, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Approach-Aversion in Calves Following Injections”. Scientific Reports 8, no 1 (2018): 9443. https://doi.org/10.1038/s41598-018-27669-7. 2.1 Introduction Injections by needle are widely used in veterinary practice for the administration of medicine, vaccination and anaesthesia. The pain caused by injections has been studied in humans (Barnhill et al., 1996; Dodd, 1945; Eland, 1981; Mitchell and Whitney, 2001; Nishioka, 2000) but little is known about how aversive these procedures are to animals. Research on acute noxious stimuli in animals often relies upon physiological indicators and behaviour measures including withdrawal movements, writhing and defensive behaviours (Bars et al., 2001; Weary et al., 2006). However, both approaches have questionable specificity (Bars et al., 2001) and there are difficulties in drawing inferences regarding affect or motivation from such responses (Mogil, 2009).  Conditioning paradigms provide an alternative approach (Li, 2013). A multitude of experimental options exists (Brush, 1971), some relying on the animals actively performing a behaviour (such as pressing a lever, pecking a key, or jumping a barrier) to avoid an aversive treatment. However, animals may find it difficult to learn active responses in a stressful environment (Rushen, 1986b). An alternative is to implement a passive avoidance paradigm in which animals are first trained to carry out a motivated behaviour (such as accessing a food reward) that is then associated with a negative event. The animal can then choose whether they are willing to pay the price of enduring the negative event to gain access to the reward. The animal’s reaction to this conflict provides insight regarding motivational balance; i.e. the relative       33 value allocated to the reward and the negative event. If the animal begins to avoid the treatment, it is reasonable to infer that the animal finds the treatment more aversive than it finds the food rewarding (Rushen, 1996). By providing control to the animal, this design also minimises other sources of distress (Rushen, 1986b).  Conditioning paradigms have been used to assess a broad range of experiences, including those induced by drugs (Tzschentke, 1998), chronic arthritis (Colpaert et al., 2001), electric shocks (Miller, 1960; Seligman and Campbell, 1965), mechanical hyperalgesia (Hummel et al., 2008) and radiation (Garcia et al., 1957). More recently, with growing public concern over animal husbandry practices (Rollin, 1995; Rushen, 2008), conditioning has been used to assess the aversiveness of procedures used for euthanasia (Cooper et al., 1998; Wong et al., 2014), cage-cleaning (Rutter and Duncan, 1992) and handling (Rushen, 1986c). To our knowledge no work to date has addressed aversion due to injections.  The purpose of this study was to assess the aversiveness of intramuscular (IM) injections, and to compare this with two possible refinements: intranasal (IN) and subcutaneous (SC). Calves were first trained to approach a milk reward. After training was completed, accessing the reward was paired with an injection and aversion was measured as an increase in the approach latency. The quantity of milk provided was gradually reduced to better assess the motivational balance between accessing the reward and avoiding the treatment. Intramuscular injections, and to a lesser extent subcutaneous and intranasal injections, were expected to increase approach latencies relative to control calves that received no injection. 2.2 Methods This study was conducted from January to June 2017 at the University of British Columbia Dairy and Education Center in Agassiz, British Columbia. The study was approved by       34 the UBC Animal Care Committee (Application A16-0310) and performed in accordance with the guidelines outlined by the Canadian Council of Animal Care (2009).  Animals, housing and treatments 24 Holstein calves were enrolled into the experiment at 22 ± 11(mean ± S.D.) days of age. Calves were housed in groups of 10 in pens measuring 4.9x7.3m bedded with 10 cm of sawdust. Ad libitum access to water and hay was provided through automatic feeders (RIC; Insentec B.V., Netherlands). A daily whole milk ration of 12 L was available through an automatic milk dispenser (CF 1000 CS Combi; DeLaval Inc., Sweden). Milk was not accessible in the 12 h before the test session began at 0900 h. The calves were returned to the group pen after the test session and received their full milk ration for the rest of the day.  Each calf was randomly assigned to one of four treatment groups: intramuscular (IM), intranasal (IN), subcutaneous (SC) or control. Each treatment group received 0.5 mL of saline solution. IM and SC groups were injected in the rump with a 0.9x40 mm and 0.9x20 mm hypodermic needle respectively (Covidien 8881251766/8881251782). The IN group received the solution in the right nostril through a vaccination nasal tip (HTI Plastics, A003-A00-06).  Apparatus  Figure 2-1. The experimental apparatus was divided in two areas: the Lobby measured 1.9 x 2.4 m (4.6 m²), had unpainted plywood walls and concrete flooring covered with 10 cm of sawdust, and was accessible through Gate 1. On the side opposite to the entrance was Gate 2, allowing access to the Test pen. At the end of the Test pen (opposite to Gate 2) was a milk bottle with the teat positioned 80 cm above the floor. The Test pen was identical to the Lobby except that walls were mounted with colored panels, either white or red depending on the experimental       35 phase. After bringing the calf into the lobby, the handler stood outside the apparatus next to the bottle and operated the gates remotely.  Figure 2-1. Experimental apparatus.  Procedure and measurements Each calf was tested individually; calves were moved gently from their home pen and brought to the lobby.  Once in the lobby, the gate to the test pen (Gate 2) was opened and the calf was allowed to enter. Approach was characterized by the latency to start drinking the milk reward after Gate 2 was opened. If the calf did not approach the bottle within 5 min, the calf was brought back to her home pen and latency was recorded as 301 s. 2.2.3.1 Habituation  During the habituation phase, 1 L of milk was placed in the bottle. Calves were allowed to approach and drink the reward without the administration of an injection. The panels mounted on the walls of the testing pen were white. Habituation ended once the calf spontaneously approached the bottle in less than 30 s and drank the full 1 L of milk, 3 d in a row.         36 2.2.3.2 Treatment phase Once the habituation was completed, the panels in the test pen were switched from white to red. This visual cue was introduced to facilitate the association. Previous work on Holstein calves has successfully used these same colors in a discrimination learning task (Neave et al., 2013). During this phase, calves received their assigned treatments as soon as they started to drink from the bottle. The milk reward was decreased every 3 d from 1 L for the first 3 sessions, to 500 ml, then to 250 ml, and finally to 0 ml for the last 3 sessions.  Statistical treatment The three latencies obtained for each reward quantity were averaged to create a mean per calf per milk reward level. Data were log-transformed to normalize residuals (Shapiro-Wilk normality test, before transformation: W = 0.89, P < 10-5; after transformation: W = 0.98, P = 0.20). The transformed data were then modeled with a linear mixed-effects model (Bates et al., 2015; R Core Team, 2015) that included as fixed effects the route of injection (3 df), the volume of milk reward (1 df) and the interaction (3 df). Calves were considered a random effect. 2.3 Results During the last three days of habituation, calves assigned to the different treatments did not differ in latency to approach the bottle (F3,20 = 0.52, P = 0.7).  The latency for calves to approach the milk bottle increased as the milk reward declined, but the magnitude of this decrease varied with treatment (Figure 2-2), as indicated by the milk reward x treatment interaction (F3,68 = 3.3, P = 0.02). On the basis of this interaction, we analyzed the effect of treatment separately for each level of milk reward, using specified contrasts to compare each injection treatment with the control. None of the injection methods resulted in an increased approach latency relative to the control when the milk reward was 1 L       37 (IM: t1,20= 1.4, P = 0.2; IN: t1,20 = 0.1, P = 0.9; SC: t1,20 = 1.1, P = 0.3) or 0.5 L (IM: t1,20= 1.5, P = 0.2; IN: t1,20 = 1.3, P = 0.2; SC: t1,20 = 1.2, P = 0.3). For the 0.25 L reward, only calves in the intramuscular treatment showed higher approach latencies compared to control calves (t1,20= 2.1, P = 0.05).When no milk reward was provided, calves in both the intramuscular and intranasal treatments showed longer approach latencies relative to the control calves (IM: t1,20= 3.4, P < 0.01; IN: t1,20 = 2.7, P = 0.01), with three animals reaching the 5 min threshold for the intramuscular group and one for intranasal. In contrast, the approach latencies for calves in the subcutaneous treatment did not differ from that of the controls (t1,20 = 1.4, P = 0.2).  Figure 2-2. Latencies of calves to approach a milk reward, depending upon size of the reward and treatment. (IM: Intramuscular, SC: Subcutaneous, IN: Intranasal). The latencies are log transformed and presented on an exponential transformed y-axis. The dotted line represents the latency limit.        38 2.4 Discussion When no treatment was administered, calves were highly and consistently motivated to access the milk reward. In contrast, groups receiving injections took longer to approach the reward, indicating a motivational shift from the approach of the milk to the avoidance of the procedure. The higher latencies recorded for the intramuscular treatment support our prediction that intramuscular injections are more aversive than the intranasal and subcutaneous routes.  The age of the animals was chosen to match that when dairy calves normally first experience injections (such as those associated with vaccination and disbudding). Adult animals also receive injections (for example, routine injections used in synchronized reproduction programs). Considering the development of thicker skin and adipose tissue as animals age, a stronger needle and greater force is required to pierce the tissue, perhaps affecting the relative aversion associated with different techniques. Similarly, different breeds and species may require different needles, forces, etc. Animals are also likely to vary in nose sensitivity affecting responses to the different treatments. Considering the frequent use of injections in veterinary practice, the literature is surprisingly sparse in regard to the aversion to the procedure and to potential refinements. Previous work has shown that 78% of dogs exhibit fear-related behaviour on the examination table of a veterinary clinic (Döring et al., 2009), and that 26% of dogs “yelp” when injected (Stanford, 1981), suggesting the need to consider less aversive methods. Aside from the obvious welfare implications of decreasing pain, better acceptance of injections may also facilitate handling, ensure a safer environment for the staff and likely improve the veterinary experience for the client (Döring et al., 2009).  As an alternative to physical restraint, positive reinforcement techniques can be used to train animals to voluntary receive injections (Videan et al., 2005). Although cooperation from the       39 animal can be achieved for intramuscular injections, only half as many training sessions are needed for subcutaneous injections (Schapiro et al., 2005).   