Land and Food Systems, Faculty of
DSpace
UBCV
Yoo, Seonpil
Doctor of Philosophy - PhD
University of British Columbia
Pain during and immediately after routine husbandry procedures has been widely studied, and mitigation strategies have been developed. However, pain experienced during the healing period—lasting pain—remains poorly understood and under-assessed. The aim of this thesis was to develop methods for assessing lasting pain and, using these methods, evaluate strategies to mitigate it. Chapter 1 reviewed existing pain assessment techniques and evaluated their potential for detecting lasting pain. It also explored factors that may influence pain during the healing period. Chapter 2 investigated a memory test paradigm as a tool for assessing lasting pain following hot-iron disbudding. No differences in memory performance were found between disbudded and control calves, suggesting that this method may not reflect lasting pain. Chapter 3 explored behavioural indicators—short-term lying bouts and half-lying bouts—and memory improvement as potential markers of lasting pain following castration. Short-term lying bouts were more sensitive to acute pain than general lying behaviour, but no reliable indicators of lasting pain were identified. Chapter 4 shifted focus to pain mitigation strategies that do not rely on pain assessment. Specifically, it examined how hot-iron application time and tip size affect wound characteristics and healing. Larger tips produced larger wounds with longer healing times, while longer application times resulted in deeper and larger wounds but did not affect healing duration. I conclude that the memory test did not prove effective for assessing lasting pain, but that refining procedural factors—such as iron tip size and application time—can reduce healing time and potentially mitigate any resulting pain. This thesis highlights the need for more sensitive and reliable indicators of lasting pain and suggests that procedural refinement may offer a practical path forward.
LASTING PAIN IN DAIRY CALVES by Seonpil Yoo B.Sc., Seoul National University, 2015 M.Sc., Seoul National University, 2019 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 2025 © Seonpil Yoo, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Lasting pain in dairy calves submitted by Seonpil Yoo in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Applied Animal Biology Examining Committee: Dr. Daniel M. Weary, Professor, Applied Animal Biology, UBC Supervisor Dr. Marina A. G. von Keyserlingk, Professor, Applied Animal Biology, UBC Supervisor Committee Member Dr. Robert Tremblay, Bovine Specialist, Boehringer Ingelheim Animal Health Supervisory Committee Member Dr. Kenneth D. Craig, Professor Emeritus, Psychology, UBC University Examiner Dr. Alexandra Protopopova, Associated Professor, Applied Animal Biology, UBC University Examiner Dr. George Stilwell, Associated Professor, Veterinary Medicine, University of Lisbon External Examiner iii Abstract Pain during and immediately after routine husbandry procedures has been widely studied, and mitigation strategies have been developed. However, pain experienced during the healing period—lasting pain—remains poorly understood and under-assessed. The aim of this thesis was to develop methods for assessing lasting pain and, using these methods, evaluate strategies to mitigate it. Chapter 1 reviewed existing pain assessment techniques and evaluated their potential for detecting lasting pain. It also explored factors that may influence pain during the healing period. Chapter 2 investigated a memory test paradigm as a tool for assessing lasting pain following hot-iron disbudding. No differences in memory performance were found between disbudded and control calves, suggesting that this method may not reflect lasting pain. Chapter 3 explored behavioural indicators—short-term lying bouts and half-lying bouts—and memory improvement as potential markers of lasting pain following castration. Short-term lying bouts were more sensitive to acute pain than general lying behaviour, but no reliable indicators of lasting pain were identified. Chapter 4 shifted focus to pain mitigation strategies that do not rely on pain assessment. Specifically, it examined how hot-iron application time and tip size affect wound characteristics and healing. Larger tips produced larger wounds with longer healing times, while longer application times resulted in deeper and larger wounds but did not affect healing duration. I conclude that the memory test did not prove effective for assessing lasting pain, but that refining procedural factors—such as iron tip size and application time—can reduce healing time and potentially mitigate any resulting pain. This thesis highlights the need for more sensitive and reliable indicators of lasting pain and suggests that procedural refinement may offer a practical path forward. iv Lay Summary Routine procedures like disbudding and castration cause pain in calves, but pain during the healing period—lasting pain—is not well understood. The research described in this thesis aimed to find ways to measure lasting pain and explore how to reduce it. I tested a memory task and calf behaviours like lying patterns to detect lasting pain, but none proved reliable. Since lasting pain was hard to measure, I studied how changing the hot-iron disbudding technique affects healing. Larger iron tips caused bigger wounds that took longer to heal, while longer application times made deeper and larger wounds but did not change healing time. In summary, lasting pain is difficult to assess, but refining procedures—like using smaller iron tips—can help wounds heal faster and may reduce pain. v Preface The use of Generative AI in this thesis was limited to the use of ChaptGPT 4o for checking sentences and paragraphs for correctness and clarity. All text was reviewed by me and my supervisors. A version of Chapter 1 has been submitted for publication as Yoo, S., von Keyserlingk, M. A. G., & Weary, D. M. Lasting pain in dairy calves: a narrative review of assessment methods and factors affecting duration. I reviewed literatures, developed the main ideas, wrote the manuscript, and revised the manuscript. D. M. Weary and M. A. G. von Keyserlingk provided input in preparation of the manuscript and revision. This chapter did not require ethics approval. A version of Chapter 2 has been published as Yoo, S., von Keyserlingk, M. A. G., & Weary, D. M. (2023). The effects of pain following disbudding on calf memory. Journal of Dairy Science, 106(12), 9507–9513. https://doi.org/10.3168/jds.2023-23604. I identified the research question, designed the experiment, collected data, performed the statistical analyses, and wrote the manuscript. D. M. Weary and M. A. G. von Keyserlingk provided assistance with the study design and input in preparation of the manuscript. This project received ethics approval from The University of British Columbia’s Animal Care Committee (certificate number: #A21-0111). A version of Chapter 3 is in press for publication as Yoo, S., von Keyserlingk, M. A. G., & Weary, D. M. Assessment of lasting pain following castration in 2-week-old calves. Animal. https://doi.org/10.1016/j.animal.2025.101719. I identified the research question, designed the experiment, collected data, performed the statistical analyses, and wrote the manuscript. D. M. Weary and M. A. G. von Keyserlingk provided assistance with the study design and input in vi preparation of the manuscript. This project received ethics approval from The University of British Columbia’s Animal Care Committee (certificate number: A18-0376 and A23-0013). A version of Chapter 4 has been submitted for publication as Yoo, S., von Keyserlingk, M. A. G., & Weary, D. M. The effects of hot-iron application method on wound characteristics and healing in dairy calves. I identified the research question, designed the experiment, collected data, performed the statistical analyses, and wrote the manuscript. D. M. Weary and M. A. G. von Keyserlingk provided assistance with the study design and input in preparation of the manuscript. This project received ethics approval from The University of British Columbia’s Animal Care Committee (certificate number: A21-0111 and A25-0081). vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ................................................................................................................. xvi Acknowledgements ................................................................................................................... xvii Chapter 1: Introduction ........................................................................................................... 1 1.1 General introduction ................................................................................................... 1 1.2 Wound healing and pain ............................................................................................. 2 1.3 Biological measures .................................................................................................... 4 1.3.1 Cortisol .................................................................................................................... 4 1.3.2 Acute phase proteins ............................................................................................... 6 1.3.3 Average daily gain .................................................................................................. 7 1.4 Behavioural measures ................................................................................................. 8 1.4.1 Lying and standing behaviours ............................................................................... 8 1.4.2 Wound-directed behaviours .................................................................................. 10 1.4.3 Social behaviour.................................................................................................... 12 1.4.4 Behavioural responses to nociceptive stimuli ....................................................... 13 1.5 Cognitive measures ................................................................................................... 15 viii 1.5.1 Judgment bias paradigm ....................................................................................... 15 1.5.2 Conditioned place preference and aversion .......................................................... 16 1.5.3 Memory testing ..................................................................................................... 17 1.6 Factors that may influence lasting pain .................................................................... 19 1.6.1 Animal factors ....................................................................................................... 19 1.6.2 Nature of wound ................................................................................................... 20 1.7 Conclusion ................................................................................................................ 21 1.8 Thesis objective ........................................................................................................ 22 Chapter 2: The effects of pain following disbudding on calf memory ............................... 23 2.1 Introduction ............................................................................................................... 23 2.2 Materials and methods .............................................................................................. 25 2.2.1 Animals and housing............................................................................................. 25 2.2.2 Apparatus .............................................................................................................. 26 2.2.3 Training phase ....................................................................................................... 27 2.2.4 Treatments............................................................................................................. 28 2.2.5 Memory tests ......................................................................................................... 29 2.2.6 Behavioural measures ........................................................................................... 29 2.2.7 Statistical analysis ................................................................................................. 30 2.3 Result and discussion ................................................................................................ 31 2.3.1 Memory ................................................................................................................. 31 2.3.2 Behaviour .............................................................................................................. 34 2.4 Conclusion ................................................................................................................ 36 Chapter 3: Assessment of lasting pain following castration in 2-week-old calves ............ 37 ix 3.1 Introduction ............................................................................................................... 37 3.2 Material and methods ................................................................................................ 39 3.2.1 Animals and housing............................................................................................. 39 3.2.2 Treatments............................................................................................................. 40 3.2.3 Clinical measurements .......................................................................................... 41 3.2.4 Lying behavior ...................................................................................................... 42 3.2.5 Spatial memory test............................................................................................... 43 3.2.5.1 Apparatus ...................................................................................................... 43 3.2.5.2 Initial training................................................................................................ 43 3.2.5.3 Memory tests ................................................................................................. 44 3.2.6 Statistical Analysis ................................................................................................ 44 3.3 Results ....................................................................................................................... 46 3.3.1 Bimodality of lying-bout duration ........................................................................ 46 3.3.2 Wound healing ...................................................................................................... 47 3.3.3 Lying behaviors .................................................................................................... 48 3.3.4 Memory ................................................................................................................. 53 3.4 Discussion ................................................................................................................. 53 3.5 Conclusion ................................................................................................................ 56 Chapter 4: The effects of hot-iron application method on wound characteristics and healing in dairy calves ............................................................................................................ 57 4.1 Introduction ............................................................................................................... 57 4.2 Material and methods ................................................................................................ 58 4.2.1 Animals and housing............................................................................................. 59 x 4.2.2 Treatments............................................................................................................. 59 4.2.3 Wound measurement ............................................................................................ 63 4.2.3.1 Wound tissue classification........................................................................... 63 4.2.3.2 Wound size and depth ................................................................................... 65 4.2.4 Statistical Analysis ................................................................................................ 66 4.3 Results ....................................................................................................................... 68 4.3.1 Disbudding parameters ......................................................................................... 68 4.3.2 Wound size and depth ........................................................................................... 68 4.3.3 Wound healing progression .................................................................................. 70 4.3.4 Wound healing duration ........................................................................................ 71 4.3.5 Horn tissue development....................................................................................... 73 4.4 Discussion ................................................................................................................. 74 4.4.1 Wound size and depth ........................................................................................... 74 4.4.2 Wound healing progression .................................................................................. 75 4.4.3 Wound healing duration ........................................................................................ 76 4.4.4 Horn tissue development....................................................................................... 78 4.5 Conclusion ................................................................................................................ 78 Chapter 5: Conclusions .......................................................................................................... 79 5.1 Thesis summary ........................................................................................................ 79 5.2 Contributions............................................................................................................. 81 5.3 Limitations ................................................................................................................ 84 5.3.1 Conceptual limitations .......................................................................................... 84 5.3.2 Limitations of the ‘holeboard’ test ........................................................................ 84 xi 5.4 Future research opportunities .................................................................................... 86 5.5 General conclusion.................................................................................................... 87 References .....................................................................................................................................88 xii List of Tables Table 1.1 Wound healing process ................................................................................................... 2 Table 3.1 Least squares means ± SE of behaviors and reference memory of the week following band and surgical castration in 2-week-old Holstein and Holstein x Angus crossbred bull calves with or without week-long meloxicam treatment ......................................................................... 51 Table 3.2 Least squares means ± SE of behaviors and reference memory of the 4th week following band and surgical castration in 2-week-old Holstein and Holstein x Angus crossbred bull calves with or without week-long meloxicam treatment ....................................................... 52 xiii List of Figures Figure 2.1 View of the hole-board test apparatus as seen from the entrance to the arena. The locations of each of 15 milk bottles are indicated......................................................................... 27 Figure 2.2 Changes in a) general working memory (GWM) scores, b) reference memory (RM) scores, and c) modified reference memory (MRM) scores following treatment [D=disbudding (n=10); D+AA=aditional analgesic treatment after disbudding (n=10); S=sham disbudding (n=10)]. Calf memory was assessed once a day for 18 d using a modified hole-board apparatus. Over the first 12 d (first phase) the locations of the rewarded bottles were kept constant; on d 13 these locations were changed and calves were tested for 6 more days (second phase). ............... 