Behavioral responses to artificial insemination and the effect of positive reinforcement training.

Dairy cattle experience a variety of stressors associated with routine farm practices, including injections, pen movements, regrouping, and artificial insemination. The primary objective of this study was to assess ear position (categorized according to 5 positions) and body movement responses of heifers during their first experience of artificial insemination, in comparison with responses during baseline observations before and after this procedure. A secondary objective was to test whether heifers exposed to positive reinforcement training (PRT) responded differently to this procedure. We tested 12 heifers (13 ± 0.7 mo. old); 7 were trained using PRT (as part of another study) and 5 had no experience with PRT. Ear axial and ear forward positions were more frequent during observations before and after artificial insemination relative to during artificial insemination (4.57 ± 0.82 vs. 0.31 ± 0.82 times/period, and 3.45 ± 0.51 vs. 0.50 ± 0.51 times/period, respectively). Backward pinned ear position was observed less frequently before and after versus during artificial insemination (0.12 ± 0.76 vs. 3.76 ± 0.76 times/period). We found no effect of period relative to artificial insemination on any of the 4 body movements assessed (leaning forward, backward, and steps taken with front legs and back legs).We found an effect of PRT on forward ear position (1.00 ± 0.51 times/PRT group vs. 2.95 ± 0.60 times/control group), and found that control heifers leaned forward and took more steps with their front legs than did PRT heifers (0.93 ± 0.57 times/PRT group vs. 3.55 ± 0.67 times/control group and 1.29 ± 0.68 times/PRT group vs. 3.85 ± 0.81 times/control group). We conclude that heifers experiencing artificial insemination for the first time express distinct ear positions that are consistent with responses to a negative emotional state. Further work is required to validate these responses and to determine the extent that they can be used to assess affective responses to this and other procedures.

Early pain experiences in dairy calves on pain sensitivity later in life.

In humans, early painful experiences can increase pain sensitivity later in life, but little is known regarding this phenomenon in cattle. This study assessed if a painful event early in life affects later pain sensitivity in 40-d-old calves. Holstein calves (n = 26) were randomly assigned to control or treatment conditions, blocked within sex and birth weight. At 9.5 ± 1.8 d old, treatment calves had 1 horn bud disbudded using caustic paste, whereas control calves had 1 horn sham disbudded; in both cases, multimodal pain control was provided. All calves had the contralateral horn bud disbudded using a hot iron 4 wk later, again with multimodal pain control. Mechanical nociceptive responses were assessed weekly using an algometer applied adjacent to both horn buds and on the rump, beginning 3 d before the first disbudding and ending 30 d after the second disbudding. Following the second disbudding, both groups of calves showed evidence of increased sensitivity (i.e., algometer pressure declined 3.69 ± 0.60 √N to 2.13 ± 0.70 √N) on the contralateral bud, but there was no difference between the control group and the treatment groups. An interaction between treatment and time, likely driven by treatment differences, was found on the rump when tested 5 h after the second disbudding event. These responses are not consistent with the hypothesis that an early pain experience results in increased sensitivity to later painful experiences.

Automated, longitudinal measures of drinking behavior provide insights into the social hierarchy in dairy cows.

Dairy cows compete for feed and water access on commercial farms. In this study we used EloSteepness to assess the summed Elo winning probabilities (i.e., dominance) of 87 cows housed in a dynamic group and compared the resulting social hierarchies based on their steepness (i.e., the average degree of differences in winning probability between adjacently ranked individuals in the group, ranging from 0 to 1). We identified a hierarchy at the drinker with a steepness of 0.55 ± 0.02 (SD), whereas the hierarchy detected at the feeder during the same time period was 0.45 ± 0.02, indicating smaller dominance differences among cows when competing for feed compared with competing for water. Individual cows' winning probabilities at the feeder and drinker were moderately correlated (rs = 0.55), and cows at the lower and upper ends of the hierarchy showed good agreement. We compared the drinker hierarchy between hot (i.e., temperature-humidity index [THI] ≥72) and normal (i.e., THI <72) periods. The hierarchy steepness was similar in both hot (0.54 ± 0.03) and normal conditions (0.56 ± 0.03), and there was a strong correlation in cows' individual winning probabilities across these periods (rs = 0.87). Cows with higher winning probability visited the drinker less frequently (hot: rs = -0.40, normal: rs = -0.33) but had a higher average daily water intake (hot: rs = 0.38, normal: rs = 0.37). We also found evidence that individual cows' drinking times differ depending on their winning probability; cows with lower winning probability shifted their drinking times to before or after the visit peak after milking. Automatically identifying cows with consistently high or low winning probabilities using drinkers may help inform grouping decisions and water provision on farms.

Access to an outdoor open pack promotes estrus activity in dairy cows.