In humans, verbal reports from patients show that intramuscular injections are more painful than the subcutaneous (Berteau et al., 2015; Schen and Singer-Edelstein, 1981; Workman, 1999) and intranasal alternatives (Suzuki et al., 2006; Wilson et al., 1997). Intramuscular injections cause tissue damage (Brazeau and Fung, 1989; Rasmussen, 1978), reach cells directly involved in inflammatory processes (Tidball, 2005), and may damage nerves (Bergeson et al., 1982; Small, 2004), whereas subcutaneous injections puncture only the epithelium. Pain is not limited to the puncture: injections of drugs such as antibiotics, anaesthetics and vaccines generally lead to more pain in humans when administered intramuscularly, which is thought to be linked to high innervation of muscle tissues (Brazeau et al., 1998). Many species are known to have highly innervated muscle, so we expect our results with saline in calves to be consistent with various drugs and species. That said, we call for study of drugs on a case-by-case basis, as the aversiveness can be modulated by factors such as pH and volume of the solution injected (Brazeau et al., 1998). This study was limited to the treatment of the same volume through the different routes. However, the use of an alternative route might require a modified protocol to achieve similar efficacy. For example, intranasal treatments are not appropriate to all compounds as they must be able to cross the nasal epithelium (Grassin-Delyle et al., 2012), but when applicable the intranasal route can provide higher efficacy (Kimman et al., 1989). A number of studies have reported similar efficacy when compounds were delivered via intramuscular and subcutaneous routes (Brown S. A. et al., 2008; Conlon et al., 1993; Kaartinen L. et al., 1995).       40  The ability of avoidance paradigms to discriminate between treatments is sometimes limited by a ceiling effect causing “all or nothing” responses (Rushen, 1996). For example, 80% of rats have been observed to completely stop moving (i.e. “freeze”) when exposed to an environment associated with electric shock (Bolles and Collier, 1976). These all or nothing responses can be prevented through experiments eliciting conflict behaviours (Miller, 1951), such that rather than only avoiding a negative event animals are also motivated to approach a positive one (Rushen, 1986b). The difficulty lies in finding a conflicting situation that does not lean too much towards the positive (leading to the “all” response) or the negative (leading to the “nothing” response). Our results indicate that starting with a highly appetitive reward and then gradually reducing this, while keeping the treatment (i.e. the injection) constant, allows for a sensitive test of differences in aversion between treatments.  Multiple pairings may be required for animals to associate treatment and location. This means a difference in approach latency might have been observed between the treatment groups at earlier rewards if more training sessions had been allowed. The current study limited number of sessions for ethical reasons (i.e. to limit the number of injections delivered to each animal), but studies using less invasive treatments may wish to consider more sessions at each reward level tested. By confounding the declining reward with time, our design made it impossible to distinguish between the effects of learning and the decline in reward size. It must emphasised that the existence of this confound was intentional; our aim was to compare treatments, not determine the precise quantity of milk calves were willing to give up to avoid each kind of injection. When the goal is to assess what quantity of reward is needed to overcome a negative event, it may be better to keep the reward constant (Ceballos and Weary, 2002).        41  Finally, our sample size (6 calves/treatment group) was justified by the ethical interest to reduce the number of calves receiving painful injections. However, this low number of subjects could cause justifiable concern over the replicability of the results.  The results of the current study suggest that subcutaneous and intranasal routes are refinements over intramuscular injections. Pharmacokinetics and dynamics will vary with route of administration, but where feasible we recommend these alternatives to minimize aversion caused by injections.       42 Chapter 3: Calf aversion to hot-iron disbudding A version of Chapter 3 has been published as Ede, Thomas, Benjamin Lecorps, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Calf Aversion to Hot-Iron Disbudding”. Scientific Reports 9, no 1 (2019): 5344. https://doi.org/10.1038/s41598-019-41798-7. 3.1 Introduction  Horned cows can be a safety concern for pen mates and their handlers, so the developing horn buds of dairy calves are typically removed via hot-iron cauterization (known as “disbudding”, or “dehorning” when done at a later age) (Cozzi et al., 2015). This procedure is painful (Fajt et al., 2011; Wikman et al., 2016) and methods to reduce or prevent this pain have gathered considerable scientific interest (Grøndahl-Nielsen et al., 1999; Stewart et al., 2009; Stock and Coetzee, 2015). The procedure can be refined by reducing stress due to handling (using a sedative such as xylazine to facilitate handling and provide weak analgesia) and by treating the intra-operative pain (using local anesthetics such as lidocaine to numb nerves serving the affected tissues (Graf and Senn, 1999)). Post-operative pain can also be partly mitigated with analgesics such as nonsteroidal anti-inflammatory drugs (NSAIDs) (Allen et al., 2013; Duffield et al., 2010) , but these are rarely provided on dairy farms (Fulwider et al., 2008; Gottardo et al., 2011; Misch et al., 2007) and are unlikely to relieve all the pain associated with the procedure (Herskin and Nielsen, 2018).  Previous work on pain associated with disbudding has primarily focused on physiological measures, especially the cortisol response: a recent meta-analysis on cautery disbudding reported that 19 out of 21 studies included cortisol measures (Winder et al., 2018). However, measures related to the hypothalamic-pituitary-adrenal (HPA) axis likely reflect arousal rather than emotional valence (i.e. whether something is experienced as positive or negative) (Rushen,       43 1986a), a key concern for animal welfare (Fraser, 2008; Yeates and Main, 2008). For example, stallions exhibit increased secretion of cortisol in response to restraint (negative event) but also in response to sexual stimulation (positive event) (Colborn et al., 1991).   Also commonly used are “pain-related” behaviours such as ear flicks, head shakes, or head rubs (Doherty et al., 2007; Duffield et al., 2010; Faulkner and Weary, 2000). Such responses can be informative regarding the acute sensory-discriminative aspect of the procedure (i.e. nociception), but similar behaviours are sometimes also expressed by anesthetized or decerebrate animals (Matthies and Franklin, 1992; Sandkühler and Gebhart, 1984; Woolf, 1983) suggesting some difficulty in drawing strong inferences regarding the affective-motivational component of the experience (Mogil, 2009).  Finally, more complex spontaneous behaviours such as feed consumption (Bates et al., 2015), movement (Theurer et al., 2012) or play (Mintline et al., 2013) have also been used. Such behaviours are usually related to activity, and their reduction following disbudding can be interpreted as an attempt to avoid stimulation of the painful area.  To assess the emotional dimension of pain, researchers are increasingly encouraged to include learnt responses, for example using conditioning paradigms (Herskin and Nielsen, 2018; Li, 2013; Weary et al., 2017). More specifically, conditioned place avoidance paradigms allow inferences regarding whether an event was experienced negatively. Such paradigms rely on the animal developing an association between a specific environment and a negative experience. For example, rats will avoid an environment associated with the ingestion of an emetic agent (Symonds and Hall, 1997) and zebrafish will avoid the side of a tank associated with anesthetic agents (Wong et al., 2014). Conditioned place avoidance has also been used to specifically assess pain. For instance, Sufka (1994) showed that rats avoid an environment where they experienced       44 inflammatory pain, but show a reduced avoidance when the pain was mitigated with an analgesic. To our knowledge, place conditioning has not yet been applied to evaluate pain in cattle.  Our aim was to use a place conditioning paradigm to assess aversion associated with hot-iron cautery (when mitigated with a sedative and local anesthesia) versus aversion to a sham procedure involving sedation only and no hot-iron cautery. We predicted that calves would show conditioned aversion to the pen where they were disbudded and allowed to recover compared to the pen where they experienced the control procedure. 3.2 Methods  Ethical statement The study was approved by the UBC Animal Care Committee (Application A16-0310) and performed in accordance with the guidelines outlined by the Canadian Council of Animal Care (2009).  Animals and housing  This study was conducted from October to December 2017 at the University of British Columbia Dairy and Education Centre in Agassiz, British Columbia. Thirteen female Holstein calves with a birthweight of (mean ± SD) 37.2 ± 5.0 kg were randomly enrolled at (mean ± SD) 35 ± 14 d of age. Four animals were excluded from the experiment (cf statistical analysis).  Animals were individually housed in sawdust bedded pens (2.1 x 1.2 m) for the first 5 d. At day 6, calves were paired in a double sized pen (2.1 x 2.4 m) with another calf of approximately the same age and weight, both calves of the pairs were enrolled. Calves were fed 4 L of whole milk twice a day using a nipple bottle (at 0800 and 1600 h) and had ad libitum access to hay, grain and water in their housing pen.       45  Apparatus  The apparatus was a 2.1 x 6.0 m plywood pen divided in three equal areas (2.1 x 2.0 m; Figure 3-1). Two ‘treatment’ pens had colored panels on the walls (either 3 red squares or 2 blue triangles on each wall) and were connected by a ‘neutral’ pen with removable gates. The visual cues (distinct colors, shapes and numbers) were intended to help calves establish an association between pen and treatment. A chute was positioned outside the apparatus, facing the entry gate to restrain the calves before they entered the apparatus.  Figure 3-1. Experimental apparatus. During Treatment sessions, calves were locked in their assigned treatment pen for 6 hours. During pre-treatment exposure and test sessions, removable gates were taken out to allow calves to freely roam between pens until they chose to lay down. Drawn with the help of Shirley Ho.        46  Protocol The experiment consisted of three phases: pre-treatment exposure (one session), treatment (two sessions: control and disbudding) and test (three sessions: 48 h, 72 h and 96 h after the second treatment).  3.2.4.1 Pre-treatment exposure  To avoid the potential influence of novelty on place conditioning (Tzschentke, 1998), calves were pre-exposed to the apparatus for a single session (Sufka, 1994). No additional pre-exposure sessions took place to avoid the weakening of the subsequent conditioning (Bardo et al., 1995).  Calves were always studied individually. During pre-exposure, at approximately 11:00 h the calf was gently brought from its home pen to the chute in front of the entrance to the apparatus (around 20 m) where she received a 0.25 L milk reward from a nipple bottle. The calf was then let into the apparatus with all compartments accessible. Time spent in each compartment was recorded (calves were considered in the compartment when both front legs were inside) for 15 min. The calf was then let out and brought back to its home pen. Treatment pen assignment was counterbalanced with the preference measured during pre-treatment exposure: half the calves were disbudded in the preferred pen and the other half was only sedated. The pen associated with disbudding (blue triangles or red squares) and order of treatment were assigned in blocks and counterbalanced.  3.2.4.2 Treatments (1 and 2) Twenty-four hours after pre-treatment exposure, the calf was brought from its home pen and injected with xylazine (0.1 mg/kg BW, Rompun, 20 mg/mL, Bayer, Leverkusen, Germany) subcutaneously on the right side of the rump while receiving a 0.25 L milk reward in the chute.       