32 Figure 2.3 The total number of a) head shakes and b) head rubs recorded from 1800 h to 2400 h, on d 0 (starting 8 h after the disbudding treatment was applied; D = disbudding, D+AA = additional analgesic after disbudding, S = sham disbudding), d 2 and d 4. Boxes illustrate the interquartile range, the central line indicates the median, the red point indicates the mean, the whiskers show the calculated minimum and maximum values, and the individual points show values beyond the minima and maxima. Different letters show differences (P < 0.05) between treatment groups............................................................................................................................ 35 Figure 3.1 Distribution of lying bout lengths during the week before and after castration. Histogram of raw data of the week a) before and b) after castration and histogram of cube root-transformed data of the week c) before and d) after castration. The arrow indicates the selected cut-off point for short-duration lying bouts. ................................................................................. 47 Figure 3.2 Healing scores over 4 weeks following castration in Holstein and Holstein x Angus crossbred calves castrated at 14 days of age (B =Band castration; S = Surgical castration). Dots xiv with black borders represent values for individual claves, dots without border represent treatment means for each week, and whiskers indicate the 2nd and 98th percentiles. ................................. 48 Figure 3.3 Least squares means (± SE) for (A) short-duration lying bouts, (B) total lying bouts, and (C) lying bouts duration during week 1, as well as (D) short-duration lying bouts during week 4, following band and surgical castration in Holstein and Holstein × Angus crossbred calves castrated at 14 days of age (B = Band castration, S = Surgical castration, NM = no meloxicam treatment, and M = meloxicam treatment). ................................................................ 50 Figure 3.4 Least squares means (± SE) of reference memory during the (A) 1st and (B) 4th weeks following band and surgical castration in Holstein and Holstein x Angus crossbred calves castrated at 14 days of age. ........................................................................................................... 53 Figure 4.1 Image of two tips (Rhinehart, Spencerville, IN, USA) used in the study. A) A front shot of large annulus tip. B) A side shot of large annulus tip. C) A front shot of small annulus tip. D) A size shot of small annulus tip. E) Outer diameter of large tip: 20.36 mm. F) Inner diameter of large tip: 14.48 mm. G) Outer diameter of small tip: 15.02 mm. H) Inner diameter of small tip: 6.99 mm. Area covered by the large annulus tip = 3.26 cm2. Area covered by the small annulus tip = 1.77 cm2. Contact area of the large annulus tip = 1.61 cm2. Contact area of the small annulus tip = 1.39 cm2. ................................................................................................................. 60 Figure 4.2 Picture of horn bud (left) and disbudding wound (right). The length of short-and long-axis were measured. Wound depth was assessed at the vertices of the axes. ............................... 62 Figure 4.3 Definitions of tissue categories used for wound assessment ....................................... 65 Figure 4.4 Effects of hot-iron disbudding of 4-wk old Holstein calves (n=24), measured immediately after the procedure. Wound dimensions (length of short and long axes) are xv presented as Mean ± SE and are shown separately for the effect of tip size, application time, and head side (a-c). * indicates P < 0.05. ........................................................................................... 69 Figure 4.5 Mean ± SE of wound length by tissue stage following hot-iron disbudding in 4-wk old Holstein calves (n=24). Fresh wound refers to the wound on the day of disbudding. Last necrotic tissue indicates the final observation of necrotic tissue. First crust represents the first observation after complete detachment of necrotic tissue, and last crust indicates the final observation of a crust. First epithelium refers to the first observation following crust disappearance, and later epithelium corresponds to 15 d after the onset of epithelialization. Values in parentheses indicate mean ± SD of d after disbudding. Asterisks (*) denote significant differences (P < 0.05) in short-axis length between adjacent tissue stages; carets (^) denote differences (P < 0.05) in long-axis length between adjacent tissue stages. .......................................................................................... 71 Figure 4.6 Mean ± SE of necrotic tissue duration and time to epithelium onset after hot-iron disbudding in 4-wk-old Holstein calves (n=24). * indicates P < 0.05. ......................................... 72 Figure 4.7 Wound images from the final measurement following hot-iron disbudding in Holstein calves (n = 24), grouped by treatment combinations: tip size (small vs. large) and application time (10 s vs. 20 s). Arrows indicate the presence of apparent horn tissue. The number at the bottom of each image represents the number of days post-disbudding. ....................................... 74 xvi List of Abbreviations ADG = Average daily gain D = Day IM = Intramuscular NSAID = Non-steroidal anti-inflammatory drug SC = Subcutaneous SD = Standard deviation SE = Standard error Wk = Week xvii Acknowledgements As I look back on my PhD journey, I realize it was shaped not only by my own effort, but also by luck, support, and dedication of many others. It was never a solitary path. First and foremost, I would like to express my deepest gratitude to Drs. Dan Weary and Nina von Keyserlingk for their guidance and mentorship. I feel incredibly lucky to have supervisors who recognized my strengths and helped me develop them, while also helping me grow into an independent scientist. Dan’s challenging questions always made me think about alternative perspectives and refine my logic. One of the most important lessons I learned from him is that study design, data analysis, and interpretation matter far more than simply achieving significant results. Nina’s guidance taught me how to communicate effectively in academia—how to format research papers and highlight key findings in presentations. I will never forget the time she reviewed my slides one by one and offered advice at her place. Their support, rather than direction, allowed me to shape my PhD journey in a way that was truly my own. I am also grateful to my committee member, Dr. Robert Tremblay, for his thoughtful and constructive feedback throughout my PhD. It was a pleasure to have him on my committee. I owe many thanks to the students who helped with data collection. Thanks to their support, I was able to spend more time with my family. To my lab mates and friends in Canada—thank you for the engaging discussions and the many memorable activities we shared. Your companionship made this journey enjoyable and meaningful. To my parents and my mother-in-law, I truly appreciate that you visited me in Canada all the way from Korea and took care of my children, even overcoming the challenge of 12-hour xviii flights. Your support and dedication allowed me to focus on my PhD and attend conferences. I believe your time here created wonderful memories for my kids, and I’m deeply grateful for everything you’ve done. Yoonju, my love, words cannot fully express my gratitude and affection for you. Without your unwavering support and dedication, this achievement would not have been possible. Thank you for preparing meals, keeping our home in order, caring for our children, staying healthy, and being my partner in life. Raising two children abroad without family support is something only you could do. This intense and challenging period has strengthened our bond and will remain a cherished chapter in our lives. I don’t know where we’ll eventually settle, but with you, the place doesn’t matter. Najun and Teaju, thank you for your emotional support, your joy, and your dedication to being happy. Your smiles are the reason I live, and playing with you helped me recharge and relieve stress. I will continue doing my best to be a good daddy, now and always. 1 Chapter 1: Introduction 1.1 General introduction Calves are often subjected to painful management practices, including castration and disbudding, for operational or safety purposes. Castration is performed to reduce aggression (Simpson, 2020) and improve meat quality (Silva et al., 2019). In the United States, 77.1% of bull beef calves are castrated before sale, primarily using surgical (49.2%) or band (47.3%) methods (USDA, 2008). Disbudding is performed to reduce the risk of injury to animals and humans, and the need for increased space required to manage horned cattle successfully (reviewed by AVMA, 2014). In the United States, 94.3% of dairy heifer calves are disbudded, most often using a hot iron (54.6%) or caustic paste (32.5%) (USDA, 2018). A large body of literature has assessed pain responses associated with castration and disbudding. However, most studies have focused on the period during the procedures and the first few days thereafter (see review related to castration, Coetzee, 2013; for disbudding see Winder et al., 2018). To our knowledge, no review has specifically addressed pain that persists for days and weeks after a procedure, herein referred to as lasting pain. Developing methods to evaluate pain that lasts days or even weeks after an injury represents an important scientific, practical, and ethical challenge. Results to date suggest that assessment of lasting pain may require different outcome measures than those used for assessing pain within the first days after a painful procedure (Marti et al., 2017; Adcock and Tucker, 2018; Drwencke et al., 2023). The aims of this narrative review were to: (1) identify the characteristics of lasting pain by drawing on insights from the literature on wound healing and pain in humans; (2) evaluate methods currently used to assess pain in calves for their potential as indicators of lasting pain, 2 based on these characteristics; and (3) explore factors that may influence lasting pain—including animal- and wound-related characteristics—to better inform strategies for minimizing lasting pain. To reduce the confounding effects of pain medication, we primarily focus on studies involving calves castrated or disbudded without analgesic treatment; when studies that include medication are discussed, treatments are specified. 1.2 Wound healing and pain The wound healing is typically divided into four phases—hemostasis, inflammation, proliferation, and remodeling—characterized by distinct cellular and molecular events (reviewed by Schultz et al., 2011; summarized in Table 1.1). The inflammatory phase generally lasts several days (Coger et al., 2019). In this paper, we refer to lasting pain as pain experienced during the proliferative and remodeling phases. To our knowledge, no previous review has focused on lasting pain in cattle. Table 1.1 Wound healing process Phase Cellular events and tissue repair Hemostasis  Vasoconstriction  Platelet aggregation and degranulation  Fibrin formation  Scab formation Inflammation  Neutrophil infiltration  Monocyte infiltration and differentiation to macrophage 3  Lymphocyte infiltration  Edema formation Proliferation  Fibroblast migration  Angiogenesis  Collagen synthesis  Extracellular matrix formation  Granulation tissue formation  Epithelialization Remodeling  Collagen remodeling  Vascular maturation and regression  Scar formation In human medicine, pain during proliferative and remodeling phases of wound healing has been categorized into four types (Vuolo, 2009): background pain (i.e., pain at rest), incident pain (i.e., pain triggered by movement or activity), procedural pain (i.e., pain during a routine, basic procedure such as dressing removal and wound cleansing), and operative pain (i.e., pain associated with any intervention such as needle injection). Procedural and operative pain are not relevant to lasting pain in calves, as they arise from wound management or interventions to control pain. Thus, our focus here is on background and incident pain during the proliferation and remodeling phases. The intensity of background and incident pain remains poorly studied regardless of species, but some work has addressed this in humans. For example, in patients with chronic leg ulcers, the mean background pain intensity was reported as 4.5 on a 0–10 scale, with 4 scores of 5–6 considered moderate pain (Leren et al., 2022). Another study found that 8 of 17 patients reported more than mild pain during a 4-wk period of wound healing (McGuire et al., 2006). Given the long healing times (e.g., 62 d to re-epithelialize after caustic paste disbudding (Drwencke et al., 2023), 114 d after hot-iron disbudding (Adcock and Tucker, 2018), and 49 d after surgical castration (Marti et al., 2018)), there is considerable scope for lasting pain in cattle. Pain during these healing phases is likely to be of mild to moderate intensity, so pain assessment methods may need to be of higher sensitivity than those used to assess more severe intra-operative pain. 1.3 Biological measures 1.3.1 Cortisol Cortisol is a well-known stress indicator. Upon exposure to a stressor, the sensory information is processed by the amygdala, the brain region that processes emotions, which in turn signals the hypothalamus. The hypothalamus secretes corticotropin-releasing hormone, which prompts the anterior pituitary to release adrenocorticotrophin (ACTH). ACTH then stimulates the adrenal glands to secrete cortisol (Lightman, 2008). As pain is known to elevate cortisol (Tennant, 2013), cortisol may be serve as a non-specific pain indicator. Cortisol in blood and hair has been assessed in numerous studies in calves following castration and disbudding. Blood cortisol levels typically peak at approximately 30 min and return to pre-procedure level within hours after surgical castration (plasma; Yoo et al., 2023a), band castration (plasma; Paull et al., 2015), Burdizzo castration (serum; Thüer et al., 2007), hot-iron disbudding (plasma; Park et al., 2020), and caustic paste disbudding (serum; Reedman et al., 5 2020). Furthermore, following this initial peak, cortisol levels in castrated and disbudded calves did not remain elevated above those of sham-treated calves during observation periods of up to 14 days (Paull et al., 2015; Yoo et al., 2023a). These findings suggest that blood cortisol is likely of limited value for assessing background. Moreover, because cortisol levels are highly influenced by sampling time, blood cortisol is also of limited value for assessing incident pain. In contrast, hair cortisol shows more promise as an indicator for lasting pain. For example, Creutzinger et al. (2017) clipped hair from the left hip the day before surgical castration and collected regrown hair from the same spot 14 d after castration, finding higher cortisol in castrated compared to sham-castrated calves. Since hair cortisol reflects integrated cortisol secretion from the time hair was initially clipped, this result likely reflects both acute and lasting pain. When analyzing cortisol in hair grown between 28 and 56 d after castration, Marti et al. (2018) found that cortisol levels were higher in band-castrated calves compared to non-castrated or knife-castrated calves. In contrast, Marti et al. (2017) found no difference in hair cortisol between band-castrated, surgically castrated, and non-castrated 2-mo-old calves when analyzing hair grown from 1 d before castration to 49 d after castration. Although the authors did not speculate on the reason for this difference between studies, it may be attributed to individual variation that could be controlled by including baseline cortisol measurements (Stalder et al., 2012). In summary, blood cortisol responses are transient and unlikely to be useful for assessing lasting pain. Hair cortisol shows more promise as a measure of lasting pain, but studies assessing hair cortisol should include samples from each animal at multiple time points, including before the procedure, to help account for individual baseline differences and to characterize the time course of this response. 6 1.3.2 Acute phase proteins Acute phase proteins, such as serum amyloid A, fibrinogen, and haptoglobin are proteins whose blood concentrations typically change in response to inflammation, infection, and tissue injury (Gruys et al., 2005). Given that the concentration of acute phase proteins is related to the extent of tissue damage (Murata et al., 2004), measuring their levels may be useful for studying lasting pain caused by this tissue injury. In cattle, haptoglobin is commonly measured in pain studies and appears to show a transient response to pain. Blood haptoglobin levels typically peak approximately 3 d after procedures like hot-iron disbudding (Park et al., 2020) and both band and surgical castration (Roberts et al., 2018), returning to baseline within 7 d. Possibly due to this transient nature, when haptoglobin was analyzed weekly over 6 wk, no difference was detected between non-castrated and castrated calves (Marti et al., 2017; 2018; 2019) or between non-disbudded and disbudded calves (Reedman et al., 2020). Serum amyloid A and fibrinogen have been measured in a limited number of studies. Two studies assessed serum amyloid A levels at d 0, 1, 2, 3, and 7 after castration, but results varied. One study found that serum amyloid A concentrations were higher in both band- and surgically castrated 2-mo-old calves on d 1, 2, and 3 compared to non-castrated calves (Meléndez et al. 2018a). A companion study reported higher serum amyloid A concentrations in surgically castrated 1-wk-old calves on d 0, 2, and 3 compared to band- and non-castrated calves, and higher serum amyloid A levels in band-castrated calves on d 7 compared to surgically and non-castrated calves (Meléndez et al., 2018b). In a study measuring serum amyloid A in calves sampled weekly from d 8 to 42 after castration, no differences were found between castrated and 7 non-castrated calves regardless of castration method (Marti et al., 2019). However, serum amyloid A levels in calves band-castrated at 1-wk of age were higher than those of surgically and non-castrated calves when measured from d 7 to 35 after the procedure (Marti et al., 2018). After dehorning (type not specified), serum amyloid A levels increased only on the day after the procedure (compared to pre-treatment levels) and not during the week that followed (Shinya et al., 2023). One study assessed fibrinogen levels after surgical castration in 5.5-mo-old calves, finding higher fibrinogen in surgically castrated calves compared to non-castrated calves in the first 2 wk of a 5-wk observation period (Earley and Crowe, 2002). Overall, due to its transient response, haptoglobin appears to be of little value for assessing background or incident pain. Serum amyloid A may be of value as an indicator for lasting pain, but this requires further investigation as results to date are inconsistent. Based on the results of a single study, changes in fibrinogen show potential as an indicator of lasting pain, but further work is required to determine if this association is robust. 1.3.3 Average daily gain Average daily gain (ADG) has often been assessed following castration and disbudding. Disbudding appears to have little effect on ADG (Adcock and Tucker, 2018; Drwencke et al., 2023), but effects are consistently noted after castration, although these effects vary over time and differ depending on castration method. For example, during the week after castration, ADG is lower in surgically castrated versus band-castrated calves (Meléndez et al., 2017; Roberts et al., 2018), but this difference reverses at 3 to 4 wk after the procedure (Roberts et al., 2018). The wound caused by surgical castration closes within days of the procedure but tissue damage persists for weeks after band castration (Marti et al., 2018; Gellatly et al., 2021), likely 8 explaining the difference between procedures in patterns of weight gain. When ADG was monitored on a weekly basis, Nogues et al. (2021) found lower ADG during the 8 wk following band castration in 1-mo-old Holstein calves compared to surgical castration, attributing the difference to lower calf starter intake in band-castrated calves. However, when ADG was assessed over a 2 mo period, no effect of castration method was observed in 2-mo-old calves (Marti et al., 2017; Gellatly et al., 2021), suggesting weekly measurement of BW may better capture changes following castration. The decrease in plasma testosterone to negligible levels within 24 h after both surgical and band castration (Fisher et al., 2001) suggests that the differences between castration methods are due to pain rather than other hormonal changes following castration. In conclusion, weekly ADG may relate to lasting pain for at least 4 wk following castration, reflecting pain patterns associated with different castration methods. Differences in ADG have not been observed following disbudding, potentially because the pain is not as severe or long lasting. Although growth can be influenced by other factors—such as nutrition, environmental temperature, and disease (Hyde et al., 2021)—in the context of a well-controlled study where these factors can be assumed to be similar across treatments, inferences about pain can be justified. 