Dairy cows have a partial preference to access an outdoor deep-bedded pack, but the effects of continuous access to an outdoor area on estrous behaviors has not been studied. Our objective was to investigate if access to an outdoor open deep-bedded pack improves the expression of estrus behaviors. We enrolled 60 lactating Holstein cows directly after calving and followed them each for 12 weeks. Cows were housed in a single freestall pen holding 36 cows at a time, with a dynamic group composition. Half of the cows were randomly assigned to the OUTDOOR treatment; these cows had access to an outdoor open pack via an automated selection gate. INDOOR cows were housed together with OUTDOOR cows but were not allowed outdoor access. All cows were fitted with an automated activity monitor (AAM) 21 ± 3 d before expected calving date. Estrous behaviors (i.e., standing to be mounted and mounting behaviors) were continuously monitored using video, and the intensity of mounting (i.e., the number of standing to be mounted and other mounting behaviors per hour) was calculated per estrus event per cow. Temperature and humidity were monitored by data loggers indoors, and these data were used to calculate the Temperature Humidity Index (THI). Following an alert from the AAM, cows were checked to detect the presence of a dominant preovulatory follicle and an absence of a mature corpus luteum (CL) by rectal ultrasonography following milking, as well as 7 d thereafter to confirm ovulation by the presence of a new CL. A total of 94 estrus events were used in the final analysis. INDOOR cows tended to have a lower mounting intensity with increasing indoor THI, but no such effect was noted for OUTDOOR cows. The total number of cows in estrus positively influenced mounting intensity. OUTDOOR cows had a longer duration of estrus, as measured by the automated monitors, compared with INDOOR cows (12.4 ± 0.7 h versus 9.9 ± 0.8 h). Estrus event number and the total number of cows in estrus were both positively associated with estrus duration. These results indicate that access to an outdoor pack tended to increase mounting behavior for freestall housed dairy cows, especially during periods of elevated THI. We conclude that providing dairy cows access to an outdoor area can help with estrus detection and may thus help improve the reproductive programs on dairy farms.

Redefining dominance calculation: Increased competition flattens the dominance hierarchy in dairy cows.

Dominance hierarchies are known for mitigating conflicts and guiding priority of access to limited resources in gregarious animals. The dominance hierarchy of dairy cows is typically investigated using agonistic interactions, often monitored at the feed bunk right after fresh feed delivery when competition is high, resulting in frequent interactions. Yet, the outcome of agonistic interactions during times of high competition may be more influenced by cows' high valuation of fresh feed than their intrinsic attributes, such that the dominance hierarchy constructed using agonistic interactions under high versus low competition times might differ. We tested how the structure of the dominance hierarchy changes in relation to different levels of competition in a dynamic group of 48 lactating dairy cows over 10 mo, with 6 cows exchanged every 16 d, for a total of 159 cows. Using a validated algorithm, we continuously detected the actor and reactor of replacement behaviors in 30 feed bins as cows competed for feed. We also calculated the percentage of occupied feed bins to characterize competition at the moment of each replacement. These data were combined to create hierarchies using Elo ratings, separately for 25 occupancy levels ranging from 13% to 100%. For each 1% rise in feeder occupancy, hierarchy steepness fell by 2.41 × 10-3 ± 9.71 × 10-5 (SE), and the percentage of dyads where both cows replaced each other rose by 0.13% ± 0.01%. At the highest feeder occupancy level in comparison to the lowest one, we observed 7.57% more dyads in which the dominant individual (those that won more interactions at the lowest feeder occupancy) started to lose proportionally more. The magnitude of decrease in the winning rate of the dominant individual in those dyads also got amplified by 1.06 × 10-3% ± 1.37 × 10-4% (SE) for each 1% increase in feeder occupancy. These findings illustrate how inferred hierarchies vary with competition, with high competition flattening the hierarchy due to increased success of subordinate animals. We suggest that during heightened competition, increased valuation of resources can affect competitive success more than the individual's intrinsic dominance attributes. We recommend against calculating dominance hierarchies based on agonistic interactions during periods of high competition alone, and more generally urge researchers to differentiate agonistic interactions based on context when constructing dominance hierarchies.

Automated monitoring of brush use in dairy cattle.

Access to brushes allows for natural scratching behaviors in cattle, especially in confined indoor settings. Cattle are motivated to use brushes, but brush use varies with multiple factors including social hierarchy and health. Brush use might serve an indicator of cow health or welfare, but practical application of these measures requires accurate and automated monitoring tools. This study describes a machine learning approach to monitor brush use by dairy cattle. We aimed to capture the daily brush use by integrating data on the rotation of a mechanical brush with data on cow identify derived from either 1) low-frequency radio frequency identification or 2) a computer vision system using fiducial markers. We found that the computer vision system outperformed the RFID system in accuracy, and that the machine learning algorithms enhanced the precision of the brush use estimates. This study presents the first description of a fiducial marker-based computer vision system for monitoring individual cattle behavior in a group setting; this approach could be applied to develop automated measures of other behaviors with the potential to better assess welfare and improve the care for farm animals.

Gene editing in animals: What does the public want to know and what information do stakeholder organizations provide?