47 The subcutaneous route was chosen as it has been found to be less aversive (Ede et al., 2018) but as effective as intra-muscular delivery (Ede et al., 2019c). The calf was then led into one of the treatment pens (and given the local block, shaved and disbudded if during a disbudding session) and locked into this pen for the next 6 h, such that the calf was able to associate with the pen the recovery from sedation and post-operative pain. The calf was then let out and brought back to its home pen. The next conditioning session took place 48 h later, during which the calf was exposed to the treatment and pen opposite to that used in the previous session.  During disbudding sessions, approximately 10 min after the injection of xylazine in the chute, sedation level was verified as defined in Ede et al. (2019c). If the calf was recumbent with a noticeable eyeball rotation, she received a local block applied on the corneal nerve by injecting 5 mL per side of lidocaine (Lido-2 [Lidocaine 2%, Epinephrine 1:100,000], Rafter8, Calgary, Canada) with a 0.9 mm x 25 mm needle (8881251782, Covidien, Dublin, Ireland) inserted in the depression between the lateral canthus of the eye and horn bud. Five minutes after the lidocaine injection, the area around the horn buds was shaved and calves were disbudded using a hot iron (X30, 1.3 cm tip, Rhinehart, Spencerville, IN, USA) preheated to approximately 500°C and applied for approximately 15 s. Although it was not done in this study, we recommend assessing the efficacy the local block prior to disbudding by testing the responsiveness of the calf to a needle prick (Sylvester et al., 1998). 3.2.4.3 Tests (1, 2 and 3) Conditioned-place-aversion testing took place in 3 sessions at 48, 72 and 96 h after the second treatment (at approximately 11:00 h). The test procedure was identical to that during pre-treatment exposure (i.e. free roaming between pens) but lasted 90 min or until the calf lay down       48 (i.e. sternal or lateral recumbency) for at least 1 min (all but one calf lay down within 90 min). Calves were then returned to their home pen.  Statistical analysis  Using the power.t.test R function (R Core Team, 2015), a sample size of 10 animals was recommended for a power of 0.8, significance level of 0.05 and effect size equal to the standard deviation for paired t-tests. Unfortunately, 4 out of the initial 13 calves were excluded from the experiment. One fell sick after the first test (lethargic, rectal temperature of 40.5°C), two jumped out of the apparatus during the first treatment session and one remained immobile during the pre-treatment exposure. These last three animals may have been particularly sensitive to the test apparatus and the associated social isolation.   Time spent in the pens was analyzed using a linear mixed-effects model using the lme4 R package (Bates et al., 2015) testing the fixed effects of treatment received in the pen (Disbudding, Control or Neutral [middle pen]; 2 df), test session (i.e. 1st, 2nd or 3rd test; 1df), their interaction (2 df), treatment order (disbudding occurred during the first versus second treatment; 1 df) and the color associated with disbudding (red or blue; 1 df). Calves were considered a random effect, resulting in 81 repeated observations: 3 (time spent in each pen) x 3 (test session) x 9 (calf). Normality and homoscedasticity of residuals were examined both statistically and graphically. Time data was transformed with a square-root transformation to achieve normality (Shapiro-Wilk normality test, before transformation: W = 0.94, P = 0.01; after transformation: W = 0.99, P = 0.9). P-values were obtained with Satterthwaite’s approximation using the lmerTest R package (Kuznetsova et al., 2017). A second similar model only focused on the time spent in the two treatment pens (Disbudding and Control; 1 df). Normality and homoscedasticity of residuals of this second model were also achieved by transforming the time data with a square       49 root transformation.  The pen in which the calves chose to lay down, in relation to treatment experienced in the pen, was analyzed using a chi-square test.  3.3 Results  Pre-treatment exposure  Time spent in the pen where the calves would experience disbudding versus time spent in the pen where they would experience the sham procedure were not found to differ during pre-treatment exposure (paired t-test, t1,8 = -0.3, P = 0.8), indicating that there was no pen bias before calves experienced the treatments.   Tests Order of treatment and color of the pen associated with disbudding had no effect on where calves spent their time (t1,6 = 0.1, P = 0.9; t1,6 = 0.6, P = 0.6, respectively). Calves spent less time in the treatment pens compared to the middle pen (Control pen: t2,67 = -3.3, P = 0.001; Disbudding pen: t2,67 = -5.4, P < 0.001; Figure 2). Compared to the first session, there was a tendency for the calves to spend more time in the disbudding pen during following sessions (t2,67 = 1.7, P = 0.1).   When only focusing on time spent in treatment pens (second model), order of treatment and color of the pen associated with disbudding also had no effect on how the calves spent their time (t1,6 < 0.1, P > 0.9; t1,6 = 0.4, P = 0.7, respectively). Calves spent less time in the disbudding pen compared to the control pen (t1,43 = -2.4, P = 0.02). When analyzing test sessions individually with paired t-tests (R Core Team, 2015), calves spent significantly less time in the disbudding pen compared to the control pen during the first session (t1,8 = -9.8, P < 0.001), tended to spend less time in the disbudding pen compared to the control pen during the second       50 session (t1,8 = -1.7, P = 0.1) and no difference was found during the third session (t1,8 = -0.03, P > 0.9). See figure 3-2.  Figure 3-2. Time (√s) that calves spent in the different pens during test sessions relative to the treatment they received in that pen during treatment sessions. (Test 1: 48 h, Test 2: 72 h, Test 3: 96 h after the second treatment). Neutral: No treatment. Control: Sedation alone. Disbudding: Sedation, local anesthesia and hot-iron disbudding. All times spent in treatment pens (control and disbudding) were lower than time spent in the middle pen (P < 0.05). The asterisk (*) represent a significant difference (P < 0.05) of the time spent in the disbudding pen compared to time in the control pen. (+) represents a tendency (P = 0.1).  Calves lay down less frequently in the pen where they had experienced and recovered from hot-iron disbudding (Figure 3-3) compared to the other pens. Across all three test sessions, a calf lay down in the pen associated with disbudding just once, compared to 10 times in the pen associated with sedation and 15 in the middle pen (χ² = 11.6, P = 0.003).       51  Figure 3-3. Pen in which calves lay down during test sessions relative to the treatment they received in that pen during treatment sessions. (Test 1: 48 h, Test 2: 72 h, Test 3: 96 h after the second treatment). Neutral: No treatment. Control: Sedation alone. Disbudding: Sedation, local anesthesia and hot-iron disbudding. In the first test only 8 of the 9 calves lay down within the 90-min limit.  3.4 Discussion  Calves spent more time (and lay down more often) in the middle pen than in either of the treatment pens, which could indicate both disbudding and the control procedure to be aversive. However, as the calf was placed in the middle pen at the start of the test, and the calves had to go through the middle pen to move from one treatment pen to another, interpreting both procedures as aversive would be questionable.   When only comparing treatment pens, calves displayed conditioned place avoidance to the pen where they had experienced and recovered from disbudding compared to the control procedure. This avoidance was likely due to calves learning to associate the pen with the pain experienced for the 6 h during and after disbudding. Calves were only provided a single conditioning session in which to associate the pain and the pen, suggesting that this pain was salient to the calves.        52  Aversion was most apparent during the first test session and waned in following sessions. This pattern was expected given that each test session served as a type of extinction training (as pens were no longer associated with any treatment) (Mackintosh, 1974).  The place conditioning paradigm used in the current study does not allow us to determine when or what part of the experience was aversive: we can only conclude that it was aversive over the 6 h period. Previous work has shown that the local anesthetic effects of lidocaine persist for approximately 1 to 2 h; after this period plasma cortisol and pain-related behaviours (e.g. head shakes, head rubs and ear flicks) increase for calves that have been disbudded (Duffield et al., 2010; Heinrich et al., 2010, 2009). The aversive experience is also likely to exceed 6 h: depending on the measure chosen, disbudded calves differ from control calves for a few hours  (Winder et al., 2018), a few days (Rushen and de Passillé, 2012; Theurer et al., 2012) or up to more than 100 days after the procedure (Casoni et al., 2019).   To our knowledge, the only previous attempts to assess the emotional impact of disbudding have been through cognitive-bias testing (Daros et al., 2014; Neave et al., 2013) ; this work found that calves showed a negative judgment bias in the hours after hot-iron disbudding (i.e. calves were more likely to respond negatively to ambiguous cues during a period when they were likely experiencing post-operative pain). Negative judgment biases are consistent with negative emotional states (Mathews and MacLeod, 2005; Mendl et al., 2009) but only allow assessments while the negative state is experienced. The current study shows that a single pairing of disbudding induced fear conditioning (i.e. avoidance of an otherwise neutral environment), providing the strongest evidence to date of the procedure’s negative emotional impact on the calves. Indeed, alpha-2 sedatives like xylazine can cause memory impairments (Hall et al., 2001), so the aversion observed in the current study (which is based on how well calves       53 remember the event), might be an underestimation of how negatively this procedure is perceived.   The design of the current study does not enable us to conclude that the aversion was due to pain per se; further work using post-operative analgesics would allow for stronger conclusions. However, the current design does allow us to rule out the experience of recovery from sedation as being the reason for the aversion, as animals experienced this recovery in both the treatment and sham conditions. In the treatment condition, calves also experienced lidocaine injections; it is possible that some elements of this procedure may also have been aversive. We did not include cornual injections in the control treatment as we feared animals who had previously been disbudded would have heightened sensitivity to this area compared to animals receiving the control treatment first, resulting in a treatment order bias.   Although we did not find an order of treatment effect, our low sample size might have not been able to detect a carry-over effect: calves who were disbudded during their first conditioning session might have still been experiencing pain 48 h later when exposed to the second conditioning session. We are aware that low sample size is a limitation of this study, especially in relation to the replicability of our results, but we wished to minimize the number of calves disbudded without post-operative analgesia.  We suggest that place conditioning could be used to investigate the efficacy of post-operative pain control strategies following disbudding (e.g. the use of nonsteroidal anti-inflammatory drugs such as meloxicam or ketoprofen; see (Winder et al., 2018), including the effects of drug, dose, route of administration, number of treatments, timing and age of the animal). The results of the current study indicate that place conditioning paradigms may be usefully applied to other species and procedures that have, until now, predominantly been tackled using physiological and pain-related behavioural measures, such as castration in lambs       54 (Paull et al., 2009), dogs (Okwee Acai et al., 2012), piglets (Leidig et al., 2009) and cattle (Coetzee et al., 2008; González et al., 2010). 