1.4 Behavioural measures 1.4.1 Lying and standing behaviours Lying (or conversely standing) behaviours, such as lying time and lying duration, have been assessed following castration and disbudding. The effect of disbudding on lying time is inconsistent. For example, a series of studies have assessed changes in lying time after hot-iron 9 disbudding and reported no differences between disbudded and non-disbudded calves for 2 d following the procedure (Sutherland et al., 2013; 2018; 2019). However, one study observed less lying time in hot-iron disbudded calves compared to non-disbudded calves for 2 d post-disbudding (Gingerich et al., 2020), and another study reported longer lying times in hot-iron disbudded calves, but only at 17 d post-disbudding (when calves were observed at 3, 10, and 17 d; Adcock et al., 2023). The reason for these discrepancies is unclear, but differences in pain management may be a factor, as only the latter two studies used local anesthetics and analgesics. Changes in lying behaviour are more commonly reported after castration, perhaps because lying down causes pain when muscular contractions associated with these movements disturb these tissues or when the injured tissues contact the surface. For example, one study found that surgically castrated 2-mo-old calves spent more time standing, compared to both non-castrated and band-castrated calves in the 3 d after castration (Gellatly et al., 2021). Similarly, another study found that 2-mo-old calves spent more time standing after surgical castration compared to non-castrated controls for 7 d following the procedure (Meléndez et al., 2017). However, these difference were not observed in 1-wk-old calves (Meléndez et al., 2017; 2018b). When compared to surgically castrated calves, band-castrated calves showed increased standing duration and decreased lying time from 7 to 42 d after castration in 4-mo-old calves, but no differences were observed in 1-wk-old calves (Marti et al., 2017). The authors also reported an increase in standing duration in the 15 to 19 d after castration in band-castrated calves compared to both non-castrated and surgically castrated calves, suggesting that this period may be particularly painful for band-castrated calves. In conclusion, results to date suggest that changes in lying and standing behaviour are of little value in assessing lasting pain following disbudding, but these measures show promise as 10 indicators of lasting pain following castration, at least in older calves. The effect of castration on these behaviours is not clear for younger calves (i.e., 2-mo-old or less); more sensitive behavioural indicators may be required for these younger animals. If contact between the castration site and the surface triggers posture changes, consistent with changes in skin sensitivity around the wound (see ‘Behavioural responses to nociceptive stimuli’ below), short-duration or interrupted lying bouts may serve as a more sensitive and specific indicator of lasting pain following castration. 1.4.2 Wound-directed behaviours Wound-directed behaviours, such as wound licking, biting, and scratching, have been used as indicators of pain in cats (Cloutier et al., 2005) and dogs (Marangoni et al., 2023). These behaviours can promote healing and relieve pain and itch (Day, 2019). In the context of lasting pain, these behaviours may reflect attempts to alleviate incident pain. In calves, wound licking has been assessed in several studies on castration. Meléndez et al. (2017) found no difference in the frequency of lesion licking between surgically and band-castrated calves in the 6 wk after castration. However, Molony et al. (1995) and Nogues et al. (2021) reported more lesion licking in band-castrated versus surgically castrated calves, at 7 and 8 wk after castration. The discrepancy between these studies likely stems from differences in observation methods. Meléndez et al. (2017) observed calves for 2 min every 10 min once a week for 9 h, Molony et al. (1995) recorded wound licking behaviour every 3 min for 3 h every 3 d, and Nogues et al. (2021) observed calves for 24 h once weekly. These differences suggest that short or intermittent observational periods may fail to detect some behavioural differences. 11 In disbudding studies, movements such as head shaking, head rubbing and ear flicking have been used as indicators of pain in the hours after the procedure (reviewed by Herskin and Nielsen, 2018). After caustic paste application, head shaking increased for about an hour post-disbudding compared to non-disbudded calves (Braz et al., 2012; Winder et al., 2017). Following hot-iron disbudding, Bučková et al. (2022) reported increased head shaking during 24 h after the procedure compared to 24 h before, and Faulkner and Weary (2000) observed a peak in head shaking at 6 h post-disbudding when lidocaine was administered for a horn nerve block. However, this result should be interpreted cautiously due to the potential confounding effect of lidocaine on head movements, as Vickers et al. (2005) found increased head rubbing and shaking from 1 to 4 h after treatment with both xylazine and lidocaine compared to xylazine alone. Heinrich et al. (2010) reported an increase in head shakes at 2, 4, 6, 9, 20, 26, 28, and 30 h after disbudding. Considering the low frequency of head behaviours observed on d 4 after hot-iron disbudding during a 6-h observation period (6.4 head shakes and 4.6 head rubs; Yoo et al., 2023b), one of the major weaknesses of using head movements as indicators of lasting pain is that they require continuous observation. Interval time sampling, such as 5-min observations every hour, may be unsuitable. Failing to capture these rare behaviours increases the relative influence of external factors—such as flies—thereby confounding the results. Analyzing video at accelerated speed may not be a solution as the short duration and intermittent nature of these behaviours make them easy to miss. In summary, wound-directed behaviours hold promise for assessing lasting pain, likely reflecting wound pain. However, further research is needed to identify practical and reliable methods for capturing these behaviours, including work on automated systems. 12 1.4.3 Social behaviour Reduced social behaviour has been observed in sick animals (reviewed by Hart, 1988). For example, induced inflammation in male rats resulted in decreased direct contact with conspecifics (Crestani et al., 1991). Similarly, calves inoculated with Mannheimia haemolytica avoided lying in proximity to other calves and spent reduced time licking other calves on the day of inoculation (Hixson et al., 2018). Ill cows spent more time behind a barrier preventing visual contact with conspecifics compared to healthy cows during the 3 d following calving (Proudfoot et al., 2014). There is some evidence that pain can have similar effects; one study found that disbudded calves spent more time in a shelter and entered the shelter more frequently when it was unoccupied for 3 d following hot-iron disbudding compared to non-disbudded calves (Gingerich et al., 2020). Play behaviour has been used to study pain after disbudding in calves. Reductions in the duration of running, head-to-head contact, and head-to-object contact have been reported 3 h after hot-iron disbudding (Mintline et al., 2013) and caustic paste disbudding (Winder et al., 2017). Rushen and de Passillé (2012) observed that calves spent less time running approximately 20 h after caustic paste disbudding compared to non-disbudded calves; however, as only disbudded calves were sedated, the persistent effects of sedation (Malviya et al., 2000) may have influenced this result. St John Wallis et al. (2025) categorized play into three types: locomotor play (individual running or trotting), parallel locomotor play (running or trotting involving two or more calves), and play fighting (one calf pushing another with its head). They found that play fighting decreased for 7 d after hot-iron disbudding, but no changes in other play types beyond the day after disbudding. 13 In summary, illness can reduce social interaction and increase time spent alone, and there is evidence that pain can have similar effects. However, long-term studies are needed to determine whether reductions in social interaction persist throughout the healing period. Play fighting appears to be a promising behaviour for assessing lasting pain following disbudding but the observed effect may be specific to wounds on the head, limiting the utility for assessing lasting pain following other painful procedures. 1.4.4 Behavioural responses to nociceptive stimuli After an injury to the skin, sensitivity to noxious stimuli increases in the injured area (primary hyperalgesia) and the surrounding uninjured skin (secondary hyperalgesia). This increased sensitivity is the result of pro-inflammatory mediators resulting in more pain signals, even in response to stimuli that would otherwise cause little or no pain (Momin and McNaughton, 2009). Mechanical nociceptive thresholds have long been used as an indicator of hyperalgesia in animals (reviewed by Pogatzki-Zahn et al., 2018). Mechanical nociceptive thresholds have been assessed near wound sites using algometers and Von Frey filaments, following both castration and disbudding. Thresholds have been compared before and after treatment, as well as between untreated and treated calves, with observed differences interpreted as evidence of pain. For example, both band and surgically castrated calves showed lower thresholds up to 9 wk after castration compared to uncastrated calves, with band castrated calves exhibiting lower thresholds between wk 3 to 5 (Gellatly et al., 2021). In disbudding studies, the mechanical nociceptive threshold was found to decrease after hot-iron disbudding and did not return to pre-disbudding levels until 7 d after the procedure (Allen et al., 2013; Stock et al., 2015). Adcock and Tucker (2018) reported decreased mechanical 14 nociceptive thresholds after disbudding, which diminished once epithelial tissue had formed. In another study, Karlen et al. (2021) found that mechanical nociceptive thresholds gradually decreased after caustic paste disbudding, with a difference detected from d 4.5 to 12.5 compared to pre-disbudding levels. Drwencke et al. (2023) observed a decrease in mechanical nociceptive threshold following caustic paste disbudding, persisting until the wounds were fully healed approximately 19 wk after the procedure. Studies comparing sham-disbudded calves and hot-iron disbudded calves have found lower mechanical nociceptive thresholds for up to 15 wk after disbudding (Stock et al., 2015; Adcock and Tucker, 2018; Casoni et al., 2019). Two studies observed lower mechanical nociceptive threshold in caustic paste disbudded calves compared to sham-disbudded calves up to 6 wk post-disbudding (Karlen et al., 2021; Drwencke et al., 2023). In contrast, a third study (Reedman et al., 2020) found no difference in mechanical nociceptive thresholds for 7 d following disbudding between caustic paste disbudded and sham disbudded calves, attributing the discrepancy to the use of different restraining methods (a metal restraint with blindfold vs. a halter). Taken together, these findings suggest that lower mechanical nociceptive thresholds may serve as an indicator of incident pain. However, it is important to note that the outcome of disbudding involves not only the creation of a wound but also the permanent removal of horn tissue. Consequently, differences observed between intact and disbudded calves, or between pre- and post-disbudding measures, may reflect the absence of horns rather than the presence of the wound itself—particularly if the horn bud area is naturally less sensitive than surrounding skin. Future studies comparing polled calves with disbudded calves are needed to confirm whether reduced skin sensitivity is attributable to wound pain. 15 1.5 Cognitive measures 1.5.1 Judgment bias paradigm Based on findings that information processing can be biased by emotions in humans, Harding et al. (2004) introduced the cognitive bias paradigm to assess affective states in animals and found that rats housed in a more stressful environment were more likely to judge ambiguous cues as negative. This paradigm has been used to assess affective states across various farm animals (Baciadonna and McElligott, 2015). In the go/no-go task, the most common paradigm used in farm animals, animals are trained to associate one cue with a positive event and another cue with a negative event (Baciadonna and McElligott, 2015). After training, they are presented with unreinforced ambiguous cues, and their responses are recorded. In calves, judgment bias has been assessed following disbudding, but to our knowledge no studies have investigated judgment bias after castration. Two different approaches have been used to assess judgment bias in calves. Neave et al. (2013) used colour cues (red or white) and recorded the rate of approach the ambiguous colours, finding that calves were less likely to approach to these ambiguous colours 6 and 22 h after disbudding (compared to pre-disbudding values). In contrast, Lecorps et al. (2019) used the location of a bottle as a cue, and again found reduced response to the bottle at the intermediate locations after disbudding. In this study there was a reduced response to even the location that was consistently rewarded, potentially indicative of a diminished experience of pleasure associated with the reward. However, as discussed in Social behaviour section, it is important to note that in both of these studies calves were sedated during disbudding, and the persistent effects of sedation may have influenced these results. 16 In summary, while judgment bias tests have shown evidence of a negative affective state in the hours after disbudding, further research is necessary to determine whether this paradigm is suitable for assessing the effects of pain in the days and weeks that follow a procedure. Also required are studies that assess whether observed differences are due to pain, versus other factors such as sedation. 1.5.2 Conditioned place preference and aversion Place conditioning was initially developed to assess the rewarding properties of morphine (Rossi and Reid, 1976) and other rewarding or aversive motivational experiences in rodents (McKendrick and Graziane, 2020). A common apparatus design for conditioning consists of three compartments: two outer compartments with distinctive characteristics (e.g., different wall colours or patterns) and a center compartment with no special characteristics that connects two outer compartments (Prus et al., 2008). Conditioned place testing involves three phases: habituation, conditioning, and testing (Cunningham et al., 2006). During the habituation phase, animals have free access to all three compartments. In the conditioning phase, a distinctive environmental cue in one of the outer compartments becomes associated with a target event (e.g., drug exposure) while another cue in the other outer compartment is associated with a neutral or control event. During the testing phase, animals are placed into the central compartment and allowed to freely explore the full apparatus; the time spent in each outer compartment is used to assess whether the animal’s memory of the experience was positive or negative. Conditioned place aversion has been used to assess pain in a series of studies by our research group. Briefly, in each study calves were confined to a visually distinctive treatment pen for 6 h following a procedure. Two days later calves were exposed to a second procedure and 17 then restrained for a second 6 h period in a visually different pen. Beginning two days after the second procedure, calves were tested for place aversion over three consecutive days by being allowed to move between the pens. In one study, calves spent less time in the pen where they had recovered from hot-iron disbudding compared to a pen where they recovered from a sham procedure (Ede et al., 2019a). In another study, when one horn was hot-iron disbudded in one pen and the other was caustic-paste disbudded in a different pen, calves avoided the pen associated with caustic paste (Ede et al., 2020). A third study found no evidence of place aversion to the pen associated with castration, compared to a sham procedure, regardless of castration method used (Ede et al., 2022a). The conditioned place aversion paradigm is not naturally suited for assessing lasting pain, as it assesses the animal’s experience at a specific point in time (that is in turn associated with the distinctive location they were kept during this experience). For lasting pain, a more suitable approach would be to compare periods with and without an effective analgesic, testing the prediction that calves prefer the location associated with analgesic administration only if they were experiencing pain. 1.5.3 Memory testing The detrimental effect of chronic pain on both working memory and long-term memory has been well-documented in humans (Mazza et al., 2018). The leading hypothesis is that patients with chronic pain devote much of their attentional capacity to pain processing, leaving fewer cognitive resources available for memory function. For example, Oosterman et al. (2011) found that patients in chronic pain show poor attention and deficits in working memory. Kuhajda et al. (2002) reported that participants experiencing pain during encoding (e.g., processing 18 information and creating a memory trace) and retrieval (e.g., accessing stored information) judged words more slowly than those without pain, suggesting that long-term memory deficits in patients with chronic pain may be attributed to limited attentional resources for encoding and retrieval. These results suggest that memory tests could be used to assess the cognitive impact of lasting pain in animals, particularly if pain is severe enough to affect attention. Indeed, pain has been shown to impair memory in animal studies. For example, rats suffering from chronic neuropathic pain exhibited longer to learn periods for spatial memory task, such as navigating a maze, compared to sham rats (Cardoso-Cruz et al., 2013). In the same study, when previously trained rats were subjected to nerve injury, their spatial memory performance deteriorated. In work with calves, Yoo et al. (2023b) investigated the effect of hot-iron disbudding on memory performance using a memory test paradigm. Calves were tested during 2 wk following the procedure. Although memory performance improved over time, no differences between disbudded and non-disbudded calves were noted. The authors attributed this lack of difference to insensitivity of the memory test paradigm, noting that factors affecting hunger and motivation—such as diarrhea and water intake—may have influenced the results. In summary, only a single study to date has used changes in memory to assess lasting pain in calves; this study found no evidence of memory impairment but we encourage further research to evaluate the effects of lasting pain on memory. In other animal models, appetite rather than hunger is often used as the primary motivation for such tests (reviewed by van der Staay et al., 2012). Identifying food items that calves consistently find highly rewarding may help improve the sensitivity of these assessments. 