3.5 Conclusion  The emotional impact of disbudding is aversive to dairy calves. We recommend the use of effective methods of mitigating this aversion, or that the procedure be discontinued (for example, by breeding for genetically hornless “polled” calves). More generally, we encourage researchers to adopt response measures specifically intended to assess the affective component of painful procedures used on animals.         55 Chapter 4: Assessing the affective component of pain, and the efficacy of pain control, using conditioned place aversion in calves A version of Chapter 4 has been published as Ede, Thomas, Marina A. G. von Keyserlingk, and Daniel M. Weary. “Assessing the affective component of pain, and the efficacy of pain control, using conditioned place aversion in calves”. Biology Letters 15, no 10 (2019): 20190642. https://doi.org/10.1098/rsbl.2019.0642. 4.1 Introduction Many approaches to study animal pain can be found in the literature, most of which rely on either nociceptive processes (e.g. hypersensitivity of injured areas (Winder et al., 2017)), activation of the hypothalamic-pituitary-adrenal axis (e.g. salivary concentration of cortisol (Caray et al., 2015)) or indirect measures of activation of the sympathetic nervous system (e.g. heart rate (Heinrich et al., 2009)). Such responses reflect the sensory component of pain and do not require processing by the central nervous system. In contrast, the affective component does require central processing as this relates to how negatively the experience is perceived to be. Nociception is generally thought to result in inelastic responses (i.e. withdrawal reflex) whereas the affective component of pain contributes to and can be affected by learning (Bateson, 1991). Thus experimental paradigms based on learned responses (such as preference, motivation and aversion tests) provide a stronger basis to investigate the affective component (Li, 2013; Weary et al., 2017).  Although a less common model than rats and mice in the study of the affective component of pain, dairy calves routinely undergo painful management procedures (Cozzi et al., 2015; Fulwider et al., 2008; Winder et al., 2016). Studying the pain associated with these routine procedures avoids the need to cause pain purely for the sake of research. One routine procedure is       56 disbudding in which horn buds are cauterized using a hot iron (heated to at least 500°C). The resulting burns are painful to calves (Stafford and Mellor, 2011), but the duration and magnitude of post-procedural pain are still unclear, making it difficult to develop pain mitigation strategies (Clutton, 2018; Rutherford, 2002).  We recently applied the principle of conditioned place aversion to study the affective impact of disbudding in dairy calves: dairy calves avoided a pen where they had been disbudded (with the use of a sedative and local anesthetic but without post procedural pain control) compared to a pen where they had only received a sham procedure (i.e. with sedation but without disbudding) (Ede et al., 2019a). This result indicates that disbudding is a negative affective experience for the calves, even when calves are provided sedation and local anaesthesia to mitigate intra-procedural pain. The observed aversion was likely a consequence of post-procedural pain emerging once the action of the local anesthetic waned in the hours after disbudding. Providing calves with post-procedural analgesics such as nonsteroidal anti-inflammatory drugs (NSAID) has shown to reduce reflex and automatic pain responses (Herskin and Nielsen, 2018; Winder et al., 2018) but no study had – to our knowledge – investigated the effect of NSAID on the affective component pain. The main objective of the current study was to identify whether providing post-procedural analgesics could mitigate the negative affective impact in dairy calves following hot-iron disbudding. We used conditioned place aversion to compare two disbudding procedures: one without post-procedural pain control and one with the use of an NSAID. A secondary objective was to evaluate two different NSAIDs commonly used in veterinary practice to alleviate post-procedural pain: meloxicam and ketoprofen. We predicted that calves would display conditioned       57 place aversion to the environment where they did not receive post-procedural pain control in comparison to the environment where they received either NSAID.  4.2 Methods  Ethics statement The study was approved by the UBC Animal Care Committee (protocol A16-0310) and performed in accordance with the guidelines outlined by the Canadian Council of Animal Care (2009). This study was conducted at the UBC Dairy Education and Research Center in Agassiz, Canada.  Animals and housing Holstein heifers (n = 34) were enrolled at 32 ± 5.9 d of age with a body weight of 68 ± 6.8 kg. Calves were assigned in blocks and balanced to two treatment groups: meloxicam and ketoprofen. See treatments section for details. We first aimed to enroll 36 calves (n = 18 per treatment group; double the sample size of 9 calves per treatment used in our previous study (Ede et al., 2019a)). Due to time and calf availability constraints we were only able to enrol 34 calves; of these three were excluded, two for not fulfilling pre-exposure criteria (see ‘Pre exposure’ section) and one for falling sick between the first and second treatment (low milk consumption, diarrhea and rectal temperature > 40 °C). Calves were housed in groups of 8, starting at 7 d of age, in pens measuring 4.9 x 7.3 m, bedded with sawdust. All calves had ad libitum access to hay and water and were provided 12 L of whole milk per day through an automatic milk feeder (CF 1000 CS Combi; DeLaval Inc., Sweden). At the time of testing, calves were brought individually from their home pen to the experimental apparatus at about 11 am.       58  Apparatus The experimental apparatus was identical to the one used in Ede et al. (2019a). Briefly, we used a plywood pen (2.1 x 6.0 m), divided into three equal-sized compartments (2.1 x 2.0 m) connected by removable gates (Figure 1). Treatment pens were on either side of the central pen; all disbudding procedures took place in the treatment pens. The walls of each treatment pen were mounted with colored plastic sheets (three red squares or two blue triangles placed on the sides of the pen). The different color patterns were used as visual cues to facilitate the association between pen and treatment. Immediately before the testing procedure the calf was placed in a holding chute positioned at the entrance of central pen.  Figure 4-1. Experimental apparatus. Calves (n = 31) received both disbudding procedures (‘Control’: without the use of post-procedural pain mitigation and ‘NSAID’ with the use of either meloxicam [n = 16] or ketoprofen [n = 15]) and spent the following 6 h in the treatment pens. During test sessions, the removable gates were taken out, allowing the calf to freely roam between pens. Drawn with the help of Chelsea Dela Rue.        59  Protocol 4.2.4.1 Pre exposure Calves were all given one session where they were provided free access to all three pens simultaneously within the apparatus before receiving treatments to avoid a potential effect of novelty. Calves were brought from their home pen and placed within the holding chute and provided a small milk reward (approximately 0.2 L). While in the chute, they were injected subcutaneously on the right side of the rump with saline (0.9% Sodium chloride, Hospira, Montreal, Canada) at 0.01 mL/kg; a volume identical to what they would receive when injected with xylazine during the treatment phase. The gate into the central pen was then opened, allowing calves to enter the apparatus. This pre-exposure session lasted 15 min, after which calves were gently brought back to their home pen. Calves that failed to explore all three pens during this pre-exposure session were excluded from the experiment (n = 2). 4.2.4.2 Treatments Calves were disbudded one bud at a time during two separate treatment sessions that took place 24 h (Treatment 1) and 72 h (Treatment 2) after pre-exposure. The two treatments were counterbalanced across the two treatment pens (i.e. if they received Treatment 1 in the pen with blue triangles, they received Treatment 2 in the pen with red squares and vice-versa). Hence, all calves were subjected to both procedures: NSAID (where they received post-procedural pain control) and Control (where they did not). During treatments, calves were kept in the pens for 6h.  NSAID procedure: calves were brought from their home pen to the chute where they received a 0.2 L of milk reward before being injected with 0.2 mg/kg of xylazine (SC, right       60 rump, Rompun 20 mg/mL, Bayer, Leverkusen, Germany). Immediately following the injection, calves were led and locked inside one of the treatment pens; the handler waited approximately 10 min for the calf to show signs of sedation (i.e. recumbency with eyeball rotation, see Ede et al. (2019c)). Once sedation was achieved, calves were injected with 5 mL of lidocaine (Lido-2 [lidocaine 2%, Epinephrine 1:100,000], Rafter8, Calgary, Canada) in the lateral canthus of one eye with a 0.9 mm x 25 mm needle (8881251782, Covidien, Dublin, Ireland).  Each calf then received one of the NSAID treatments according to recommended use (meloxicam group: SC, neck, 0.5 mg/kg, Metacam, 20 mg/mL, Boehringer Ingelheim, Burlington, Ontario, Canada; ketoprofen group: SC, neck, 3 mg/kg, Anafen, 100 mg/mL, Boehringer Ingelheim, Burlington, Ontario, Canada). 10 min later, the horn bud on the side of the lidocaine injection was shaved, tested for pain-reflex with a needle-prick and disbudded by placing a 500°C hot-iron (X30, 1.3 cm tip, Rhinehart, Spencerville, IN, USA) over the horn bud for approximately 15 s. The calf was then positioned in sternal recumbency and left in the treatment pen for the next 6 h before being returned to their home pen.  Control procedure: The procedure was identical to that described above for the NSAID procedure but instead of receiving an NSAID, calves were injected with saline (0.9% Sodium chloride, Hospira, Montreal, Canada, with a volume similar to the NSAID injection).  Order of treatment (Control or NSAID first), treatment pen associated with NSAID (Red squares or Blue triangles), first horn disbudded (Left or Right) and pre-exposure preference (i.e. treatment associated with the pen in which calves spent the most time during pre-exposure) were balanced across treatments.       61 4.2.4.3 Place aversion testing Testing occurred 48 h, 72 h and 96 h after the second treatment (Tests 1, 2 and 3 respectively). The test procedure was similar to pre-exposure: the calves were brought to the apparatus where the removable gates had been withdrawn. During testing, calves were allowed free access to all three pens until they lay down (for at least 1 min, which ended the session) or after 60 min had passed; they were then returned to their home pen.  Statistical analysis Time spent in each of the treatment pens (s) during testing was analyzed with a mixed linear model using the lme4 R package (D. Bates et al., 2015; R Core Team, 2015). The model’s fixed factors were the treatment received in the pen (Control or NSAID, 1df), which NSAID was used (meloxicam or ketoprofen, 1 df), their interaction (1df), the test session number (1, 2 or 3, 1 df), which treatment was received first (Control or NSAID, 1df), which treatment pen was associated with the NSAID procedure (blue triangles or red squares, 1df) and which bud was first disbudded (Left or Right, 1 df). Calf was considered a random factor (n = 31). The total number of pseudo-repeated observations of time spent in pens was 186 (31 calves x 3 sessions x 2 pens). Normality and homoscedasticity of residuals were scrutinized statistically and graphically. Square root transformation was conducted to improve residuals normality (Shapiro-Wilk normality test, before transformation: W = 0.86, P < 10-11; after transformation: W = 0.97, P < 0.001). P-values were calculated with Satterthwaite’s approximations using the lmerTest R package (Kuznetsova et al., 2017). The effect of treatment on which pen calves chose to lie down in was analyzed with a chi-square test.        62 4.3 Results Three calves were excluded from the study: two for not fulfilling pre-exposure criteria and one for falling sick between the first and second treatment (low milk consumption, diarrhea and rectal temperature > 40 °C; this calf later recovered), which resulted in 16 calves in the meloxicam group and 15 in the ketoprofen group. During pre-exposure, calves did not differ in the time spent in the two different pens (paired t-test, t30 = -0.3, P = 0.7).  After conditioning, time spent in the pen was not affected by test session number, order of treatment, color of treatment pen and side of first bud disbudded (respectively: t1,152 = 0.4, P = 0.7; t1,26 = 0.6, P = 0.5; t1,26 = 0.5, P = 0.6; t1,26 = -0.3, P = 0.8) but there was an interaction between treatment received in the pen (Control or NSAID) and the NSAID used (meloxicam or ketoprofen) (t1,152 = 4.4, P < 0.001). We therefore repeated the analysis of treatment separately for the two NSAIDs. In the meloxicam group, calves spent more time in the pen where they had received the NSAID compared to the control pen where no NSAID was provided (t1,78 = 2.6, P = 0.01). In the ketoprofen group, calves spent less time in the NSAID pen compared to the control pen (t1,73 = -3.7, P < 0.001, Figure 2a). Out of the 93 tests (31 calves x 3 tests), there were 5 in which calves did not lie down within the 60 min session (distributed among 3 calves: 2 in the meloxicam group, 1 in ketoprofen). Among the remaining tests, calves in the meloxicam group lay down more frequently in the NSAID pen (X² = 6.8, P = 0.009), and calves in the ketoprofen group showed the opposite pattern (X² = 10.5, P = 0.001, Figure 2b).       63  Figure 4-2. Time (√s) that calves spent in test pens (a) and the pen in which calves eventually lay down (b) during test sessions. Treatments were control (sedation, local anesthesia and hot-iron disbudding) and NSAID (sedation, local anesthesia, NSAID and hot-iron disbudding); calves received either meloxicam or ketoprofen as the NSAID. (*) represents a significant difference (P < 0.05).  4.4 Discussion Aversion to the pen where calves had been disbudded varied depending upon the type of NSAID used. As predicted, calves that received meloxicam showed more aversion to the pen where they had been disbudded without the NSAID. Surprisingly, calves that had received       64 ketoprofen avoided the pen where they received the NSAID relative to the pen with NSAID treatment. We suggest that this difference in place conditioning might be explained by the differences in duration of action of the two drugs. Meloxicam has an elimination half-life of approximately 25 h (Dumka and Srivastava, 2004); whereas, ketoprofen’s half-life is about 3 h (Plessers et al., 2015). This difference means that only meloxicam was likely effective in preventing post-procedural pain for the entire 6 h conditioning period while calves were kept in the treatment pen. The shorter duration of action of ketoprofen means that the calves were likely experiencing pain at the end of conditioning session. This is in accordance with studies reporting a rise in plasma cortisol 3 to 4 h after disbudding in calves provided with ketoprofen (Stafford et al., 2003; Sutherland et al., 2002). We hypothesize that the aversion to the ketoprofen pen was related to this timing of analgesic effects and its importance on place conditioning. In the control condition calves only received a local block, the effect of which likely waned at about the time calves recovered from the xylazine sedation. This implies that during the majority of the conditioning session, the post-procedural, inflammatory pain was untreated. In the ketoprofen condition calves likely received some analgesic benefit from the drug, but the protective effect of this treatment likely diminished over the course of the session. There is evidence in the literature that a worsening pain trajectory is especially aversive. For example, calves given a local block had higher plasma cortisol 4 h after disbudding (i.e. after the local block is expected to have worn off) compared to control calves that have not received a local block (Winder et al., 2018). Humans have been reported to focus more on the final moments of an event in their recall of painful experiences, rather than the total duration of pain (Kahneman et al., 1993; Redelmeier and Kahneman, 1996). We encourage researchers to consider the time course of the pain experience when assessing aversion using       65 place conditioning paradigms. Calves provided ketoprofen 2 h before disbudding and again 2 h and 7 h after the procedure showed fewer behavioural events indicating pain (including ear-flicks and head-shakes) in the 24 h after disbudding compared to control calves (Faulkner and Weary, 2000). Thus, future work could attempt more frequent treatments with ketoprofen, or the use of other drugs with different durations of analgesia, to more directly test the hypothesis that conditioned place avoidance varies according to the time trajectory of the pain. Ketoprofen can have a number of negative effects (e.g. on the digestive system, central nervous system, cardiovascular system, etc.) when administered to humans (Cusano et al., 1987; Schattenkirchner, 1991; Tosti et al., 1990; Valsecchi et al., 1983). We did not observe any evidence of such effects, and consider them unlikely following a single dose, but these direct effects are impossible to rule out in the current study. As our experiment did not include a sham treatment in which calves were not disbudded, our results do not provide evidence that meloxicam eliminates the post-procedural pain associated with disbudding, only that it makes the memory of the procedure less aversive.  We also note that the post-procedural pain associated with disbudding is thought to exceed the 6 h duration of focus in this study (Casoni et al., 2019; Mintline et al., 2013; Winder et al., 2018). Conditioned place avoidance (as well as other measures of affective states, see (Ede et al., 2019b; Herskin and Nielsen, 2018) for details) could be used to better assess the time course of this experience by conducting separate conditioning trials at different times relative to the procedure and analgesic treatments. Although we did not observe an effect of factors such as treatment order, it is possible that our sample size did not allow us to detect the impact of the lingering pain from the first treatment during the second disbudding. Our low sample size could also hinder the replicability       66 of our results. There are other factors worthy of attention regarding pain associated with horn removal, including the method used (e.g. amputation (Stafford et al., 2003), caustic paste (Winder et al., 2017), clove oil (Sutherland et al., 2018)) and the age at time of the procedure (Adcock and Tucker, 2018). Place conditioning seems to be an appropriate paradigm to study aspects of the affective component of pain in calves and other animals; we recommend adopting similar approaches to develop more effective methods of reducing pain in animals.  4.5 Conclusion Meloxicam treatment made hot-iron disbudding less aversive during the 6 h following the procedure, but ketoprofen treatment made the experience more aversive. Further research is needed to determine the number and timing of analgesic treatments required to effectively control post-procedural pain when disbudding. We recommend the use of place conditioning to explore the affective impact of pain on animals.        67 Chapter 5: Conditioned place aversion of caustic paste and hot-iron disbudding in dairy calves A version of Chapter 5 is in press for publication in Journal of Dairy Science. 5.1 Introduction Disbudding is common on dairy farms (Cozzi et al., 2015; USDA, 2018). The use of an iron rod heated at a high temperature (500-600°C) to cauterize horn tissue is the most common procedure, with around 70-80% of North American and European farms using this method (Cozzi et al., 2015; USDA, 2018; Winder et al., 2016). Hot-iron disbudding (or ‘dehorning’ if performed on older calves with developed horns) is a painful procedure, but this pain can be mitigated with local anesthetics and post-procedural pain control (for reviews, see Stafford and Mellor, 2011; Herskin and Nielsen, 2018; Winder et al., 2018).  An alternative method of disbudding is the application of a caustic paste (frequently, a mix of calcium hydroxide and sodium hydroxide) that results in a chemical burn that damages the developing tissue. Caustic paste is less commonly used than hot-iron disbudding; approximately 10-30% of farms in North America and Europe use this method (Cozzi et al., 2015; Gottardo et al., 2011; Staněk et al., 2018; USDA, 2018; Winder et al., 2016); however, the proportion of heifers in the United States disbudded with caustic paste increased from 12% to 32% between 2007 and 2014 (USDA, 2018, 2009).  Studies have reported pain-related behaviours (such as ear flicks and head shakes), increased cortisol, and increased pressure sensitivity in the hours following caustic paste application, and that local anesthesia and a non-steroidal anti-inflammatory drugs (NSAID) can mitigate these responses (Stilwell et al., 2009; Winder et al., 2017). Two studies directly       68 compared caustic paste to hot-iron disbudding, but the results of these comparisons were inconsistent. Morisse et al. (1995) found that chemically disbudded calves showed a higher cortisol response than cauterized calves. In contrast, Vickers et al. (2005) found more pain-related behaviours following hot-iron disbudding than following caustic paste disbudding. In previous applications of conditioned place aversion, calves spent less time and were less likely to lie down in a pen associated with cautery disbudding compared to a pen where they were only sedated (Ede et al., 2019a). Calves also showed less aversion to cautery disbudding if they received the NSAID meloxicam (compared to not receiving it) in addition to sedation and local anesthesia (Ede et al., 2019d). The objective of this study was to use conditioned place aversion to compare the experience of caustic paste to hot-iron disbudding, in both cases with sedation, local anesthesia and an NSAID.  5.2 Methods  Ethics statement This study was approved by University of British Columbia’s Animal Care Committee (Protocol A16-0310) and was conducted at University of British Columbia’s Dairy Education and Research Centre in Agassiz, Canada from October to December 2019.    Animals and housing 15 Holstein bull calves were enrolled at (mean ± SD) 7 ± 2 days of age, weighting 51 ± 4 kg. Calves were individually housed in 2.1 x 2.4 m pens bedded with sawdust, fed 4 L of whole milk twice a day (at approximately 9 am and 4 pm) and given ad libitum access to water, hay and grain.        69  Apparatus The apparatus was similar to the one described in previous studies (Ede et al., 2019a, 2019d). Briefly, a 2.1 x 6.0 m pen with plywood walls was divided in three pens (two ‘treatment’ pens and a ‘central’ pen, 2.1 x 2.0 m each), bedded with sawdust and separated by removable gates (Figure 5-1). As visual cues to aid place association, colored sheets (either blue triangles or red squares) were fixed on the walls of treatment pens. A holding chute mounted with a bottle holder was positioned in front of the entry gate leading into the central pen.   Figure 5-1: Experimental apparatus. During treatments, calves were confined to one of two treatment pens for 6 h, while recovering from either hot-iron or caustic paste disbudding (1st horn bud); this conditioning procedure was repeated 2 days later when calves recovered from the other disbudding method (2nd horn bud). For all treatments, calves received sedation (xylazine, 0.2 mg/kg), local anesthesia (lidocaine, 5 mL) and analgesia (meloxicam, 0.5 mg/kg). At 48 h, 72 h and 96 h after the last treatment, gates separating pens were removed and calves were tested for place aversion. Illustration by Ann Sanderson.   Protocol 5.2.4.1 Pre exposure Calves were individually pre-exposed to the apparatus prior to enrollment. During pre-exposure, calves were brought from their home pen (at approximately 11:00 h) to the holding       70 pen, placed in front of the apparatus, and given a milk reward (approximately 0.3 L of whole milk). Calves were then let into the apparatus (gates had been removed, allowing access to all pens). Time spent in each treatment pens (i.e. with both front legs in the pen) was recorded for 15 min, after which calves were brought back to their home pen. To avoid including animals with a strong pre-existing bias, one calf that did not enter both treatment pens during pre-exposure was excluded, resulting in a total of 14 subjects. 