19 1.6 Factors that may influence lasting pain Understanding the factors that contribute to pain is crucial for effectively measuring and managing pain (Mills et al., 2019). Since lasting pain refers to the pain experienced during the healing period, the duration of this period is likely to influence the amount of lasting pain experienced. In addition, prolonged healing can increase the risk of complications (e.g., due to infection) that can accentuate pain (Gould et al., 2015). To our knowledge, factors influencing healing time have not been thoroughly addressed in any farm animal species, including cattle. This section explores factors related to healing time, focusing on characteristics of the animal and the nature of wound. 1.6.1 Animal factors Age is known to affect healing, likely due to age-related changes in tensile strength, wound healing factors, and inflammatory response (Thomas, 2001). In calves, studies examining the influence of age on pain and healing have yielded mixed results, complicating interpretation due to confounding variables. For instance, calves surgically castrated before 8 d of age healed faster than calves castrated at 11 wk, a difference that may be influenced by the smaller wound size in younger calves (Norring et al., 2017). Adcock and Tucker (2018) found no difference in healing time between calves disbudded at 3 versus 35 d of age, but differences in the size of the hot-iron tip (0.6 cm for younger calves vs. 1.3 cm for older calves) may have confounded the result. Both studies assessed skin sensitivity around the wounds but reported inconsistent findings. While Norring et al. (2017) observed greater sensitivity around the wound in younger calves for 6 wk following castration, Adcock and Tucker (2018) failed to note any differences in mechanical nociceptive thresholds between calves disbudded at 3 and 35 d for 3 wk following 20 the procedure. However, given the lack of differences in skin sensitivity between caustic paste–disbudded and sham-treated calves when procedures were performed at 3 and 9 d of age (Reedman et al., 2020; Karlen et al., 2021), skin sensitivity does not appear to be a suitable measure for assessing pain in young calves. Studies that report comparisons of skin sensitivity between younger and older calves should be interpreted with caution. Marti et al. (2017) observed shorter lying times in band-castrated versus non-castrated and surgically castrated 4-mo-old calves, but not in calves castrated at 1 wk or 2 mo of age, when calves were observed from d 7 to 42 after castration. Nutritional state is another factor related to healing. Nutritional deficiencies can impede wound healing and increase the risk of infection, contributing to lasting pain (Stechmiller, 2010). In calves, Reedman et al. (2022) reported that calves receiving 6 L/d of milk showed prolonged healing times after hot-iron disbudding compared to those receiving 15 L/d. 1.6.2 Nature of wound The methods used for disbudding and castration can influence both lasting pain and healing time. For example, wounds from hot-iron disbudding took 6 to 13 wk to re-epithelialize (Adcock and Tucker, 2018), while wounds from caustic paste disbudding required 6 to 32 wk for re-epithelialization and 9 to 34 wk for full contraction (Drwencke et al., 2023). Similarly, surgically castrated calves fully healed within 4 weeks, while band castrated calves did not fully heal within 8 wk (Nogues et al., 2021). In the latter study, band castrated calves showed higher skin temperature at the lesion site and more wound licking behaviour, and spent less time lying down for 8 wk after the procedure, suggesting greater lasting pain following band versus surgical castration. 21 Infection can affect healing. For instance, Roche et al. (2012) found that infected wounds in pigs took longer to heal than did non-infected wounds. Other work has shown that the biofilm produced by bacteria delays wound healing in rats (Zhao et al., 2010), but Reedman et al. (2022) found no evidence that purulent discharge (indicative of infection) was related to wound healing after disbudding in calves. Wound size and depth are associated with healing time. Larger wounds tend to heal slower, as seen in a study on diabetic foot ulcers in humans (Margolis et al., 2003). Similarly, Taniguchi and Matsumoto-Oda (2018) found that larger wounds took longer to heal in baboons. Additionally, deeper burn wound takes more time to heal (Merz et al., 2010). To our knowledge, no published studies have addressed the impact of wound size and depth on healing time in cattle. 1.7 Conclusion This narrative review has examined parameters used to study pain in calves, focusing on their potential as indicators of lasting pain. Wound-directed behaviours and skin sensitivity are likely useful for assessing incident pain during healing. The substantial time required for detailed observations is a likely to be a limitation of using wound-directed behaviours as a lasting pain indicator. Lying behaviour and weekly ADG appear to serve as general indicators of lasting pain following castration, although more sensitive lying parameters may be needed for young calves. Hair cortisol and social behaviours also seem promising, but the use of social behaviour as an indicator of lasting pain after castration remains to be tested. Memory and judgment bias tests warrant further exploration. This review also explores what factors relating to healing time have 22 been studied, including procedural methods and wound size. The identified lasting pain indicators and related factors can help inform strategies for mitigating lasting pain. 1.8 Thesis objective The primary objectives of this thesis were to 1) assess potential indicators of lasting pain following castration and disbudding in dairy calves, and 2) investigate factors influencing healing time and lasting pain following these procedures. To address these objectives firstly, I evaluated the spatial memory test as an indicator of lasting pain following hot-iron disbudding and castration as pain models. Secondly, I evaluated short-duration lying bouts and half-lying bouts as an indicator of lasting pain following castration. Thirdly, I explored the impact of two procedural factors on wound characteristics and healing following hot-iron disbudding: application time and tip size. 23 Chapter 2: The effects of pain following disbudding on calf memory A version of Chapter 2 has been published as Yoo, S., von Keyserlingk, M. A. G., & Weary, D. M. (2023). The effects of pain following disbudding on calf memory. Journal of dairy science, 106(12), 9507–9513. https://doi.org/10.3168/jds.2023-23604. 2.1 Introduction Hot-iron disbudding is the process of destroying horn-producing cells through cauterization. Disbudding causes pain that can be mitigated using a combination of sedatives, local anesthetics, and analgesics (reviewed by Stafford and Mellor, 2011). For example, the benefits of nonsteroidal anti-inflammatory drugs (NSAIDs; typically used in combination with local anesthesia) on pain related to disbudding have been demonstrated in various studies (reviewed by Herskin and Nielsen, 2018). Studies on pain due to disbudding have focused on pain mitigation during and in the hours following the procedure. For example, Stilwell et al. (2012) found that calves that received local anesthesia and carprofen before disbudding showed a decreased frequency of ear flicking for several hours after disbudding compared with calves that received only local anesthesia. Additional ketoprofen administration at 2 h before disbudding, and at 2 h and 7 h after disbudding, reduced the number of ear flicks and head shakes in the hours after disbudding (Faulkner and Weary, 2000). Additional ketoprofen administration before disbudding also decreased the total frequency of pain behaviours in the hours after disbudding (Duffield et al., 2010). One study found that meloxicam administration mitigated the typical increase in plasma cortisol, heart rate, and respiration rate for up to one day after disbudding (Heinrich et al., 2009), 24 and another found that this treatment mitigated ear flicks for 2 d after disbudding (Heinrich et al., 2010). More recent work on disbudding has shown evidence of longer lasting pain. For example, Casoni et al. (2019) found higher pain scores and reduced mechanical nociceptive thresholds (MNT) in disbudded versus control calves for approximately 3 mo after disbudding. Another study (Adcock and Tucker, 2020) reported that disbudded calves did not avoid a lidocaine-paired stimulus, but sham calves were averse to the stimulus 20 days after disbudding, indicative of continued pain. Disbudded calves treated with lidocaine at 11 d after disbudding showed a decreased frequency of head shaking compared with disbudded calves treated with saline (Adcock and Tucker, 2020). Pain is known to affect cognitive process in humans and animals, including attention (reviewed by Moore et al., 2019), decision-making (Apkarian et al., 2004; Tamburin et al., 2014), executive function (Morogiello et al., 2018), and memory (reviewed by Mazza et al., 2018). In rodents, attention (Millecamps et al., 2004; Boyette-Davis et al., 2008), emotional decision-making (Pais-Vieira et al., 2009; Ji et al., 2010), and spatial memory (Henderson et al., 2015) are disrupted by inflammatory pain, and spatial memory is affected by neuropathic pain (Cardoso-Cruz et al., 2013). In dairy calves, hot-iron disbudding is reported to affect perception (Ede et al., 2019a; Adcock and Tucker, 2020) and decision-making (Neave et al., 2013), but the effect of pain on memory has not been assessed. Previous work has used a T-maze to test learning ability in calves (Horvath et al., 2017; Horvath and Miller-Cushon, 2020). In the current study we used a modified hole-board test, as this was able to provide measures of both working and reference memory, as opposed to just reference memory in the T-maze test. 25 The hole-board test was developed to study exploratory behaviours in rats (Oades and Isaacson, 1978), and over the years it has been used to measure spatial learning and memory in various species including cats (Steigerwald et al., 1999; McCune et al., 2008), chickens (Tahamtani et al., 2015), fish (Durán et al., 2010), gerbils (Wappler et al., 2009), mice (Kuc et al., 2006; Li et al., 2009), pigs (Gieling et al., 2012; Roelofs et al., 2017), rats (Heim et al., 2004; Woldeit and Korz, 2010), and treeshrews (Keuker et al., 2004). Recently, Lecorps et al. (2022) modified the hole-board apparatus for use in dairy calves. The objective of our paper was to assess how pain, in the days and weeks after disbudding, affects measures of working and reference memory; our hope was these cognitive measures would be sensitive to longer lasting pain following the time that acute pain behaviours such as head shaking and rubbing are typically detected. 2.2 Materials and methods The study was approved by The University of British Columbia’s Animal Care Committee (# A21-0111) and carried out in accordance with the guidelines outlined by the Canadian Council of Animal Care (2009). The study was conducted at The University of British Columbia Dairy Education and Research Centre (Agassiz, British Columbia, Canada) from February to July 2022. 2.2.1 Animals and housing Thirty Holstein heifer calves were enrolled at 4 d of age. Calves were blocked by birth date and within block randomly assigned to one of three treatments. On the day of enrollment, calves were moved to a sawdust-bedded 35 m2 group pen from an individual pen. The group pen housed 26 8 to 10 calves and pen composition did not change during the study. Calves were housed in the group pen for 10 d before disbudding. Calves were offered a total of 12 L/d of whole pasteurized milk using an automated calf feeder (CF 1000 CS Combi, DeLaval Inc.). Milk consumption in the home pen was not measured. Calves were provided ad libitum access to calf starter (Hi-Pro Medicated, Hi-Pro Feeds Inc.) through the automated calf feeder. Hay and water were provided ad libitum. Once training began (see below) the milk feeder did not provide milk between 2200 h and the beginning of the trail the next morning. 2.2.2 Apparatus The apparatus was as described by Lecorps et al. (2022). Briefly, the sawdust-bedded arena was surrounded by four walls (height: 1.2 m) with three of the four walls holding five milk bottle holders (i.e. 15 holders in total). The fourth wall contained a gate through which the calves entered the pen. Bottle locations were labeled clockwise, starting from the gate (Figure 2.1). The distance between two adjacent bottles was 65 cm (measured center-to-center) and nipples were positioned 0.9 m above the floor. 27 2.2.3 Training phase Training started when calves were 9-d old. On the first and second day of training, calves were brought to the front of the apparatus and gently pushed into the arena where they were fed 2 L of milk from a single bottle (location # 8 in the apparatus). On the third day, four bottles (each containing 0.5 L of milk) were placed at locations # 5, 6, 7, and 8 with the remaining positions empty. On the fourth day, 4 bottles containing 0.5 L of milk were placed at locations # 3, 8, 10, and 13 with the remaining locations empty. On the fifth day, the rewarded bottles were in the same locations and 4 empty bottles were provided at locations # 2, 7, 11, and 14. On the sixth day the locations of rewarded bottles and empty bottles were switched. The purpose of this initial training phase was to familiarize calves with the concept that some bottles were rewarded and others were not. We switched the location of rewarded and unrewarded bottles at the end of this phase so that calves would begin the next phase with no knowledge of the locations of the rewarded bottles. When the calves were first brought to the apparatus, they were provided with 0.5 L of milk. They Figure 2.1 View of the hole-board test apparatus as seen from the entrance to the arena. The locations of each of 15 milk bottles are indicated. 28 were given another 0.5 L of milk when they were returned to their home pen, such that calves received a total of 3 L/d before, during, and immediately after these sessions. 2.2.4 Treatments After the initial training phase calves were assigned to 1 of 3 treatments (n=10 calves/treatment): disbudding (D), disbudding with additional analgesic (D+AA), and sham disbudding (S). The sample size was determined based on the results of Lecorps et al. (2022) who showed memory improvement over time using 9 calves; we added one calf per group to account for possible exclusions. All calves were sedated using xylazine (SC, 0.2mg/kg, right rump, Nerfasin 20TM, 20 mg/ml, Dechra, Northwich, United Kingdom). Once calves were recumbent with eyeballs rotated (Ede et al., 2019b), 5 ml of lidocaine (Lido-2 [lidocaine 2%, Epinephrine 1:100,000], Rafter8, Calgary, Canada) was injected caudal to the lateral canthus of eyes, using a 20-gauge x 25-mm needle. Ten min after the lidocaine was administered, the hair around the horn buds was clipped, and calves were tested for pain reflex using a needle-prick (no calf responded). Cautery disbudding was conducted by applying a 500°C hot-iron (X30, 1.3 cm tip, Rhinehart, Spencerville, IN, USA) to the horn bud for approximately 15 s. Sham calves were also sedated and received 5 ml lidocaine and the unheated iron was placed lightly against the horn bud for 15 s. Within a minute after iron application, all calves were treated with ketoprofen (SC, 3 mg/kg, right neck, Anafen, 100 mg/mL, Boehringer Ingelheim, Burlington, Ontario, Canada). At the same time, calves in the disbudding with additional analgesic were also treated with meloxicam (SC, left rump, 0.5 mg/kg, Metacam, 20 mg/mL, Boehringer Ingelheim, Burlington, Ontario, Canada). Calves in this treatment then continued to receive injections of meloxicam every other day until the end of their trial. 29 2.2.5 Memory tests The memory test consisted of two test phases. The first phase started the day after disbudding and continued for 12 d. Each day, from 0800 to 1000 h, calves were brought individually to the apparatus with 4 rewarded bottles and 11 empty bottles. Rewarded milk bottles contained 0.5 L milk and were positioned at locations 1, 4, 9, and 12; empty bottles were placed at the other 11 locations. The second phase started on d 13 with rewarded bottles moved to locations 3, 7, 11, and 14, and empty bottles were placed at the other 11 locations. The trial ended when the calf drank milk from all 4 bottles or after 5 min had elapsed, whichever came first (following Lecorps et al., 2022). To reduce the risk that calves could use olfactory or visual cues, we washed all nipples after each calf was tested. Trials were video recorded using a Network Video Recorder (NVR8-7250, Swann). A visit was recorded whenever the calf touched a bottle with its nose or mouth. SY and two other observers (blind to treatment) scored 3 d of video (13 trials) to calculate inter-scorer reliability (percent agreement = 95%). SY then scored all of the remaining videos. 2.2.6 Behavioural measures We monitored acute pain behaviours during the period when others have reported these behaviours (Heinrich et al., 2010). Calf behaviour in the group pen was recorded using the same video recording system. Three observers blind to treatment recorded head shakes (i.e. calf turns its head rapidly from one side to the other side; a shake was recorded once the head returned to a resting position) and head rubs (i.e., moving the rear leg in contact with the upper head, or moving the head in contact with some part of the pen; a head rub was counted when the movement stopped). The frequency of these behaviours was recorded for 6 h (from 1800 to 2400 h) on the 30 day of disbudding, and on days 2 and 4 after disbudding. Inter-scorer reliability (85.6 %) was calculated using 36, 20-min video clips. 2.2.7 Statistical analysis We constructed linear mixed models for behaviour and memory data using the lme4 package in R version 4.2.1 (R Core Team, 2022). Normality and homogeneity were confirmed by visually assessing the residual plots from the mixed models. The fixed effects were treatment, time, and treatment x time interaction. Calf was the experimental unit and specified in our models as a random effect. When an interaction was detected, post hoc Tukey’s HSD tests were used to investigate. The pen footage was missing for 2 d such that 4 calves were excluded from the behaviour analysis: one calf in S, one calf in D, and two calves in the D+AA treatment. The results of the memory test allow for assessments of general working memory (GWM; i.e., the number of different bottles visited / the total number of visits to all bottles), working memory (WM; i.e., number of baited bottles visited / total number of visits to baited bottles), reference memory (RM; i.e., the total number of visits to baited bottles / the total number of visits to all bottles), and modified reference memory (MRM; i.e., number of baited bottles visited / total number of bottles visited). We calculated MRM to more specifically investigate longer term memory, as RM has a working memory component. Two calves (one in D and one in D+AA treatments) did not drink milk for 4 out of 6 trials in the second test phase; the reasons for this were not clear and these animals were excluded from the analysis of memory during the second test phase. Given that both GWM and WM assess short-term memory and the results showed a similar pattern, we only presented the results for GWM. 31 2.3 Result and discussion 2.3.1 Memory GWM (F11,294.4 = 4.10, P < 0.001), RM (F11,294.4 = 10.7, P < 0.001), and MRM (F11,293.9 = 8.40, P < 0.001) improved over the first test phase, indicating calves learned the locations of milk bottles, but we detected no effect of treatment and no treatment by time interaction. Similarly, during the second test phase, both GWM (F5,122.7 = 6.53, P < 0.001), RM (F5,122.6 = 5.08, P < 0.001), MRM (F5,122.6 = 3.97, P < 0.001) improved, but again we detected no effect of treatment or treatment by time interaction (Figure 2.2). 32 Figure 2.2 Changes in a) general working memory (GWM) scores, b) reference memory (RM) scores, and c) modified reference memory (MRM) scores following treatment [D=disbudding (n=10); D+AA=aditional analgesic treatment after disbudding (n=10); S=sham disbudding (n=10)]. Calf memory was assessed once a day for 18 d using a modified hole-board apparatus. Over the first 12 d (first phase) the locations of the rewarded bottles were kept constant; on d 13 these locations were changed and calves were tested for 6 more days (second phase). 