5.2.4.2 Treatments At 24 h and 72 h after pre-exposure (to allow a 2 d recovery from disbudding), calves were disbudded (one horn bud at a time, one in each treatment pen, such that all calves received both treatments). During hot-iron disbudding, calves were brought to the holding chute where they received a milk reward (approximately 0.3 L) as well as a subcutaneous injection of sedative (xylazine 0.2 mg/kg, Rompun 20 mg/mL, Bayer, Leverkusen, Germany), which was expected to sedate calves for approximately 1 to 2 h (Ede et al., 2019c). We did not expect xylazine to prevent calves from learning to associate place with treatment as calves have previously showed evidence of conditioned place aversion following xylazine treatments (Ede et al., 2019a, 2019d). Calves were then led to one of the treatment pens (with the gate mounted, confining the calf to that pen). Once the calves were sedated (recumbency and eyeball rotation, 5-10 min after injection), they were injected with a local cornual nerve block (5 mL, lidocaine 2%, epinephrine 1:100,000, Lido-2, Rafter8, Calgary, Canada) in the lateral canthus of the eye of the side to be disbudded. Immediately after the local block, calves were injected with a NSAID subcutaneously in the neck (meloxicam 0.5 mg/kg, Metacam 20 mg/mL, Boehringer Ingelheim, Burlington, Canada). At 10 min after injection of the local block, the horn bud was shaved, tested for pain-reflex with a needle-prick (no calf reacted) and cauterized with a pre-heated hot-iron       71 (X30, 1.3 cm tip, Rhinehart, Spencerville, IN, USA) for approximately 10 s (no calf reacted to disbudding). Calves were then positioned in sternal recumbency and left in the treatment pen for 6 h, a duration long enough to allow calves recovery from sedation and local anesthesia which are both expected to be effective for approximately 1 to 2 h (Ede et al., 2019c; Winder et al., 2018). Calves were then brought back to their home pen. For caustic paste disbudding all aspects of the procedure were identical, but instead of using a hot-iron, a thin layer of disbudding paste (calcium hydroxide 24.9%, sodium hydroxide 21.5%, Dr. Naylor, Morris, USA) was applied onto the horn bud (as a circle of around 2 cm in diameter) and a ring of petroleum jelly (Original Vaseline, Unilever, Toronto, Canada) was applied around the horn bud.  Order of treatment, color of treatment pen, side of horn bud, and pre-exposure preferences were counterbalanced among calves by Latin square design. 5.2.4.3 Place aversion testing At 48 h, 72 h and 96 h after the second disbudding treatment, calves were tested for place aversion. During tests, gates were removed allowing calves access to all pens. Time spent in each treatment pen (i.e. with both front legs in that pen) was recorded live from video (camera: WV-CP310, Panasonic Canada, Ontario) until the calf chose to lie down for at least 1 min, ending the session. All calves lay down within 60 min of the test start.  Statistical analysis A minimum sample size of 10 animals was calculated using R’s ‘power.t.test’ function for a statistical power of 0.8, given a mean difference in time spent between treatment pens equal to the standard deviation of the difference.        72 Before conditioning. The proportion of time spent in the pen to be associated with hot-iron disbudding (of total time spent in both treatment pens) during pre-exposure was compared to the null expectation (50%) using a one-sample t-test; normality of the differences was confirmed graphically.  After conditioning. The proportion of time spent in the hot-iron pen was analyzed with a linear mixed model (Bates et al., 2015) , testing the fixed effects of test session number (1, 2 or 3; 1 df) order of treatment (hot-iron or caustic paste first, 1 df), their interaction (1df), color of the pen associated with hot-iron disbudding (blue triangles or red squares, 1 df), horn side disbudded by cautery (left or right, 1 df) and latency to lie down (continuous, 1 df) with calf specified as a random effect. Normality and homoscedasticity of residuals were confirmed statistically and graphically (Shapiro-Wilk normality test: W = 0.98, P = 0.6). Reported estimates for fixed factors reflect the difference in proportion of time spent in the hot-iron pen between factor levels. 95% confidence intervals were obtained with R’s ‘confint’ function. An effect of test session was found, but no other factor was significant (see Results section). An additional model that did not include non-significant factors was also conducted (fixed effect: test session number, random effect: calf). Fit of the full and simplified models was compared by ANOVA, and the results from both models were reported. The effect of treatment on the pen calves chose to lie down in was analysed using a chi-square test, separately for each of the three post-conditioning test sessions. 5.3 Results Before conditioning, the proportion of time spent in hot-iron pen did not differ from the 50% value expected by chance (47.3%, 95% CI [32.9, 61.7]).        73 After conditioning, calves took on average of 31.0 (±8.6) min to lie down. Calves initially tended to spend more time in the hot-iron pen (intercept: 80.6%, 95% CI [39.5, 120.7]). Proportion of time spent in the hot-iron pen did not vary in relation to treatment order or its interaction with test number (hot-iron first: -2.4%, 95% [-35.7, 30.7]; interaction with test number: +5.0%, 95% [-9.4, 19.6]), color of the pen associated with hot-iron (red squares: -10.3%, 95% CI [-28.5, 7.9]), side of horn bud treated with hot-iron (right horn: -2.3%, 95% CI [-20.8, 16.2]) and latency to lie down (per minute: +0.01%, 95% CI [-0.8, 0.9]). However, the proportion of time spent in the hot-iron pen did vary in relation to test session; preference for this pen declined over the 3 test sessions (per session: -13.3%, 95% CI [-23.5, -3.2]) (Fig. 2a).  A simplified model only including test session number as fixed effect and calf as random effect was conducted. Excluding non-significant covariates did not hinder the model’s goodness of fit (ANOVA: χ² = 3.0, P = 0.7). Calves initially spent more time in the hot-iron pen (intercept: 73.5%, 95% CI [56.5, 90.5]) and preference for this side declined over the 3 sessions (per session: -10.8%, 95% CI [-18.1, -3.6]). Fewer calves lay down in the pen associated with caustic paste disbudding during the first test session (23% of calves, χ² = 3.8, P = 0.05), but we saw no evidence of preference in the second or third session (respectively, 33% of calves, χ² = 1.3, P = 0.2; 50% of calves, χ² = 0, P = 1; Figure 2b).       74  Figure 5-2. Place aversion results comparing hot-iron disbudding to caustic paste. Figure 2a (top) shows proportions of time spent in the pen where calves (n = 14) were previously disbudded with hot-iron (dotted line indicates the proportion expected by chance, 50%). Figure 2b (bottom) shows the number of calves lying down in pens where they were previously disbudded with either hot-iron or caustic paste (some calves lay down in the central pen; these lying events are not shown here).  Place aversion tests took place 48h, 72h and 96h after the last disbudding treatment.  5.4 Discussion After disbudding with hot-iron and caustic paste in distinct pens, calves tended to spend less time and lay down less frequently in the pen associated with caustic paste compared to hot-      75 iron, especially during the initial place aversion testing. These results suggest that, despite using the identical pain control protocol thought to be effective in mitigating the pain associated with both methods of disbudding (Stilwell et al., 2009; Winder et al., 2017), the caustic pain procedure was initially more aversive to calves.  We found no evidence of aversion in the second test session; reduced place aversion over time is expected when animals are tested repeatedly, as the learned association between treatment and pen is weakened every time the animal experiences the pen without painful stimulus (Brush, 1971). Reduced evidence of place aversion with multiple tests was also reported in a previous study investigating calf aversion to disbudding (Ede et al., 2019a). Motivation to spend more time in a pen they had previously avoided could also potentially be an expression of curiosity (Byrne, 2013) but this interpretation is speculative and we suggest that further work is required to determine if this result is replicable. Comparisons across studies are rendered difficult due to differences in calf age and pharmacological treatments. Also, the results of previous work are inconsistent. Vickers et al. (2005) compared calves disbudded by hot iron (treated with a sedative and local anesthesia) with calves disbudded by caustic paste (treated with a sedative only) and found that calves in caustic paste treatment showed less evidence of pain (as evidenced by reduced numbers of head shakes) in the first 4 h after the procedure. In contrast, Morisse et al. (1995) reported a weaker reaction to hot-iron disbudding than to caustic paste, although treatment was confounded with calf age. A series of studies on goat kids reported evidence of higher pain sensitivity (Hempstead et al., 2018c), behavioural (Hempstead et al., 2018a) and physiological (Hempstead et al., 2018b) responses to chemical versus hot-iron disbudding, but in all cases no pain control was provided. These results, combined with the mixed results from the current study, make it difficult to draw       76 firm conclusions. However, the consistent results of the studies by Hempstead and colleagues, combined with aversion in the pen calves chose to lie down in during the initial test session of the current study, provides a tentative basis for recommending the hot-iron procedure over caustic paste. Even with a sedative, local anesthetic and a NSAID, both methods of disbudding were likely aversive to calves, underlying the importance of developing further refinements for disbudding, and where feasible, avoiding this practice (for example, by using polled sires). Our study had several limitations. First, despite selecting our sample size based on a power analysis, our ability to detect differences and the potential replicability of results was still limited by the number of calves tested. A second limitation is that aversion tests were based upon the calves’ memory of the 6 h following the procedure, which is unlikely to encompass the full duration of pain caused by disbudding (Casoni et al., 2019; Winder et al., 2018). The long duration of post-operative pain means that during the second disbudding event calves may have been still in pain from the first event. That said, we saw no evidence that this ongoing pain influenced aversion, as this would have been expected to result in an effect of treatment order. Thirdly, we did not explore state dependent effects (i.e. that the calves’ current pain state influences their aversion; see Tzschentke, 1998). Such effects have not been investigated in calves, but it is possible that calves avoided the pen associated with caustic paste not only because of their aversive memory of the procedure, but also because they were still feeling pain during testing. Fourthly, our conclusions are limited to the overall aversiveness of the experience, and we cannot make inferences about more limited time frames (e.g. whether caustic paste was more aversive throughout conditioning or during only the last hours). Finally, our results are also limited to the specific combination of pain control methods provided; the drugs used in this study are based upon a substantial body of research on hot-iron disbudding, but       77 much less is known about pain control for caustic paste; it is likely that the ideal multi-modal approach will differ for these two procedures.  