33 To our knowledge, the current study is the first to assess the effects of pain on learning and memory in dairy calves. Contrary to our prediction, we found no effect of treatment during either the first test phase or the second test phase when the location of the rewarded bottles was changed. Like Lecorps et al. (2022), we found that the modified hole-board test was able to assess changes in GWM and RM, how performance improves with training, and how performance is affected when the locations of the rewarded stimuli were changed. It is possible that calves may have detected olfactory or visual cues that would aid in detecting the correct positions, but we believe that this was unlikely given that we washed all bottles between calves and given the drop in performance we detected when the location of the rewarded bottles was changed. Previous work has shown negative effects of lasting pain on memory in humans (Mazza et al., 2018) and rats (Johnston et al., 2012). It is not clear why, in the current study, disbudding had no effect, but one possible explanation is that none of the treatments resulted in lasting pain during the experiment. This interpretation is consistent with the behavioural results described in the next section, showing no consistent evidence of pain (i.e. neither head rubbing nor head shaking was elevated in disbudded calves relative to the sham-treated calves). For humane reasons all calves received a sophisticated, multi-modal pain treatment; this treatment protocol may have been sufficiently effective as to render both disbudding treatments indistinguishable from the sham treatment and to negate any benefit of the supplementary NSAID. It is also possible that there were differences in the pain experienced by calves in the different treatments, but our assessment methods were not sufficiently sensitive to detect these differences. Anderson et al.'s (2021) research on humans showed that higher pain intensity is associated with lower WM performance. Johnston et al. (2012) reported intense long-lasting inflammatory pain 34 caused by Complete Freund’s adjuvant (CFA) resulted in learning impairment. In the current study, any pain experienced during the days and weeks following disbudding may not be have been enough to impair memory. 2.3.2 Behaviour We found a treatment by time interaction (F4, 46 = 4.28, P < 0.01) on head-shaking behaviour (Figure 2.3a), driven by more head shaking of D+AA calves on d 4 after disbudding compared to the D calves. We found no effect of time or treatment on head rubbing behaviour (Figure 2.3b). 35 While head shaking decreased over time in D and S calves, it did not decline in the D+AA calves. This result may be explained by the combination of post-burn pruritus and pain habituation. The hot-iron disbudding process causes burn wounds that take about 9 wk to heal (Adcock and Tucker, 2018). The healing process may have been associated with itching (pruritus); over 90% of Figure 2.3 The total number of a) head shakes and b) head rubs recorded from 1800 h to 2400 h, on d 0 (starting 8 h after the disbudding treatment was applied; D = disbudding, D+AA = additional analgesic after disbudding, S = sham disbudding), d 2 and d 4. Boxes illustrate the interquartile range, the central line indicates the median, the red point indicates the mean, the whiskers show the calculated minimum and maximum values, and the individual points show values beyond the minima and maxima. Different letters show differences (P < 0.05) between treatment groups. 36 human burn patients report itching at the early healing phase (Carrougher et al., 2013). Humans repeatedly exposed to identical painful stimuli over several days report decreased pain intensities over time (Bingel et al., 2007; Rennefeld et al., 2010; Maeoka et al., 2015). Similarly, when the pruritus occurred, D calves might feel less intensity of itch than D+AA calves because D calves had adapted to unpleasant sensations on the disbudding wound while pain in D+AA calves was controlled. Further work is required to determine the extent that head shaking and rubbing may be associated with itch. We noted that some calves appeared to rub their head on their back and on the floor of the pen, but we could not score these behaviours from video as we recorded pens from above and this angle made it impossible to reliably distinguish between licking the back and rubbing the head on the back. For this reason we likely missed some head rubbing. We encourage future studies to include these behaviours in their assessments of head rubbing, perhaps using video recorded from different angles. 2.4 Conclusion Calves in all three treatments learned the locations of 4 rewarded bottles in a modified hole-board test. Memory performance declined as expected when the locations of the rewarded bottles were changed, but performance again improved with repeated testing with the new locations. We found no effect of disbudding, with or without the provision of supplementary analgesics, on either working or reference memory, perhaps because the intensity of lasting pain was too minor to cause differences in learning and memory. 37 Chapter 3: Assessment of lasting pain following castration in 2-week-old calves 3.1 Introduction Band and surgical castration are commonly performed on cattle (Moggy et al., 2017; Greaves, 2019). While both methods cause pain, the duration and timing of the pain is likely different. Previous research has shown that surgically castrated calves spend less time lying (Meléndez et al., 2017) and gain less weight (Roberts et al., 2018) compared to band-castrated calves during the 1st week following castration. In contrast, band-castrated calves spent less time lying down compared to surgically castrated calves from days 7 to 42 following castration (Marti et al., 2017) and consumed less feed compared to sham calves during the 4th week after castration (Repenning et al., 2013). Marti et al. (2017) also found that post-operative swelling peaked 3 weeks after band castration versus just 1 week after surgical castration, suggesting that differences in pain be associated with differences in wound healing. Pain following an injury can be classified as evoked (i.e., triggered by an external stimulus, such as tissue palpation) or non-evoked (i.e., spontaneous or occurring without an identifiable stimulus). These two types of pain may have distinct mechanisms and require different treatments (Segelcke et al., 2019). Studies on pain following castration generally have not distinguished between evoked and non-evoked pain, and some of the responses used to assess pain are likely affected by both. For example, lying behaviors, such as lying time and number of lying bouts, often used to assess post-castration pain (Marti et al., 2017; Meléndez et al., 2017; Cho et al., 2024), can be influenced by both evoked and non-evoked pain. By 38 distinguishing between these types of pain, we may be better able to identify and treat pain in animals. Lying behaviors have limitations for assessing pain after castration, as evidenced by variability across studies. For example, one study (Meléndez et al., 2017) reported fewer lying bouts on days 2 and 3 following both band and surgical castration, whereas the same research group did not observed this difference in a subsequent study (Meléndez et al., 2018b). This discrepancy is not surprising, given that factors including calf health can also affect lying behavior (Studds et al., 2018; Swartz et al., 2020; Guevara-Mann et al., 2023). However, no differences in lying behaviors were detected between surgically and band-castrated calves when castration was performed at one week or two months of age (Marti et al., 2017). Given that early-age castration is recommended (NFACC, 2013; AABP, 2019), there is need for more sensitive and specific indicators of post-castration pain in young calves. We hypothesized that when castrated calves lie down, pressure applied to the wound from contact with the ground evokes pain, prompting the calf to move to alleviate the pain (e.g., by standing up). Following this logic, we predicted that short-duration lying bouts (i.e., when animals lie down but quickly stand up again, sooner than would normally occur) would increase following castration. Similarly, we predicted an increase in the frequency of incomplete lying bouts (i.e., when calves attempt to lie down but keeps their hindquarters elevated, similar to a "play bow" in dogs), reflecting anticipation of evoked pain at the castration site when changing posture. Pain has been shown to negatively impact memory in humans, likely because it reduces attentional resources available for memory tasks (Mazza et al., 2018). In rats, previous work has shown that lasting pain can induce spatial memory deficits (Hu et al., 2010; Henderson et al., 39 2015). These results suggest that memory tasks could be used to assess lasting pain following castration. The aims of the current study were to assess 1) short-duration lying bouts and incomplete lying bouts as indicators of evoked pain, and 2) changes in spatial memory as an indicator of lasting pain, over a 4-week period following castration. We focused on the 1st and 4th weeks post-castration, periods during which pain intensity was expected to differ between band and surgical castration. To allow for stronger inferences regarding pain, we included pain control groups in which meloxicam, a nonsteroidal anti-inflammatory drug, was administered. As the primary objective of this study was to evaluate potential indicators of lasting pain following castration, meloxicam was provided only during the focal periods—specifically, the 1st and 4th weeks after castration. Additionally, total lying time and number of lying bouts were measured, as these have commonly been used to assess pain in previous studies. We also measured healing scores, given our hypothesis that pain intensity would correlate with wound healing status. 3.2 Material and methods The experiment was performed at The University of British Columbia Dairy Education and Research Centre located in Agassiz, BC, from July 2022 to March 2023. 3.2.1 Animals and housing Forty male calves (24 Holstein and 16 Holstein x Angus cross-bred) were enrolled in the study at 4 days of age. The first 20 calves were randomly assigned unique 3-digit identification numbers using a computer-generated random sequence (RAND function in Google Sheet), blocked by birth order into groups of four (regardless of breed). Within block, treatment was 40 assigned based on the calves’ assigned number: the calf with the lowest number was assigned to band castration; the second lowest to surgical castration; the second highest to band castration with week-long meloxicam treatment, and the highest to surgical castration with week-long meloxicam treatment. The next 20 calves were pseudo-randomly assigned to one of the four treatments, balancing for breed and weight (six Holstein calves and four Holstein x Angus cross calves per treatment group). The sample size was based on findings from Lecorps et al. (2022) showing effects on measures of the hole-board test with nine calves per group; we added one additional calf (n=10 per treatment) in the event that any animals were later excluded. Once enrolled, the calves were moved into spacious group pens with sawdust bedding. They were given 12 L of pasteurized milk each day through automatic feeders and had constant access to water, hay, and calf starter feed. During training and testing days, they got a bit less milk from the automated feeder (9 L/day) but were given an additional 3 L around the training and testing sessions as described in the next section. 3.2.2 Treatments At 14 days of age, after the training for the spatial memory test was complete (see below), calves were castrated according to their assigned treatment: band castration with or without week-long meloxicam treatment or surgical castration with or without week-long meloxicam treatment administered during the 1st and 4th weeks post-castration. All calves were sedated with xylazine (subcutaneous, 0.25 mg/kg, right neck, Rompun, 2%, Bayer Inc.). Once calves were fully sedated (approximately 10 min after administration of xylazine), they were injected with a local anesthetic (Lurocaine, 2% lidocaine, Vetoquinol N.A. Inc., Lavaltrie, Quebec, Canada; 1.5 ml into each testis and 2 ml subcutaneously at the base of the scrotum). 41 Castration was conducted 10 min after local anesthetic injection. Band castrations were performed by applying a latex band around the upper neck of the scrotum using an elastrator. Surgical castrations were performed by excising the distal third of the scrotum and removing the testes. Within a minute after castration, 3 mg/kg of ketoprofen (Anafen, Boehringer Ingelheim, Burlington, Ontario, Canada) was injected subcutaneously on the neck. Calves in the meloxicam treatment received 0.5 mg/kg of meloxicam (Metacam, Boehringer Ingelheim, Burlington, Ontario, Canada) subcutaneously at the neck after castration and every other day throughout the memory-testing phases. 3.2.3 Clinical measurements Healing scores were measured to describe differences between surgical and band castration. Castration sites were photographed with a smartphone camera (Galaxy 21, Samsung Electronics, Seoul, South Korea) from a distance of approximately 0.5 m on the day before castration (day-1) and weekly. S. Yoo and one other observer assessed the healing stage of wounds using the pictures on a 5-point scale following Meléndez et al. (2017). Briefly, healing scores for surgical castration were based on the length of the incision: for example, a visible incision extending the full length of the scrotum was scored as 1, while a completely healed incision with no visible scar was scored as 5. Healing scores for band castration were based on the condition of the scrotum: for instance, a necrotic scrotum without purulent exudate was assigned a score of 2, and the detachment of the necrotic scrotum was scored as 4. Inter-rater agreement was 78%, and discrepancies were resolved through consensus. Both observers were blinded to meloxicam treatment status but not to castration method, as wound appearance allowed visual identification of the procedure type. 42 3.2.4 Lying behavior Lying behavior was monitored using data loggers (Hobo Pendant G; Onset Computer Corp., Bourne, Massachusetts, USA), programmed to record the x-axis orientation every 15-second. Loggers were placed vertically on the right front leg, just above the fetlock joint, using self-adhesive bandages when the calves were 4 days old. On the day of castration (14 days of age), the devices were replaced and then exchanged weekly, alternating between the left and right front legs. Data from the loggers were imported into Microsoft Excel and processed according to the procedure outlined by UBC Animal Welfare Program (2013). To validate logger accuracy, data were compared with video footage of two calves recorded using overhead cameras (WV-CW504SP, Panasonic, Kadoma, Osaka, Japan), positioned 8 m above each pen. From the first 2 calves, errors were only observed in lying bouts lasting 2 min or less. Therefore, for subsequent calves, only these shorter bouts were reviewed. Corrections were made when discrepancies were observed—for example, if a calf was recorded as standing while the video showed bent hind limbs, the bout was reclassified as lying. The corrected data were used to calculate the number of short-duration lying bouts. Total lying time and total number of lying bouts were also calculated as reference measures. Video footage was also used to identify incomplete lying postures, defined as positions in which the calf’s hindquarters remained elevated, resembling the “play bow” posture commonly observed in dogs. 43 3.2.5 Spatial memory test 3.2.5.1 Apparatus Calves were trained and tested in a 4 x 4 m sawdust-bedded pen, enclosed with plywood walls 1.2 m high. One wall had a gate in the middle and each of the other walls held five milk bottle holders; bottle positions were numbered clockwise starting to the left side of the gate and ending at the holder nearest the right side of the gate. The distance between the bottles was 0.5 m and the height of the nipple was 0.9 m above the pen surface (see Yoo et al., 2023b). 3.2.5.2 Initial training Calves were trained daily from 9 to 14 days of age. All training and test sessions were conducted individually once a day, between 0800 to 1000 h. During these sessions calves were brought to the apparatus and provided with 0.5 L of milk. Calves were also given an additional 0.5 L of milk when returned to their home pen. In addition, a total of 2 L/d of milk was provided during the task, resulting in a total of 3 L of milk per session. On the 1st day of training, a full 2-L bottle of milk was placed in holder position # 8, and calves were led to this bottle and allowed to drink. On the 2nd day this procedure was repeated but calves were allowed to find the bottle on their own. On the 3rd day, four 2-L bottles, each containing 0.5 L of milk, were placed on holders # 5, 6, 7, and 8. On the 4th day, this procedure was repeated but bottles were placed in positions # 3, 8, 10, and 13. On the 5th day, this procedure was repeated with the addition of 4 empty bottles placed in positions # 2, 7, 11, and 14. On the 6th day, the locations of bottles containing milk and those without were switched. 44 3.2.5.3 Memory tests Two, week-long daily memory tests were conducted. In the 1st week of testing (starting the day after castration, when the calves were 15 days old), 2-L bottles containing 0.5 L of milk were placed in positions #1, 4, 9, and 12, while all other positions contained empty bottles. The locations of the milk-filled bottles remained unchanged throughout the test phase. During the second week of testing (starting 22 days after castration, when calves were 36 days old), the test procedures were identical, but the bottles containing milk were placed in positions #3, 7, 11, and 14. Trials were terminated when the calf drank milk from all 4 milk bottles or after 5 min had elapsed, whichever came first. Trials were video recorded using a camera (NVR8-7250, Swann Communications U.S.A. Inc., Santa Fe Springs, California, USA) positioned above the test apparatus, and visits to the bottles were assessed from the video. A 'visit' to a bottle was recorded when the calf approached the nipple and touched it with its nose. If the calf did not move to another bottle and repeatedly touched the same bottle, this was recorded as a single 'visit'. Whether the calves drank milk or emptied the bottles was noted by live observation. Reference memory (RM) was calculated by dividing the total number of visits to the milk-containing bottles by the total number of visits to all bottles. SY and two other observers, blinded to treatment, recorded visits from 12 randomly selected trials, with an inter-observer agreement of 93%. 3.2.6 Statistical Analysis Calves were excluded from the analysis if they drank less than 4 L/day of milk in the home pen for four consecutive days, as this was taken as a sign of illness; this resulted in the loss 45 of one calf from the surgical castration without meloxicam treatment group. Missing data noted for healing scores at week 2 after castration for six calves (two in the band castration without meloxicam treatment group, two in the band castration with meloxicam treatment group, and two in surgical castration with meloxicam treatment group), and for lying behavior for five calves (three in band castration without meloxicam treatment group, one in band castration with meloxicam treatment group, and one in surgical castration with meloxicam treatment group). These calves were excluded from the respective analyses. Additionally, one calf in the band castration with meloxicam treatment group did not interact with any bottles during the second phase of the memory test (starting 22 days after castration) and was excluded from analysis for that measure. Statistical analyses were conducted using PROC GLIMMIX in SAS software (version 9.4, SAS Institute Inc., Cary, North Carolina, USA). The distribution of residuals was visually assessed using quantile-quantile plots of conditional studentized residuals to ensure model assumptions were met. The number of short-duration lying bouts, number of daily lying bouts, daily lying time, and memory scores during the 1st and 4th weeks after castration, and weekly healing scores over 4 weeks after castration were analyzed using mixed models. Fixed effects included castration method, meloxicam treatment, time (day for daily measures; week for healing score), their interactions, and breed. Calf ID was included as a random effect. The covariance structure that minimized the Akaike Information Criterion was selected from among compound symmetry, first-order autoregressive, and first-order ante-dependence structures. Due to the infrequent observation of incomplete lying bouts, with occurrences on only a few days each week, this measure was calculated as a weekly cumulative value. A separate 46 statistical model was used to analyze these weekly counts of incomplete lying bouts. This model included the fixed effects of castration method, meloxicam treatment, and their interaction to assess their impact on the outcome. Results are presented as least square means and standard errors unless otherwise specified. 