5.5 Conclusion Calves tended to spend less time and laid down less often in a pen associated with caustic paste disbudding compared to hot-iron, but only in the initial test session; results from following test sessions were ambiguous. Our results suggest caustic paste is initially more aversive than hot-iron disbudding, but this finding should be considered tentative and warrants further investigation.         78 Chapter 6: General discussion 6.1 Thesis summary In the first chapter of this thesis, we reviewed the literature on the assessment of affective states in dairy cattle. We noted that most studies relied on physiological and spontaneous behavioural responses which typically reflect arousal (i.e. how ‘excited’ the animal is) rather than valence (whether something is experienced as positive or negative). We suggested that the use of learned responses (such as approach, aversion, preference and motivation), mood assessments (cognitive biases, anhedonia) and inclusion of pharmacological treatments were promising yet still under-used paradigms to study cattle affective states. Considering the broad scope of this topic, I focused this thesis on the use of learned aversion and pharmacological treatments to study pain in dairy calves, which I believe to be a priority area for welfare research. In the second chapter, we evaluated the aversiveness of injection methods by allowing calves to avoid injections by giving up access to a valued reward (milk). Calves were more hesitant (i.e. had a longer latency) to access their reward if it was associated with an intramuscular injection compared to intranasal or subcutaneous. We concluded that intramuscular injections were the most painful of the studied routes. In the third chapter, we studied the pain caused by hot-iron disbudding (cauterisation of horn producing tissues, intended to prevent future horn growth). Our previous ‘trade-off’ paradigm to study injections relied on repeated exposure to an acute and short-term aversive event, which was not suitable to study disbudding. Instead, we adopted a conditioned place aversion paradigm allowing calves to establish an association between pen and treatment over a 6 h period. At one month of age, calves were subjected to disbudding and control treatments in distinct pens and were then allowed free access to both pens two days after the last treatment.       79 Calves avoided the pen associated with disbudding (i.e. they spent less time and were less likely to lay down in it). Given that calves had been treated with a local anesthetic (implying any intra-operative pain should have been mitigated), we concluded that aversion was caused by the post-operative pain. In the fourth chapter, we aimed to verify this hypothesis by testing the effect of post-operative pain control (non-steroidal anti-inflammatory drugs: NSAID) on aversion. The paradigm was similar to that described for Chapter 3, but in this case, calves were disbudded in both treatment pens (one horn bud in each): once with an NSAID and once without (all procedures included local anesthesia). We tested two NSAIDs commonly used in veterinary medicine: meloxicam and ketoprofen. For meloxicam, calves displayed more avoidance to the pen where they did not receive the analgesic, which was expected and in accordance with our hypothesis that aversion was caused by post-operative pain. More surprisingly, calves treated with ketoprofen exhibited the opposite response by avoiding the pen where they had received the analgesic. Ketoprofen’s shorter duration of effect (Dumka and Srivastava, 2004; Plessers et al., 2015) implies that the efficacy of the analgesic waned during the time spent in the treatment pen. This may have impacted calves’ memory of the treatment by worsening the end of the experience, a factor known to influence the recall of painful experiences (Kahneman et al., 1993) (for a more detailed discussion, please see Chapter 4 and ‘memory of pain’ section of the general discussion). The fifth and final empirical chapter focused on the practical issue of which of two common disbudding methods (hot iron and caustic paste) was more aversive. Using a paradigm similar to Chapter 4, calves were disbudded in both treatment pens, one horn by hot iron and the other by caustic paste (in both cases with sedation, local anesthesia and meloxicam).       80 Immediately after conditioning calves tended to avoid the pen associated with caustic paste, but this tendency reversed over subsequent test sessions. We cautiously concluded that chemical disbudding was more aversive, but that further investigation was required. 6.2 Contributions  Despite growing interest by both scientists and the general public, the assessment of animal subjective states in animals remains a challenge. The field of animal pain has been (and to some extent, still is) dominated by research focusing on reflex responses. The aim of this thesis was to expand the study of calf pain beyond this reflex component and explore the emotional aspect. Rather than concentrating on responses during or shortly following painful procedures – the more common method of studying animal pain – the work presented in this thesis was centered around calves’ memory of the experience. Allowing calves to ‘vote with their feet’ by looking at how much they would avoid a place where they experienced pain proved to be a successful approach. This avoidance provides evidence of pain beyond its arousing quality and indicates that calves perceive pain as negative, forming potent memories of the event and adapting their behaviour accordingly.  The progress of cognition-based assessments is encouraging, yet paradigms based on animal memory remain under used. The results of this thesis indicate that place conditioning is a powerful and reliable research method for detecting differences between treatments. One strength of place conditioning designs is their suitability to intra-individual comparisons. The work described in this thesis also shows that place conditioning is a practical approach, requiring minimal animal training in comparison to other cognition-based assessments; in contrast, judgment bias training has been reported to require between 10 to 20 training sessions with 10 to 30 trials per session (Lecorps et al., 2019; Neave et al., 2013). Thus, this thesis emphasizes the       81 potential of place conditioning to further our knowledge in this field and I encourage other researchers to consider place conditioning in future studies of animal emotions.  Applications of this concept presented in this thesis allow for the presentation of the following recommendations for veterinarians and farmers regarding injections and disbudding in calves:  1. When possible, favor subcutaneous or intranasal injections over the intramuscular route  2. The use of a sedative and local anesthetic is not sufficient in controlling disbudding pain. 3. Combining sedation, local anesthesia and post-operative analgesia is recommended, although further research is needed to determine the most appropriate post-operative analgesia protocol 4. Hot-iron should be tentatively favored over caustic paste disbudding. 6.3 Limitations and future research areas  Methodological limitations Some criticisms previously expressed over judgment bias tests (Kremer et al., 2020) can be applied to place conditioning. In most cases, farm or laboratory animals are housed in barren environments with few opportunities for cognitive stimulation. By providing access to a novel environment and a learning opportunity, the paradigm itself might be rewarding (Meagher et al., 2020) and this could influence the animals’ affective state. This potential confounder could be mitigated by providing a more complex, stimulating housing environments, reducing the significance of experimental paradigms as sources of cognitive challenge. Another flaw common to place conditioning and judgment bias is the loss of novelty and experimental relevance after the first test. In the case of judgment bias, tests rely on the reaction of animals to ambiguous cues (Mendl et al., 2009). Whether this ambiguity is rewarded, non-      82 rewarded or punished will influence the animals’ response in subsequent tests. This implies that after the initial first test, judgment biases do not only evaluate mood, but also conditioning to ambiguous cues. Place conditioning relies on the association between environmental cues and previous experiences. As animals are exposed to these cues in the absence of treatment, they gradually loose the association and the paradigm shifts to an extinction trial (Shadmehr and Mussa-Ivaldi, 2012). Thus, similar to the potential flaw of judgment bias, place conditioning tests focus on an additional cognitive process after the initial test. The extinction process and the animals’ capacity to quickly recover their conditioning with re-exposure to treatment (Shadmehr and Mussa-Ivaldi, 2012) are interesting research paths to assess the potency of negative memories caused by treatments. Finally, as place conditioning relies on the animals establishing a learned association, these paradigms are influenced by learning abilities. In all our experiments, we witnessed some degree of variability between individuals in response to identical treatments. We focused the statistical approach on the response of the sample as a whole, assuming differences in aversion were linked to differences in affective states. However, it is possible that observed variability was due (at least partially) to differences in calves’ capacity to establish the place-treatment association. A supplementary test such as a simple discrimination task (for an example in calves, see Meagher et al., 2015) could be valuable in accounting for differences in cognitive capacities.  Disbudding and long-term pain One applied goal of this thesis was to identify the extent of pain during hot iron disbudding and how to minimize it. We restricted our study to the 6 h following the procedure. However disbudding impacts calves for more than 6 h, indeed, it likely causes pain for 24 h or longer (Adcock and Tucker, 2020; Casoni et al., 2019; Lecorps et al., 2019; Mintline et al.,       83 2013). One way to investigate disbudding pain past the 6 h window would be with a self-administered analgesia paradigm. As mentioned in the first chapter, this type of paradigm has been applied to lame chickens, who were found to prefer a feed laced with analgesics (in contrast, healthy chickens tended to avoid the laced feed; Danbury et al., 2000). In theory, animals should self-administer the analgesia as long as they are experiencing pain and analgesia is effective. However, there are few published studies using this paradigm, perhaps because it is difficult for animals to learn the association between eating the laced feed and the analgesic effect. Drug induction time is likely to be delayed when administered through feed, and this could interfere with learning as the stimulus and reward become separated in time (Lattal, 2010). To overcome this challenge, calves could be provided a rapid acting analgesic. One idea would be to provide access to ice-packs; the premise being that calves could initiate contact with the pack for immediate pain relief. Cold-therapy is expected to provide some degree of analgesia with minimal delay, as shown in work on humans (Algafly & George, 2007; Auley, 2001), but it is also possible that contact with the pack may be more aversive than any analgesia provided by the cold.  Another self-administration method would be one familiar to hospital patients: placement of an intravenous catheter delivering an analgesic drug under patient control (i.e. receiving a dose in response to an operant task such as a button press). This method could provide valuable information on the intensity and duration of pain. Operant conditioning has been applied to the study of pain in rats (Lyness et al., 1989) and I suggest there are potential benefits in implementing this approach when pain in cattle, including longer duration ailments like lameness (Laven et al., 2008); metritis (Stojkov et al., 2015) and mastitis (Leslie and Petersson-Wolfe, 2012)).       