3.3 Results 3.3.1 Bimodality of lying-bout duration Short-duration lying bouts were identified using the distribution of all lying bout durations during the 1st week after castration. First, the left-skewed distribution was normalized using a cube root transformation. The transformed frequency distribution revealed two distinct peaks with a nadir between peaks at 11.4 min (Figure 3.1d). In contrast, this bimodal pattern was not observed when lying bout durations from the week before castration were transformed (Figure 3.1b). Bouts shorter than this value were identified as short-duration lying bouts. 47 3.3.2 Wound healing There were effects of castration method (F1,87 = 76.6, P < 0.001), time after the procedure (F3,87 = 167.8, P < 0.001), and their interaction (F3,87 = 16.4, P < 0.001) on wound healing, but we found no effect of the meloxicam treatment (F1,87 = 0.01, P = 0.94, Figure 3.2) on this measure. By week 4 post-castration, 18 of 19 surgically castrated calves were fully healed, but none of the band-castrated calves had done so. Figure 3.1 Distribution of lying bout lengths during the week before and after castration. Histogram of raw data of the week a) before and b) after castration and histogram of cube root-transformed data of the week c) before and d) after castration. The arrow indicates the selected cut-off point for short-duration lying bouts. 48 Figure 3.2 Healing scores over 4 weeks following castration in Holstein and Holstein x Angus crossbred calves castrated at 14 days of age (B =Band castration; S = Surgical castration). Dots with black borders represent values for individual calves, dots without border represent treatment means for each week, and whiskers indicate the 2nd and 98th percentiles. 3.3.3 Lying behaviors On the day after castration, the number of short-duration lying bouts increased (time effect, F6,180 = 9.64, P < 0.001), with band-castrated calves showing more bouts compared to surgically castrated calves (castration x time interaction, F6,180 = 2.64, P = 0.02, Figure 3.3a). This difference was not observed beyond the first day after castration. During the 4th week after castration, only band-castrated calves treated with meloxicam showed an increased frequency of short-duration lying bouts over time (castration x meloxicam x time interaction, F6,180 = 3.34, P = 0.003, Figure 3.3c). No effect of castration method, meloxicam treatment, or their interaction was detected on incomplete lying bouts during either week 1 or week 4 after castration (Table 3.1 and 3.2). 49 The total number of lying bouts also increased on the day after castration and then decreased the following day (time effect, F6,180 = 8.59, P < 0.001, Figure 3.3b). No other effects of castration method, meloxicam, or their interaction were detected on total lying bouts during the week after castration. During week 4, no effects of castration method, meloxicam, time, or their interaction were detected. For lying time, no effects of castration method, meloxicam, time, and their interaction were detected during either period. 50 Figure 3.3 Least squares means (± SE) for (A) short-duration lying bouts, (B) total lying bouts, and (C) lying bouts duration during week 1, as well as (D) short-duration lying bouts during week 4, following band and surgical castration in Holstein and Holstein × Angus crossbred calves castrated at 14 days of age (B = Band castration, S = Surgical castration, NM = no meloxicam treatment, and M = meloxicam treatment). 51 Table 3.1 Least squares means ± SE of behaviors and reference memory of the week following band and surgical castration in 2-week-old Holstein and Holstein x Angus crossbred bull calves with or without week-long meloxicam treatment Treatment Band castration Surgical castration P-value (treatment df, error df) Variables NM M NM M C M C x M T T x C T x M T x C x M Number of short-duration lying bouts, no./day 3.39 ± 0.74 4.07 ± 0.65 3.54 ± 0.65 5.10 ± 0.65 0.38 (1, 180) 0.10 (1, 180) 0.52, (1, 180) < 0.001 (6, 180) 0.002 (6, 180) 0.53 (6, 180) 0.55 (6, 180) Number of incomplete lying bouts, no./week 1.86 ± 1.09 1.00 ± 1.01 2.11 ± 0.96 2.55 ± 0.96 0.38 (1, 28 0.84 (1, 28) 0.52 (1, 28) - - - - Lying time, h/day 16.75 ± 0.35 17.14 ± 0.31 16.87 ± 0.31 17.23 ± 0.31 0.74 (1, 180) 0.25 (1, 180) 0.96 (1, 180) 0.07 (6, 180) 0.12 (6, 180) 0.74 (6, 180) 0.48 (6, 180 Number of lying bouts, no./day 17.41 ± 1.2 17.93 ± 1.07 17.93 ± 1.07 20.54 ± 1.07 0.16 (1, 180) 0.16 (1, 180) 0.35 (1, 180) <0.001 (6, 180) 0.16 (6, 180) 0.61 (6, 180) 0.96 (6, 180) Reference memory 0.23 ± 0.02 0.23 ± 0.02 0.21 ± 0.02 0.20 ± 0.02 0.30 (1, 210) 0.92 (1, 210) 0.99, (1, 210) < 0.001 (6, 210) 0.17 (6, 210) 0.96 (6, 210) 0.80 (6, 210) Abbreviations: NM = Without meloxicam treatment; M = With meloxicam treatment; C = Castration effect; M = Meloxicam effect; T = Time effect. 52 Table 3.2 Least squares means ± SE of behaviors and reference memory of the 4th week following band and surgical castration in 2-week-old Holstein and Holstein x Angus crossbred bull calves with or without week-long meloxicam treatment Treatment Band castration Surgical castration P-value (treatment df, error df) Variables NM M NM M C M C x M T T x C T x M T x C x M Number of short-duration lying bouts, no./day 3.23 ± 0.73 3.61 ± 0.65 2.77 ± 0.65 3.88 ± 0.65 0.89 (1, 180) 0.27 (1, 180) 0.59 (1, 180) 0.45 (6, 180) 0.73 (6, 180) 0.65 (6, 180) 0.004 (6, 180) Number of incomplete lying bouts, no./week 0.61 ± 0.35 0.50 ± 0.33 0.75 ± 0.31 0.30 ± 0.31 0.92 (1, 28) 0.40 (1, 28) 0.26 (1, 28) - - - - Lying time, h/day 16.41 ± 0.36 17.10 ± 0.31 16.90 ± 0.31 17.25 ± 0.31 0.32 (1, 180) 0.11 (1, 180) 0.61 (1, 180) 0.33 (6, 180) 0.52 (6, 180) 0.60 (6, 180) 0.59 (6, 180) Number of lying bouts, no./day 16.24 ± 1.43 18.28 ± 1.26 17.50 ± 1.26 19.66 ± 1.26 0.31 (1, 180) 0.11 (1, 180) 0.96 (1, 180) 0.19 (6, 180) 0.47 (6, 180) 0.95 (6, 180) 0.75 (6, 180) Reference memory 0.26 ± 0.02 0.28 ± 0.03 0.25 ± 0.03 0.26 ± 0.02 0.52 (1, 204) 0.59 (1, 204) 0.71, (1, 204) < 0.001 (6, 204) 0.52 (6, 204) 0.51 (6, 204) 0.59 (6, 204) Abbreviations: NM = Without meloxicam treatment; M = With meloxicam treatment; C = Castration effect; M = Meloxicam effect; T = Time effect. 53 3.3.4 Memory Reference memory improved over time during both week 1 and 4 (time effect; F6,210 = 6.39, P < 0.001 and F6,204 = 4.15, P < 0.001, respectively; Figure 3.4). There was no effect of castration method, meloxicam, or their interaction on this measure. Figure 3.4 Least squares means (± SE) of reference memory during the (A) 1st and (B) 4th weeks following band and surgical castration in Holstein and Holstein x Angus crossbred calves castrated at 14 days of age. 3.4 Discussion This study investigated the effects of castration method (band vs. surgical) and supplemental nonsteroidal anti-inflammatory drug treatment on pain and memory in dairy calves. 54 By week 4, the wounds on most surgically castrated calves were fully healed, whereas the majority of band-castrated calves still presented with necrotic scrotal tissue and purulent exudate. These results align with previous research on calves castrated at 1 and 4 weeks of age (Meléndez et al., 2018b; Nogues et al., 2021), and indicate that surgical castration allows for improved wound healing. Despite the concern that NSAIDs may delay wound healing by suppressing inflammation (Darby and Weller, 2017), we found no evidence that meloxicam affected healing. This result aligns with Marti et al. (2018), who reported that a single dose of meloxicam did not hinder healing after either band or surgical castration. We observed distinct peaks in the distribution of lying bout durations in the 1st week after castration; this pattern was absent before castration. Band-castrated calves showed more short-duration lying bouts on the day following castration compared to surgically castrated calves, but we found no such effect for the total number of lying bouts. Interestingly, during the 4th week post-castration, band-castrated calves that received meloxicam showed an increase in short-duration lying bouts over time, while untreated band-castrated calves showed a decrease. One possible explanation for this pattern is that meloxicam delayed the inflammatory response associated with tissue necrosis, postponing the peak of pain to later in this week. Contrary to our hypothesis, we found no effect of either treatment on the frequency of incomplete lying bouts. We note that there was considerable variation among subjects in this outcome measure (11 out of 34 calves showed no incomplete lying bouts during the 1st week following castration). More work is required to understand why calves exhibit this posture. We found no effect of castration method on lying time or total lying bouts. Meléndez et al. (2017) showed age-dependent changes in lying behavior, with 2- and 4-month-old calves exhibiting more pronounced differences in lying time between castration methods, while 1-week-55 old calves did not. We speculate that the 2-week-old calves in this study were not old enough to show clear differences in lying time and lying bouts in response to castration method. We found time effects, regardless of method, on lying bouts during the 1st week, with the highest number of lying bouts on the day after castration. These results are consistent with previous studies in 1-week-old calves (Meléndez et al., 2017; 2018b). These findings suggest that while the multimodal analgesia used in this study may have controlled pain on the day of castration, it was likely insufficient to alleviate pain on the following day. Further work is needed to develop more effective methods for controlling pain on the day after castration. Regarding spatial memory, we found no effects of castration method or meloxicam treatment. This result is consistent with our findings on disbudding (Yoo et al., 2023b). The absence of a treatment effect may reflect several possibilities: pain-related differences may have been too subtle to affect cognition, or the spatial memory task may have lacked the sensitivity to detect mild impairments. We used food as a motivator, but hunger can also influence memory, as shown in both rats (Vawter and Van Ree, 1989) and calves (Lecorps et al., 2023). We withheld milk before testing to motivate performance in the hole-board task, but calves at this age vary considerably in their consumption of solid feed and this could have introduced variation in satiety and hence calf motivation. Additionally, gastrointestinal disorders can affect cognitive performance (Kennedy et al., 2014), but we cannot draw conclusions regarding this effect, as calf health was not systematically recorded during the experiment. Overall, the circumstances associated with the current study may have limited the use of the spatial memory test as a measure of lasting pain. A limitation of this study is the time period we focused on. We hypothesized that the 1st and 4th week after castration would be optimal for detecting differences between surgical and 56 band castration, but the timing of pain, especially following band castration, might vary considerably between individuals. Daily assessments of castration wounds may better elucidate the relationship between healing progression and pain. 3.5 Conclusion We found evidence of pain on the day after castration, suggesting that the multimodal protocol we used (a combination of sedation, a local nerve block, and ketoprofen) was insufficient. To our knowledge, this is the first report of bimodality in lying bout duration following castration. We also demonstrated that short-duration lying bouts increased post-castration, likely reflecting evoked pain associated with posture changes. Short-duration lying bouts may serve as a sensitive and practical indicator for assessing post-castration pain in future research. 57 Chapter 4: The effects of hot-iron application method on wound characteristics and healing in dairy calves 4.1 Introduction Hot-iron disbudding is the most commonly used method for disbudding dairy calves (USDA, 2018). Research has focused on pain experienced during and shortly after the procedure (reviewed by Stafford and Mellor, 2011; Winder et al., 2018), but some recent work has begun to examine pain throughout the healing period, as well as the healing process itself. Adcock and Tucker (2018) reported that epithelium was first observed 62 d following hot-iron disbudding, with increased skin sensitivity observed around the wound once new epithelial tissue appeared. Similarly, Adcock et al. (2019) reported that it took on average 54 d to re-epithelialization and a gradual decrease in wound length over 56 d after disbudding. Drwencke et al. (2025b) reported 42 d to re- epithelialization and also noted increased skin sensitivity around the wound following epithelialization. Several factors influencing healing time following hot-iron disbudding have been investigated. Adcock et al. (2019) compared two brands of hot-irons with similar tip size and found no effect on wound healing. Adcock and Tucker (2018) also found no difference in time to re-epithelialization between calves disbudded at 3 and 35 d of age. Reedman et al. (2022) observed delayed healing in calves provided 6 L/d of milk compared to those given 15 L/d. Post-procedural treatments have also been studied. Ridgway et al. (2022) found that oxytetracycline treatment immediately after disbudding promoted healing, and Huebner et al. (2017) reported improved healing in calves treated with aluminum-based aerosol bandages. More recently, 58 procedural factors related to caustic paste disbudding—such as paste dose and presence of hair—have been examined (Drwencke et al., 2025b), but to our knowledge the effects of procedural factors specific to hot-iron disbudding—such as tip size, heating time, and application duration—have not been studied. Wound size and depth are known to influence healing time. For example, larger foot ulcers in humans require more time to heal (Margolis et al., 2003), and wound size has been shown to affect healing duration in baboons (Taniguchi and Matsumoto-Oda, 2018). Additionally, deeper burn wounds in human patients are associated with longer healing times (Merz et al., 2010). The objective of this study was to investigate the effects of hot-iron application time and tip size on wound characteristics and healing duration. We hypothesized that longer application times would result in deeper wounds, larger tips would produce larger wounds, and that both factors would result in longer healing times. 4.2 Material and methods The study was approved by The University of British Columbia’s Animal Care Committee (# A21-0111; # A25-0081) and performed in accordance with the guidelines outlined by the Canadian Council of Animal Care (2009). The study was conducted at The University of British Columbia Dairy Education and Research Centre (Agassiz, British Columbia, Canada) from March to September 2025. 59 4.2.1 Animals and housing Twenty-four Holstein heifer calves were used in this study. At birth, calves were moved from the maternity pen to individual, sawdust-bedded pens (1.2 m x 2.0 m) and were fed 4 L of colostrum within the first 12 h. An additional 4 L of colostrum was provided for the following meal. Calves remained in individual pens for the first 6 d of life, receiving 4 L of milk twice daily at 08:00 and 16:00 h. At 7 d of age, calves were moved to a sawdust-bedded 35 m2 group pen, where they were provided access to 12 L/d of whole pasteurized milk via an automated calf feeder (CF 1000 CS Combi, DeLaval Inc.), and ad libitum access to calf starter (Hi-Pro Medicated, Hi-Pro Feeds Inc.), hay, and water. At 7 wk of age, daily milk allowance was reduced by 1 L/d until reaching 6 L/d; this allowance was then maintained for 15 d. Thereafter, milk was again reduced by 1 L/d until complete weaning. Once all calves in a pen were fully weaned, they were moved to another building and housed in a sawdust-bedded pen, where they had unrestricted access to a mixed ration and water. 4.2.2 Treatments To ensure comparability with existing literature, application times were selected based on studies the same hot-iron model as in our study. Previous research reported application durations ranging from 10 to 20 s (Adcock et al., 2019; Gingerich et al., 2020; Ede et al., 2023; Drwencke et al., 2025a), so we chose to compare the minimum (10 s) and maximum (20 s) durations reported. The hot-iron manufacturer offers five different tip sizes and shapes, but only two have been used in published calf studies. These two tips are also the largest and smallest circular tips. 60 Therefore, we selected them for comparison to align our methods within the existing literature on this topic. For treatment assignment, a unique 3-decimal number was generated for each calf using a random number generation function (RAND function) in Google Sheets. Calves were paired by birth order and, and within a pair, the calf with a lower number was assigned to the 20-s hot-iron application time; the other calf was assigned to the 10-s application time. The assignment of hot-iron tip sizes was determined by the last digit of the generated number: if the last digit was odd, the large annulus tip (outer diameter: 20.36 mm; contact area: 138.81 mm2) was used on the left horn and the small annulus tip (outer diameter: 15.02 mm; contact area: 138.81 mm2) on the right (Figure 4.1); if even, the reverse was applied. Figure 4.1 Image of two tips (Rhinehart, Spencerville, IN, USA) used in the study. A) A front shot of large annulus tip. B) A side shot of large annulus tip. C) A front shot of small annulus tip. D) A size shot of small annulus tip. E) Outer diameter of large tip: 20.36 mm. F) Inner diameter of large tip: 14.48 mm. G) Outer diameter of small tip: 15.02 mm. H) Inner diameter of small tip: 6.99 mm. Area covered by the large annulus tip = 3.26 cm2. Area covered by the small annulus tip = 1.77 cm2. Contact area of the large annulus tip = 1.61 cm2. Contact area of the small annulus tip = 1.39 cm2. 61 Two hot-irons (X50A, Rhinehart, Spencerville, IN, USA) equipped the large and small tips were used. The large and small tips were alternated between devices for each pair of calves to control for potential hot-iron effects Although the manufacturer recommends approximately 10 minutes of preheating, we observed that the large tip heated up more quickly but had a lower maximum temperature compared to the small tip. After testing, we determined that 15 minutes of preheating resulted in similar temperatures for both tip sizes. Therefore, hot-irons were preheated for at least 15 min before use. Temperatures were measured at the time of disbudding using an infrared thermal camera (T650sc, Teledyne FLIR, Wilsonville, OR, USA). Temperature recordings were taken at least 10 s, and the average was calculated from three randomly sampled time points that were spaced at least one second apart, using the recorded footage. At 28 d of age, calves were weighted using a digital scale (GSE 355, Illinois Tool Works, Glenview, IL) and moved to individual pens where they were sedated with an intramuscular injection of xylazine (0.2 mg/kg, right neck, Rompun, 20 mg/ml, Bayer Inc., Mississauga, Ontario, Canada). For nerve block, 5 mL of 2% lidocaine (Lurocaine, Vetoquinol N.A. Inc., Lavaltrie, Quebec, Canada) was administered perpendicularly using a 20-gauge × 13-mm needle at the midpoint between the horn bud and the lateral canthus of the eye. Right after lidocaine administration, the horn bud length was measured in two directions (Figure 4.2; short-axis: 14.4 mm (SD = 2.67 mm); long-axis: 16.8 mm (SD = 2.76 mm)) using a 15 cm digital caliper (Kynup, Shenzhen, China). Ten minutes after lidocaine administration, the hair around the horn buds was clipped to a length of 0.7 mm using a clipper (Bravura lithium clipper, Wahl, Sterling, IL, USA), and pain reflex was assessed using a needle-prick test (to which no calves responded). The calf’s head was blindfolded and positioned with the horn bud facing upward. The disbudding procedure was performed by the first author, who applied the hot-iron 62 perpendicularly to the horn bud for the assigned duration without additional pressure, so the theoretical force used for disbudding was 6.18 N (weight of hot-iron x acceleration of gravity: 0.63 kg x 9.81 m/s2). Disbudding was consistently performed from the left to the right horn bud, and the procedure was recorded using the infrared thermal camera to calculate the actual application time. The mean application times for the 20-s and 10-s treatments were 19.7 s (SD = 0.56 s) and 9.7 s (SD = 0.41 s), respectively. The temperature of the right horn bud was recorded after the procedure until it decreased to 70°C, a temperature known to cause tissue damage within seconds (Ong, 2005). Wounds were photographed using a digital camera (Nikon D3200, Toyko, Japan), and wound size and depth were measured (see Wound Measurement below). Following these measurements, calves received subcutaneous meloxicam (left neck, 0.5 mg/kg, Metacam, 20 mg/mL, Boehringer Ingelheim, Burlington, Ontario, Canada). Calves were returned to their home pen 4 h after disbudding. Figure 4.2 Picture of horn bud (left) and disbudding wound (right). The length of short-and long-axis were measured. Wound depth was assessed at the vertices of the axes. 63 4.2.3 Wound measurement Starting 3 d after disbudding, calves were blindfolded and gently led to a head-restraint apparatus located outside their pen every 3 d, guided by a bottle containing electrolyte solution. While restrained, the hair surrounding the wound was clipped to a uniform length of 0.7 mm using an electric clipper. After measurements were completed, calves were unblindfolded and returned to their pen, drinking the electrolyte solution (a total of 0.5 L of electrolyte was provided per session). 4.2.3.1 Wound tissue classification To ensure consistency, the camera’s focusing ring was always fully turned counterclockwise, and focus was manually adjusted by changing the distance between the camera and the wound. Measurement was terminated when they were moved to the heifer barn or if only light pink tissue on white skin, or opaque gray tissue with or without light pink on black skin, was observed for two consecutive sessions, which comes later. To determine wound healing phases—including the necrotic tissue phase (defined as the duration from disbudding to the last observation of necrotic tissue) and the crust phase (defined as the duration from necrotic tissue detachment to the last observation of crust)—the timing of complete necrotic tissue detachment, crust appearance, and first observation of epithelial tissue was assessed by the first author , who was blinded to treatment groups during scoring (Figure 4.3). The Intraclass Correlation Coefficient for all 48 wounds with another observer was 100 % for the day necrotic tissue fell off, 93.2 % for the last day of crust observed, and 92.1% for the first day epithelial tissue observed. Inter-observer agreement was 88.3% for crust. Although the first author did not intentionally memorize which wounds corresponded to which treatments, the visibly larger 64 necrotic tissue resulting from the large tip prevented the scorer from being fully blinded to treatment allocation. During the experiment, we observed the development of hairless, bumpy, or rough tissue surrounding some wounds. We referred to this tissue as horn tissue and monitored its presence throughout the study. Due to time constraints, the presence of horn tissue was assessed only in a subset of calves (n = 11) approximately 5 mo after disbudding. White skin Black skin Tissue Description Detached necrotic Any portion of the wound edge outside the copper ring that has separated from the skin. Crust Any part of the wound covered by dried exudate; crust color may be dark red, yellow, black, or a mixture of these colors. 65 Epithelium Wound surface appears light pink, opaque gray, or a combination of both colors. Horn tissue Rough, bumpy tissue observed near the disbudding wound. Figure 4.3 Definitions of tissue categories used for wound assessment 4.2.3.2 Wound size and depth The hairless disbudding area was measured using a 15-cm digital caliper. As wounds typically contracted into an oval shape, with the long axis running from the crown toward the region between the ear and eye, both long and short axes were recorded (Figure 4.2). When necrotic tissue began to detach, an additional circular boundary formed outside the original wound; the edge of this necrotic tissue was also measured. Wound depth was assessed at 4 locations using the depth rod of the caliper (Figure 4.2). Depth measurements continued until the necrotic tissue had detached. If depth measurement appeared to cause detachment of necrotic tissue, it was skipped for that session. 66 4.2.4 Statistical Analysis This study employed a split-plot design to evaluate the effects of two hot-iron application times (10 s vs. 20 s; whole-plot factor) and two tip sizes (small vs. large; sub-plot factor). A total of 24 calves were used, with 12 replicates per application time. Each calf received only one application time but had their different horn buds assigned to different tip sizes, resulting in one treated with the small tip and the other with the large tip. Thus, the experimental unit for application time was the calf, while the horn bud served as the experimental unit for tip size. Due to the lack of published estimates for both whole-plot (calf-level) and sub-plot (horn-level) errors, sample size estimation was based on data from Adcock and Tucker (2018). In their study, hot-iron disbudding wounds in 3-d-old calves treated with the small tip healed in 60 ± 2 d, while wounds in 35-d-old calves treated with the large tip healed in 64 ± 2 d (n = 12 per group), suggesting a SD of approximately 7 d. Given that application time and age were controlled in the present study, a lower SD of 4 d and a 6-d difference in healing time between groups were assumed. Under these assumptions, a sample size of 12 per group provided statistical power >0.8 for detecting differences in both factors. Hot-iron temperature of 2 horns was missing (One in 10-s application and small tip; one in 20-s application and large tip). Two wounds treated with the 20-s application and large-tip lost necrotic tissue partially before complete detachment. Measurements of necrotic tissue length and depth during this period were excluded from analysis. All statistical analyses were conducted using the PROC GLIMMIX procedure in SAS software (version 9.4, SAS Institute Inc.). Outcome variables—including tip temperature, short and long axes of wound, depth of the wound, the duration of necrotic tissue phase and crust phase, and the day epithelium tissue firstly observed (the next measurement day following the 67 last observation of crust)—were analyzed using mixed models with application time (10 vs. 20 s), tip size (small vs. large), and their interaction as fixed effects. Horn side (left vs. right) was included as an additional fixed effect to account for potential systematic differences. For time to reach 70 °C, no random effect was included in this model as it measured once per calf. The normality of residuals was assessed graphically using Q-Q plots and residual plots. To evaluate wound size change over time, 6 wound type categories were used: fresh wound, last necrotic tissue, first crust, last crust, and first epithelium (defined as the first observation following the disappearance of the crust), and later epithelium (16 d of epithelium phase). Short-and long-axes were analyzed using a mixed model with wound type and side as a fixed effect and horn of each calf as a random effect. Mean depth of the four locations were analyzed using a mixed model with wound type (fresh wound and last necrotic tissue) and side as a fixed effect and horn of each calf a random effect. Tukey’s Honest Significant Difference test was applied for pairwise comparisons among least-squares means. Group comparisons are presented as estimated means ± SE, while descriptive statistics are reported as mean (SD). As horn tissue occurrence was a binary outcome (presence or absence), these data were analyzed using a generalized linear mixed model with a logit link and binomial distribution, including fixed effects of time, tip size, their interaction, and side, and a random intercept for calf. To estimate the effect of horn bud size and wound size differences on horn tissue occurrence, a binary logistic regression was conducted using PROC LOGISTIC in SAS 68 4.3 Results 4.3.1 Disbudding parameters The temperature of the small tips was higher than that of the large-tip (508 ± 2.9 vs. 492 ± 2.9 °C; F1,19 = 14.7, P = 0.001). The temperature of the hot-irons was higher for right side than the left side (506 ± 2.9 vs. 493 ± 2.9 °C; F1,19 = 9.75, P = 0.006). Following 20-s application, wound temperature decreased to 70 °C in 49.3 ± 3.32 s compared to 31.8 ± 3.32 s after 10-s application (F1,20 = 13.9, P = 0.001). 4.3.2 Wound size and depth Regardless of tip size, wounds were larger than the tip size used (large tips: short axis: 46.6 % (SD = 32.0 %); long axis: 31.6 % (SD = 22.6 %) and small tips: short axis: 16.4 % (SD = 31.4 %); long axis: 6.7 % (SD = 20.0 %)). Wounds resulting from the large-tip were approximately 27 % longer in both the short long axis compared to the small tip (Short axis: F1,21 = 159.7, P < 0.001; long axis: F1,21 = 197.0, P < 0.001; Figure 4.4a). Wounds resulting from 20-s application were approximately 5 % longer in the short axis and 8 % longer in the long axis compared to 10-s application (Short axis: F1,21 = 4.8, P = 0.04; long axis: F1,21 = 17.0, P < 0.001; Figure 4.4b). Wounds on the right side of the head averaged approximately 5 % longer on the long axis than did wounds on the left (F1,21 = 7.3, P = 0.01; Figure 4.4c), but no such difference was apparent on the short axis (F1,21 = 2.19, P = 0.15). The 20-s application time created deeper wounds compared to 10-s application (2.50 ± 0.07 vs. 1.97 ± 0.07 mm; F1,22 = 24.5, P < 0.001) and the small tip created deeper wounds than did the large tip (2.43 ± 0.06 vs. 2.03 ± 0.06 mm; F1,21 = 53.1, P < 0.001). When the small tip was applied for 20 s, wounds were approximately 38 % deeper than those created with the 10-s 69 application (2.82 ± 0.08 vs. 2.05 ± 0.08 mm) while the large tip applied for 20 s produced wounds only 15 % deeper than those created with 10-s application (2.17 ± 0.08 vs. 1.89 ± 0.08 mm, interaction; F1,21 = 21.0, P < 0.001). Wounds on the right side of the calf’s head tended to be deeper (by 5 %) compared to those on the left (F1,21 = 4.0, P = 0.06). Figure 4.4 Effects of hot-iron disbudding of 4-wk old Holstein calves (n = 24), measured immediately after the procedure. Wound dimensions (length of short and long axes) are presented as Mean ± SE and are shown separately for the effect of tip size, application time, and head side (a-c). * indicates P < 0.05. 70 4.3.3 Wound healing progression The outer ring of the wound began detaching 9.31 d (SD = 2.57 d) after disbudding and the size was 5.54 mm (SD = 2.74 mm) and 4.99 mm (SD = 1.39 mm) longer in the short-and long-axes, respectively, than the inner ring (e.g., the wound directly damaged by hot-iron). Wound type related to wound size, as measured using both short- and long-axis lengths (F5,233 = 451.8, P < 0.001; F5,233 = 70.1, P < 0.001, respectively; Figure 4.5). Compared to wounds immediately after disbudding, the last observed necrotic tissues were 5.68 ± 0.37 mm shorter in short axis (t = 15.4, P < 0.001) and 4.29 ± 0.44 mm shorter in long-axis (t = 9.8, P < 0.001), while wound depth increased by 0.62 ± 0.11 mm (F1,38 = 33.8, P < 0.001). After complete necrotic tissue detachment, the revealed wound was 2.18 ± 0.44 mm longer in long-axis than the last necrotic tissue (t = -5.0, P < 0.001), with no difference observed in short-axis (t = -1.48, P = 0.14). During the crust phase, the short axis decreased by 7.03 ± 0.36 mm (t = 19.4, P < 0.001), while the long-axis increased by 1.67 ± 0.43 mm (t = 3.9, P < 0.001). The first observed epithelium was 1.13 ± 0.43 mm longer in long-axis (t = -2.6, P = 0.001), but no difference was observed in short-axis compared to the last crust (t = 0.63, P = 0.53). Fifteen days after first signs of the epithelium, the short-axis decreased in by 1.54 ± 0.36 mm (t = 4.9, P < 0.001), while the long-axis increased by 2.64 ± 0.43 mm (t = -6.1, P < 0.001). 71 Figure 4.5 Mean ± SE of wound length by tissue stage following hot-iron disbudding in 4-wk old Holstein calves (n = 24). Fresh wound refers to the wound on the day of disbudding. Last necrotic tissue indicates the final observation of necrotic tissue. First crust represents the first observation after complete detachment of necrotic tissue, and last crust indicates the final observation of a crust. First epithelium refers to the first observation following crust disappearance, and later epithelium corresponds to 15 d after the onset of epithelialization. Values in parentheses indicate mean ± SD of d after disbudding. Asterisks (*) denote significant differences (P < 0.05) in short-axis length between adjacent tissue stages; carets (^) denote differences (P < 0.05) in long-axis length between adjacent tissue stages. 4.3.4 Wound healing duration Necrotic tissue persisted 3.5 ± 1.43 d longer when calves were disbudded with the large versus small tip (F1,21 = 6.0, P = 0.02; Figure 4.6), and 2.0 ± 0.76 d longer when the iron was applied for 20 s versus 10 s (F1,21 = 7.00, P =0.02; Figure 4.6). The crust phase tended to be shorter in the small-tip group by 4.6 ± 2.60 d (F1,21 = 3.2, P = 0.09), with durations of 25.9 ± 72 2.02 d vs. 21.3 ± 2.02 d. When the epithelium was assessed based on tissue description (Figure 4.3), no effects of application time (F1,21 = 0.1, P = 0.77), tip size (F1,21 = 0.7, P = 0.43), and their interaction (F1,21 = 0.00, P = 0.95) were detected on the day epithelial tissue was first observed. However, when epithelium was assessed in the absence of a crust, it occurred 6.6 ± 2.67 d earlier in the small-tip group compared to the large-tip group (F1,21 = 6.1, P = 0.02; Figure 4.6). Figure 4.6 Mean ± SE of necrotic tissue duration and time to epithelium onset after hot-iron disbudding in 4-wk-old Holstein calves (n = 24). * indicates P < 0.05. 73 4.3.5 Horn tissue development Out of 48 horn bud sites, 18 developed apparent horn tissue after disbudding, with 15 occurring in the small-tip treatment and 3 in the large-tip group (F1,21 = 11.3, P = 0.003; Figure 4.7). Additionally, horn buds that sustained larger wounds were less likely to develop horn tissue (Short axis: coefficient = -0.41, P = 0.001; long axis: coefficient = -0.25, P = 0.02). Among 22 horn sites inspected 5 mo after disbudding, horn tissues were observed during measurements in 8 sites, and these tissues remained visible during the later inspection. Additionally, 3 more sites showed horn tissue during the later inspection (2 in the large-tip group and 1 in the small-tip group). 74 Figure 4.7 Wound images from the final measurement following hot-iron disbudding in Holstein calves (n = 24), grouped by treatment combinations: tip size (small vs. large) and application time (10 s vs. 20 s). Arrows indicate the presence of apparent horn tissue. The number at the bottom of each image represents the number of days post-disbudding. 4.4 Discussion 4.4.1 Wound size and depth Overall, both application time and tip size influenced wound size. Interestingly, wounds were larger than the size of the tips regardless of tip size, suggesting that the hot iron damaged surrounding tissue beyond the direct contact area. By estimating from the figure provided by Adcock et al. (2019) it appears that the wounds in their study made by the same sized small tip used in the current study were approximately 19 mm following a 20-s application. This is larger 75 than both the long and short axes observed in our study under the same conditions (17.5 ± 0.04 mm and 16.4 ± 0.03 mm, respectively). If hot iron temperature is considered, the lower operating temperature reported by Adcock et al. (2019) (433 °C vs. 508 °C in our study) would be expected to result in smaller wounds. Thus, we speculate that the larger wounds reported by Adcock et al. (2019) were due to other factors not measured, possibly the application of pressure during disbudding. In our study, the hot iron was positioned without applying additional pressure. Drwencke et al. (2025a) measured across the frontal plane and reported a wound diameter of 19.0 ± 0.5 mm 4 d after a 13–15 s application using an 18 mm outer diameter tip. This wound is smaller than that produced by our larger tip (20.4 mm diameter). The broader range of wound sizes reported by Drwencke et al. (15.2 to 27.6 mm vs. 15.2 to 20.0 mm in our study) may be due to repeated placement of the hot iron for wound inspection, potentially causing more tissue damage than the consistent application used in our study. Interestingly, we found that the small tip created larger wounds than the large tip when applied for a longer duration. Given that the wound depth for the 20-s application was 2.8 mm compared to 2.1 mm for the 10-s application, one possible explanation is the shape of the hot iron. The diameter of the inner hole narrows from 7.0 mm to 6.6 mm over a depth of 2.2 mm, indicating that the contact surface increases until the wound depth exceeds 2.2 mm. Beyond this point, the surface area remains constant, potentially allowing the wound to deepen more rapidly. 4.4.2 Wound healing progression We observed that necrotic tissue shrank in both axes while wound depth increased over time. As necrotic tissue began detaching, it became drier and sometimes folded, which appeared to shrink the tissue and deepen the wound. The longer wound revealed after necrotic tissue 76 detachment suggests that healing continued beneath the necrotic layer. While some studies classify wounds as fully healed based on scar thickness (Reedman et al., 2022; Drwencke et al., 2023, 2025a), we chose not to do so, as some wounds did not form a thin line even 108 d after disbudding, and tissue remodeling beneath the scar can continue for up to a year (reviewed by Reinke and Sorg, 2012). 4.4.3 Wound healing duration Initially, we defined epithelium based on wound color (see Figure 3), adopting descriptions from other studies (Adcock and Tucker, 2018; Reedman et al., 2022); using this method no treatment effects were detected for the timing of first epithelial observation. However, when the absence of crust was used as the criterion for epithelialization onset, an effect of tip size was clear. Wound healing typically progresses through 4 stages: hemostasis, inflammation, proliferation, and remodeling (reviewed by Schultz et al., 2011). During the proliferation phase, granulation and epithelialization occur. Since crust formation ceases once epithelial cells fully cover the granulation tissue, the absence of crust can serve as a milestone for epithelialization. The lack of treatment effects based on wound color may be due to our exclusion of wounds with reddish spots inside light pink tissue from the epithelialized category. To accurately study healing time, it is essential that wound definitions allow for reliable detection of the first appearance of each tissue type. In this context, Cohen’s kappa—calculated from a sample of wound images—is insufficient to evaluate the quality of wound classification. We recommend using a series of wound photographs ordered by measurement date to determine the first observation of each tissue type and reporting the Intraclass Correlation Coefficient to better reflect the consistency between observers in identifying healing milestones. 