84  The ethics of disbudding refinement An applied concern that was not addressed in the thesis was the actual valence of the experiences studied. In the studies described in this dissertation, we compared two procedures both likely to be aversive (sedation or disbudding, disbudding with or without analgesia, disbudding by hot-iron or caustic paste), limiting our ability to conclude how far from a ‘neutral’ experience the most refined procedure was. As we found differences in calf aversion (hot-iron vs caustic paste, both with sedation, local anesthesia and post-operative analgesia - see Chapter 5), it is likely that even the ‘gold standard’ method does not eliminate the pain of disbudding. This raises the ethical question of how refined a procedure needs to be in order to be acceptable. Ethics of animal use are usually presented within a cost/benefit framework where a procedure is ethically justified if its benefits outweighs its costs (Ferdowsian and Beck, 2011; Orlans, 1997). In the case of disbudding, the main benefit used as justification are usually the safety of handlers and other animals, similar to justifications used for teeth clipping of piglets (Fraser and Thompson, 1991) and beak trimming of chicks (Glatz, 2000). The safety argument is often mentioned but rarely supported by empirical evidence. In a qualitative survey conducted in Europe (Kling-Eveillard et al., 2015), authors reported that most farmers thought the presence of horns made cows more aggressive and that hornless cows were more docile, quoting a participant saying ‘cows are consciously aware of their horns’ and referring to horns as the ‘weapons of the cows’. Interestingly, comparison of horned and hornless cattle have shown that horned animals were calmer than hornless when handled (Fordyce et al., 1988). It was also reported that horned cows do not display more agonistic behaviours than hornless cows; rather, horned animals are more likely to engage in threats without any physical       85 contact (Lutz et al., 2019) and to keep greater inter-individual distances (Knierim et al., 2015). These results suggest horns may play more of a role in intimidation rather than physical fighting. Rather than horn status, factors such as space allowance, management and human-animal interactions were emphasized as most relevant to cattle aggression (Knierim et al., 2015; Lutz et al., 2019). Farmers who rear horned cattle underline the importance of respectful management and calm composure to reduce animal aggression and agitation (Kling-Eveillard et al., 2015). Clearly, the accepted long-term welfare benefits of disbudding need to be scrutinized when considering the ethical cost/benefit balance of the procedure. Another approach to avoiding the harms associated with disbudding is breeding of genetically hornless (or ‘polled’) animals (Prayaga, 2007). This alternative is not widely adopted in the dairy industry due to concerns regarding trade-offs with production and reproduction traits, and a low number of polled bulls increasing inbreeding levels (Götz et al., 2015; Windig et al., 2015). Progress has been made in recent years in the quantity and quality of polled sires (Götz et al., 2015; Windig et al., 2015). The willingness of farmers to integrate polled genetics in their herds (Kling-Eveillard et al., 2015), and consumers apparent support for hornlessness for animal welfare reasons (McConnachie et al., 2019) may encourage adoption of polled genetics as an alternative to disbudding. In summary, rather than being forced into the ethically difficult utilitarian reasoning about the benefits and costs of disbudding, I suggest that efforts be focused on avoiding disbudding, either via development of management practices allowing for horned animals, or the promotion of hornless genetics. In this way, disbudding may no longer be considered a routine procedure, and rather only be conducted in exceptional circumstances.       86  Social aspect of pain In every study undertaken in this thesis, calves experienced pain isolated from social partners. I made the choice to avoid any influence of social partners on the calf’s memory of the experience. However, social contact can have important effects on coping in cattle and other social species (Ishiwata et al., 2007; Rault, 2012). To explore this concern, a project using a design similar to that described in Chapters 4 and 5 could be conducted: calves would be assessed for conditioned place aversion comparing recovery from disbudding with or without a social partner. I expected that the presence of a conspecific would make the disbudding experience less aversive. I also expect that this ‘social buffering’ (Kikusui Takefumi et al., 2006) would be correlated with calf personality traits; previous work on pigs has shown that the benefits of social support are linked to coping style (Reimert et al., 2014).  Memory of pain  So far, I have focused on gaps related to applied issues, but more basic research questions also remain. As all experiments in this thesis relied on learned aversion, we were not evaluating pain per se, but rather the animals’ memory of pain. This distinction might appear as semantic, but consequential differences between pain and memory of that pain have been noted in humans (Erskine et al., 1990; Mazza et al., 2018). For example, a discrepancy was found in chronic pain patients between their initial baseline pain ratings and their memory of that baseline pain after 3 to 11 weeks of treatment (Linton and Melin, 1982). Additionally, post-partum reports of labour pain were inconsistent with reports during labour (Lowe and Roberts, 1988). Deviations from an initial rating could be due to an ‘updated’ evaluation, interpreting this within the totality of the experience (Erskine et al., 1990; Lowe and Roberts, 1988) (e.g. pain initially viewed as severe could be retrospectively devalued if followed by worse pain).       87  As mentioned in Chapter 4, the final part of a painful experience is a potent factor in the memory of it in humans (Kahneman et al., 1993; Redelmeier and Kahneman, 1996). For example, consider two aversive procedures: submerging your hand in cold water (14°C) for 1 min and submerging your hand in 14°C water for 1 min but with an additional 30 s in slightly warmer water (15°C). After being exposed to both of these procedures human participants were then asked which they would rather repeat; surprisingly, most subjects chose to repeat the second trial, the one with more pain. The authors concluded that evaluations of aversive experiences were often dominated by the discomfort felt in the final moments (Kahneman et al., 1993).  To my knowledge, such a process has not been investigated in non-human animals. To assess the relative influence of early and later phases of pain experiences on conditioned place aversion in calves, a design similar to Kahneman et al.’s (1993) could be conducted. As described in Chapters 4 and 5, calves would be disbudded twice (one bud in each treatment pen) but in one pen restricted for 6 h; whereas, in the other pen restricted for 7 h, but with the last hour following treatment with a fast-acting analgesic (e.g. ketoprofen). If calves showed a conditioned place preference for the pen associated with the extra hour, it would suggest that they prefer a less painful ending even when this results in more pain in total, similar to Kahneman et al.’s (1993) observations in humans.   Another interesting, but overlooked memory process in animal research, is mood congruency bias. In humans, there is evidence that memory retrieval is facilitated if the on-going mood matches the affective valence of the memory (Blaney, 1986). For example, depressed individuals display improved memory of negatively valenced words compared to non-depressed subjects (Watkins et al., 1996). Surprisingly, memory biases have seldom been explored in animals. In one instance, Takatsu-Coleman et al. (2013) trained mice to avoid an aversive       88 environment (bright light and loud noise). Mice were then put through an extinction trial where control mice stopped displaying aversion 30 days after training. In contrast, mice subjected to social isolation (which induced depressive-like behaviours) extended their avoidance of the negative environment. The authors suggested that a negative affective state in the isolated mice facilitated retrieval of negative memories. In a more recent rat study using social status as a proxy for affective state (subordinate as negative and dominant as positive), animals were trained to associated maze arms with positive (food pellets) or negative (quinine soaked pellets) events. Rats in both the negative and positive states failed to show improved memory of negative or positive events (Burman and Mendl, 2018). The results of this study are not consistent with previous results reported in humans and mice, but sample size was low (5 subordinates compared to 4 dominants) and dominance is likely to be imperfectly associated with affect.   A conditioned place aversion paradigm could be applied to assess the influence of pain on memory bias in calves following disbudding. Similar to that described in Chapters 3 and 4, calves would be assessed for learned aversion following disbudding. However, in this case subjects would be divided into two groups: one receiving an analgesic before the test sessions and a control group that was given a saline injection. I hypothesize that during aversion testing, control calves would still be in pain from disbudding, improving their memory of the negative event and thus increasing their display of aversion relative to calves that are still benefiting from analgesia. If this hypothesis was confirmed, it would have both basic and applied implications: it would provide evidence that calves display mood-congruency memory bias, and it would show that disbudding has an affective impact for a longer period than that assessed in this thesis (at least 48 h after disbudding in this case).       89 6.4 General conclusion The collective evidence arising from the research described in my thesis suggests that calves experience an affective response to painful procedures and remember these as negative. Learned aversion tests proved to be a useful tool for assessing the affective component of pain in dairy calves. Calves showed an affective response to both acute and longer-term pain (over 6 h). Calves displayed varying approach-aversion responses depending on methods of injection: the more painful the injection was, the more calves were willing to give up milk.  Calves also displayed conditioned place aversion to compartments associated with painful disbudding procedures. Disbudding was more aversive than a sham procedure, even with the use of local anesthesia. When exploring multi-modal pain control (by combining sedation, local anesthesia and an NSAID), calves’ memory of disbudding was dependent of the NSAID used: meloxicam made the experience less aversive; whereas, ketoprofen had the opposite effect. Further research is needed to explore this result, but we hypothesized the NSAIDs’ different durations of action were critical. Finally, the results of the last empirical chapter tentatively suggested that calves find caustic paste more aversive than hot-iron disbudding, even with ‘gold standard’ pain mitigation.  Collectively the results also provide a scientific basis for recommendations to the dairy industry that can be used to reduce the pain experienced by dairy calves undergoing disbudding. Specifically, the results indicate that subcutaneous and intranasal injections should be favored over intramuscular injections when the injected substance allows it. Calves subjected to disbudding should be provided multi-modal pain control (i.e. sedation, local anesthesia and post-procedural analgesia) and hot-iron is likely preferable to caustic paste. Even with these refinements, calves are likely to still experience pain. I recommended conducting more research       90 to explore refinements, especially to address long-term pain and potential alternatives to disbudding.  Lastly, I encourage fellow researchers to consider memory processes to assess pain and other affective states both in cattle and other species. Paradigms based on animals’ memory of an experience proved to be powerful and practical for understanding their emotional responses, suggesting that they could be of great benefit in futures studies.       91 References Adcock, S.J.J., Tucker, C.B., 2020. 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