77 Throughout the healing process, wounds created with the small tip healed faster than those created with the large tip. Adcock and Tucker (2018) used the same size tips, but reported no difference in healing time. However, their study involved calves of different ages (3 d vs. 35 d), and milk allowance varied with age (3.8 L/d for the first 14 d, 4.8 L/d for the next 10 d, and 5.8 L/d thereafter). In our study, epithelial tissue appeared earlier (51.9 ± 2.04 d) in the large-tip group than calves disbudded at 35 d of age with the large tip in Adcock and Tucker’s study (64 ± 2 d), possibly due to higher milk allowance in our study (12 vs. 5.8 L/d); this interpretation is consistent with Reedman et al. (2022) who found that calves fed 15 L/d healed faster than those fed 6 L/d. Longer application times resulted in deeper wounds, and resulted in the tissue remaining above 70 °C (the temperatures known to cause third-degree burns within seconds; Ong, 2005) for a longer duration. However, the longer application time did not result in a longer time to re-epithelialization. One possible explanation is that the depth difference (0.5 mm) was insufficient to influence healing duration. Deeper wounds created with application times longer than 20 s or by applying additional pressure may require more time to heal. However, this hypothesis should be tested with caution, as prolonged application durations have been shown to cause brain damage in goats (Hempstead et al., 2021). Alternatively, wound depth may not play a key role in determining healing time. Merz et al. (2010) found that burn wound depth did prolong healing, but in that study the impact of burn wound depth on healing was assessed based on burn classification rather than depth measurements. Since hot-iron disbudding is intended to produce a third-degree burn, small differences in wound depth may not meaningfully affect the healing process. 78 4.4.4 Horn tissue development Horn tissue development was observed in only 3 out of 24 horn sites disbudded using the large tip; a finding consistent with Drwencke et al. (2025a). In contrast, horn tissue was observed in 15 out of 24 horn sites treated with the small tip. Given that wound size was similar to the horn bud size, these results suggest that a 15 mm diameter tip (small-tip) may be insufficient to fully prevent horn growth in 4-week-old calves. Furthermore, the observation of additional horn tissue 5 months post-disbudding indicates that the timing of horn tissue development may vary among individuals. Although these tissues did not appear capable of causing harm to pen-mates, long-term observation is needed to determine whether they grow to a degree that could negatively impact other animals housed in the same pen. 4.5 Conclusion Application time, and especially tip size of the hot iron, affect wound size and healing. A 20-s application created deeper and larger wounds compared to 10-s application, but this effect was only observed until necrotic tissue phase. The large-tip created shallower and larger wounds compared to small-tip, and effect observed throughout the healing process. As 10 s application did not result in more horn tissue, 10 s application may be enough for preventing horn growth. 79 Chapter 5: Conclusions 5.1 Thesis summary In Chapter 1, I reviewed the literature on pain assessment in dairy cattle to identify indicators of lasting pain and factors influencing this pain. I concluded blood cortisol and acute phase proteins are not suitable for studying lasting pain due to their transient nature, whereas hair cortisol shows promise as a lasting pain indicator. ADG and changes in lying behavior appear to be useful, although non-specific, for assessing lasting pain following castration, but not following disbudding. Wound-directed behaviours and behavioural responses to nociceptive stimuli appear to be used for studying lasting pain, but collecting these data requires considerable time making these approaches less practical. Among cognitive measures, memory tests show promise for assessing lasting pain. Wound size appears to be an important factor affecting lasting pain and healing. In Chapter 2, building on the findings from Chapter 1, I investigated whether hot-iron disbudding affects memory in calves. A spatial memory test was used to assess learning and memory over an 18-d period. Calves were trained to find 4 milk-containing bottles among 15 bottles positioned in a U shape with 65 cm between bottles. Memory performance was assessed based on the calf’s ability to correctly identify which bottles contained milk. Three treatments were compared: disbudding with standard pain control, disbudding with supplemental pain control, and sham disbudding. Although I noted no differences among treatments, memory performance improved over time in all groups, and when bottle locations were changed on d 13 after disbudding, memory scores dropped, followed by a gradual recovery showing the ability of the calves to relearn these locations. These findings suggest that hot-iron disbudding, with or 80 without supplemental pain control, did not impair spatial memory in calves under the conditions of this study. The lack of treatment differences may be due to 1) the pain resulting from hot-iron disbudding not being severe or prolonged enough to affect cognitive performance in this context, or 2) that the spatial memory test used may lack the sensitivity required to detect subtle cognitive impairments. In Chapter 3, building upon the findings of Chapter 2, I investigated whether band and surgical castration influence memory in calves differently over time, and whether short-lying and half-lying bouts could serve as behavioral indicators of pain in young calves. A 2 × 2 factorial design was employed, incorporating castration method (band vs. surgical) and the presence or absence of additional pain control. Memory testing and behavioural observations were conducted at wk 1 and 4 following castration. While memory performance improved over the two time periods, I did not observe any differences between treatments. On day 2 following castration, calves subjected to band castration exhibited a higher frequency of short-lying bouts compared to those that underwent surgical castration. Taken together, these findings suggest that the memory test paradigm used in this study may not have been suitable for assessing long-term pain in calves but short-lying bouts may serve as a behavioral indicator for young calves. In Chapter 4, following the challenges encountered in Chapters 2 and 3 in identifying a reliable method for assessing lasting pain, I shifted my focus to wound healing. Specifically, I investigated the effects of hot-iron application time and tip size on wound characteristics and healing outcomes. The results of this chapter showed that the large-tip iron produced larger wounds compared to the small-tip, and these larger wounds took longer to heal. I also found that longer application times resulted in deeper wounds, but this did not affect healing time. Also, in some instances the disbudding procedure failed to result in the elimination of all horn producing 81 cells; evidence of horn growth was observed more frequently in wounds created using the small-tip iron. The smaller tip may not have always damaged the horn-producing cells sufficiently to prevent all horn growth. These findings underscore the importance of procedural factors in influencing wound severity and healing, and highlight the need for further refinement of disbudding techniques to minimize lasting pain in calves. 5.2 Contributions One of the main contributions of my thesis is the use of a memory test paradigm to assess lasting pain in calves. While other cognitive tests have been used to study pain in calves, these methods often lack the ability to compare between treatment groups. For instance, the conditioned aversion paradigm infers aversion based on the time calves spend in a room associated with a negative experience (Ede et al., 2019a, 2020, 2022a). If calves spend less time in that room, they are considered averse to it. However, this method is inherently designed to determine whether an experience is good or bad—not to quantify the degree of aversion—making it unclear whether time spent in the aversive environment reliably reflects the intensity of the negative experience. For instance, if hot-iron disbudded calves spend 30% of the trial in the disbudding room and caustic paste disbudded calves spend 20%, it may be inappropriate to interpret this as stronger aversion to caustic paste disbudding. Similarly, the judgment bias test uses latency to respond to ambiguous cues as an indicator of negative emotional states (Neave et al., 2013; Lecorps et al., 2019). Yet, longer latency may not necessarily reflect greater pessimism or emotional distress. In contrast, the hole-board test paradigm used in my study has been applied to compare multiple group based on age (Weiss et al., 1998; McCune et al., 2008), disease severity (Van Der Kooij et al., 2010), and drug dosage (Kuc et al., 2006; Sontag et al., 82 2008). This framework offers an objective method for assessing the cognitive impact of pain and analgesia in calves. Another contribution of the research described in this thesis is the identification of short-lying bouts as a more sensitive behavioral indicator of pain in calves. Previous studies have utilized parameters such as the duration of individual lying bouts, overall lying frequency, and total lying time to assess pain following castration (Mintline et al., 2014; Meléndez et al., 2017; Marti et al., 2019). However, these measures have failed to detect differences between band and surgical castration in 1-wk-old calves (Meléndez et al., 2017; Marti et al., 2018), suggesting the need for more sensitive behavioral indicators in this age group. In my study, calves subjected to band castration exhibited a higher frequency of short-lying bouts compared to those that underwent surgical castration, even though no differences were observed in general lying behavior. One advantage of using short-lying bouts as a pain indicator is its practicality. It can be measured using the same data collection methods for collecting general lying behavior. I also employed accelerometers, a technology previously used when studying lying behaviors in calves (Meléndez et al., 2017; Nogues et al., 2021; Bajus et al., 2023; McNeil et al., 2024), to monitor lying behavior and extracted short-lying bouts from the dataset. Because short-lying bouts are derived from general lying behavior, the results can be directly compared with the existing literature, enhancing the interpretability and relevance of this metric. The third contribution of this thesis was the proposal to standardize hot-iron disbudding using application time rather than visual cues. Previous studies have described applying the iron until a “copper-colored ring” appears (Adcock and Tucker, 2018; Reedman et al., 2022; Ridgway et al., 2022; Drwencke et al., 2025a). This method requires repeated inspection of wound coloration, often leading to multiple applications of the hot-iron. Such repetition introduces 83 variability in tissue damage, increasing the risk that some calves experience more severe injury (and arguably pain) than others. Although application time has been reported in previous studies (Stock et al., 2015; Adcock and Tucker, 2018; Ede et al., 2019a; Drwencke et al., 2025a), it typically reflects the total duration of the disbudding procedure—including inspection time—rather than the direct contact time between the iron and tissue, resulting in inconsistent damage levels. In contrast, I standardized the procedure by applying the hot iron continuously and using contact time as the endpoint. This approach produced more uniform wound sizes and allowed for the detection of subtle differences due to reduced variation. These findings support the use of controlled application time to improve the consistency of disbudding outcomes. The final contribution of my dissertation is the emphasis on healing outcomes following hot-iron disbudding. If the application of the hot iron fails to prevent horn growth, the procedure cannot be considered true disbudding. Unfortunately, most published studies on disbudding do not report horn regrowth, likely due to the extended observation period required to detect scur formation. In the present thesis, horn tissue was observed within a few weeks after disbudding; the tissue continued to grow into a scur about 5 months later. Therefore, the identification of horn tissue after disbudding may reduce the required observation period for detecting scurs. To better position ongoing disbudding work within the current literature, I also encourage future disbudding studies to report the disbudding method in detail, as well as monitor and report horn growth. By consistently documenting these factors, researchers can contribute to identifying the most effective disbudding procedures that minimize tissue damage and reduce the risk of lasting pain. 84 5.3 Limitations 5.3.1 Conceptual limitations A major conceptual limitation of this thesis lies in the definition and assessment of lasting pain, which is understood herein as pain experienced during wound healing. Evidence of lasting pain following hot-iron disbudding includes increased skin sensitivity around the wound site (Adcock and Tucker, 2018; Casoni et al., 2019; Drwencke et al., 2023), indicative of hyperalgesia. However, the intensity of the evoked pain remains unclear, and it is also unclear from these data if animals experience pain when tissues are not manipulated. In human studies, pain associated with wound healing has been more thoroughly characterized. For example, patients with chronic leg ulcers reported an average pain score at rest of 4.5 on a 0–10 numeric rating scale, with scores of 5–6 considered moderate pain (Leren et al., 2022). Similarly, McGuire et al. (2006) found that 9 out of 17 patients reported mild or less than mild pain, on average, during 4 wk following gastric bypass surgery. When comparing the wound size and level of tissue damage from hot-iron disbudding to that of gastric bypass surgery, it is plausible that the lasting pain following disbudding is also mild or less than mild, suggesting that it may be difficult to identify reliable indicators of lasting pain using current behavioral and cognitive approaches. 5.3.2 Limitations of the ‘holeboard’ test The memory test paradigm used in Chapters 1 and 2 of this thesis was adapted from the holeboard test and, as such, shares some of this test’s limitations. Results of the holeboard test can vary with individual personality traits. For example, high-anxiety rats have been shown to exhibit longer latencies to engage with the board and reduced hole exploration (Ohl et al., 2001), 85 and even when anxiety levels are controlled rodent responses to the test remain inconsistent—showing increased (Takeda et al., 1998), decreased (Pellow et al., 1985), or unchanged performance (Casarrubea et al., 2009). Stress is another confounding factor known to affect spatial memory, with chronic stress impairing cognitive function (Krugers et al., 1997; Keuker et al., 2004). Additionally, food deprivation—commonly used to motivate exploration—can itself act as a stressor, with its effects on activity levels varying depending on the degree of deprivation (reviewed by Wang et al., 2006). Although all calves in my study were housed under similar environmental conditions, individual differences in coping styles and personality likely led to varying levels of anxiety and stress (reviewed by Nogues et al., 2023). These uncontrolled variables may have confounded the effects of lasting pain on memory performance, limiting the suitability of the spatial memory test for assessing pain-related cognitive changes in calves. Another limitation of the test was the use of milk as a reinforcer. To increase motivation to engage in the test, milk was withheld for 10 h before testing. This approach did not guarantee a consistent level of hunger, as calves still had access to hay and grain, and the timing of their last milk consumption varied. In rodent studies, food deprivation is typically achieved by reducing feed intake to 85% of free-feeding weight (reviewed by van der Staay et al., 2012). However, it remains unclear whether food deprivation is necessary to motivate animals to engage in hole-board-type tasks. In fact, successful performance without deprivation has been reported in rodents (Ohl et al., 2003), chickens (Nordquist et al., 2011), pigs (Arts et al., 2009), and Tupaias (Keuker et al., 2004), where the attractiveness of the bait alone was sufficient to elicit participation. To date, no bait has been identified that reliably motivates calves in a similar manner, which presents a challenge in adapting this test for use in calf research. 86 5.4 Future research opportunities As discussed in the limitations section, two key challenges were identified in the memory test paradigm used in Chapters 1 and 2: the use of hunger as a motivational driver and the difficulty in controlling motivation levels across individuals. These areas both warrant further investigation to improve the reliability and sensitivity of cognitive assessments in calves. In other animal species, a variety of appetitive reinforcers have been successfully used to motivate participation in cognitive tasks (Rats: sugar pellets (Post et al., 2011), chocolate chips (Nocjar et al., 2007), cereals (Callaway et al., 2004), and sunflower seeds (Wappler et al., 2010); chickens: mealworms (Nordquist et al., 2011); pigs: chocolate raisins (Arts et al., 2009) and chocolate candies (Roelofs et al., 2017)). In calves, sugar water has been used for testing anhedonia following disbudding (Lecorps et al., 2020). During my Phd, I often observed calves showing strong motivation for electrolyte solution such as running toward the restraint apparatus as soon as the door was opened. However, liquid-based rewards may influence satiety levels, potentially affecting motivation and performance consistency. The ideal food reinforcement for calves in cognitive testing should be dense in nature (to minimize satiety effects), small (to allow repeated trials without overfeeding), and odorless (to avoid confounding olfactory cues). Future research should explore and validate alternative reinforcers that meet these criteria to enhance the effectiveness of cognitive testing paradigms in calves. In addition to refining appetitive motivation, alternative motivational paradigms should be considered. For instance, in rodents the Morris Water Maze has been widely used to study memory and learning; animals are motivated by aversion to water and a desire to escape (reviewed by D’Hooge and De Deyn, 2001). Translating this concept to calves, who are known to be socially motivated and prefer proximity to conspecifics (Ede et al., 2022b), a maze that 87 ends in access to their home pen or social group could serve as a novel and ethically sound motivational framework. This approach may reduce reliance on hunger and improve consistency in cognitive performance across individuals. Finally, I encourage continued research into procedural factors affecting wound healing following other painful procedures. In hot-iron disbudding, the temperature of the iron may influence wound severity and healing outcomes and is worth investigating. In caustic paste disbudding, while factors such as hair removal and paste quantity have been studied (Drwencke et al., 2025b), other variables like paste thickness and application area size remain unexplored. In castration, the use of multiple rubber rings may accelerate testicular necrosis and promote faster healing. In surgical castration, the direction of the incision may affect wound healing dynamics. Collectively, accumulating data from these studies could lead to the development of optimized procedures that minimize tissue damage and reduce the risk of lasting pain; thereby, improving animal welfare. 5.5 General conclusion Throughout the research described in this thesis, I have focused on the concept of lasting pain in calves—specifically, how it can be assessed and mitigated. The spatial memory test paradigm evaluated in Chapters 2 and 3 was found to be unsuitable for investigating long-term pain responses in calves. This finding highlights the need for further research to identify reliable indicators of lasting pain. In the final chapter, I demonstrated that procedural factors, such as application time and tip size, influence wound size and healing duration following hot-iron disbudding. Continued investigation into procedural variables is warranted to refine painful husbandry practices and reduce the incidence of lasting pain in calves. 88 References Adcock, S.J.J., B.C. 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Thesis/Dissertation
eng
Applied Animal Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Weary, Daniel M.
Lasting pain in dairy calves
Text