Organizations involved with gene editing may engage with the public to share information and address concerns about the technology. It is unclear, however, if the information shared aligns with what people want to know. We aimed to understand what members of the public want to know about gene editing in animals by soliciting their questions through an open-ended survey question and comparing them with questions posed in Frequently Asked Question (FAQ) webpages developed by gene editing stakeholder organizations. Participants (338 USA residents) asked the most questions about gene editing in general and animal welfare. In contrast, FAQ webpages focused on regulations. The questions survey participants asked demonstrate a range of knowledge and interests. The discrepancy between survey participant questions and the information provided in the FAQ webpages suggests that gene editing stakeholders might engage in more meaningful public engagement by soliciting actual questions from the public and opening up opportunities for real dialogue.

Effects of group size on agonistic interactions in dairy cows: a descriptive study.

Group-housed cattle may engage in agonistic interactions over resources such as feed, which can negatively affect aspects of welfare. Little is known about how contextual factors such as group size influence agonistic behaviour. We explored the frequency of agonistic interactions at the feeder when cattle were housed in different-sized groups. We also explored the consistency of the directionality of agonistic interactions in dyads and of the number of agonistic interactions initiated by individuals across the group sizes. Four replicates of 50 cows each were assessed in two group-size phases. In Phase 1, cows were kept in one group of 50. In Phase 2, these same cows were divided into five groups of 10, maintaining stocking density (i.e., ratio of animals to lying stalls and feed bunk spaces). We measured agonistic replacements (i.e., interactions that result in one cow leaving the feed bin and another taking her place) at an electronic feeder using a validated algorithm. We used these data from Phase 1 to calculate individual Elo-ratings (a type of dominance score). Cows were then categorised into five dominance categories based upon these ratings. To ensure a consistent Elo-rating distribution between phases, two cows from each dominance category were randomly assigned to each small group of 10 cows. The mean ± SE number of replacements per cow was similar regardless of whether the cows were housed in groups of 50 (34.1 ± 2.4) or 10 (31.1 ± 4.5), although the groups of 10 were more variable. Further, 81.6 ± 7.7% (mean ± SD) of dyads had the same directionality across group sizes (i.e., the same individual won the majority of interactions in the dyad) and individuals were moderately consistent in the number of replacements they initiated (intraclass correlation coefficient = 0.62 ± 0.11; mean ± SD). These results indicate that the relationship between group size and agonistic behaviour is complex; we discuss these challenges and suggest new avenues for further research.

Are regulations addressing farm animal welfare issues during live transportation fit for purpose? A multi-country jurisdictional check.

Growing animal welfare concerns have pushed some jurisdictions to strengthen regulations addressing live farm animal transportation, but whether they provide satisfactory levels of protection for animals remains to be shown. Using the recent peer-reviewed literature, we identified four major risk factors associated with live animal transportation (fitness for transport, journey duration, climatic conditions and space allowances) and explored how regulations were structured to prevent animal welfare issues in five English-speaking Western jurisdictions (Australia, Canada, New Zealand, the EU and the USA). All legally binding federal regulations were systematically reviewed and compared. Whether these rules were fit for purpose was assessed using the relevant peer-reviewed scientific literature. Our findings indicate the majority of regulations in most jurisdictions are often insufficient or too vague to be deemed fit for purpose. All five jurisdictions fall short in guaranteeing adequate protection to livestock during transport. Using recent changes as well as future policy proposals under discussion, we identify future directions that could form the basis for regulatory changes that may significantly improve the welfare of farm animals during transportation.

Attitudes of western Canadian dairy farmers toward technology.

Dairy farms have become more reliant on technology. The overall aim of this study was to better understand how dairy farmers view technology and its effects on animal care, including their views on the prospect of integrating gene-editing technology in the future. Virtual-semistructured interviews were conducted with dairy farmers (n = 11) from British Columbia and Alberta. To facilitate discussion, the participants were asked to develop and discuss a timeline describing when and why various technologies were adopted on their farm. Although farmers defined technology broadly and affecting multiple aspects of farm management, this paper focuses on their views regarding how technology can affect animal care. Following thematic analysis of the data, the following 3 themes emerged: (1) the changing role of the farmer (including intergenerational considerations and learning new technology), (2) the effect of technology on the cow and her relationship with the farmer and, (3) technology as the future of the farm. The discussions also highlight the concerns that some farmers have regarding challenges associated with reduced human-animal interactions and effective use of the large amounts of data that are collected through technology. We also specifically asked the participants their views about gene editing as a potential future technology. Most of the participants did not specifically address their views on gene editing, but they spoke about the effect on genetic technologies more generally, often making references to genomic testing. However, some questioned how this technology may affect farmers more generally and spoke about how it could affect human-animal relationships. These results illustrate differences among farmers in the way they view technology and how this can affect the dairy cattle they care for.

Perspective: Prolonged cow-calf contact-A dilemma or simply another step in the evolution of the dairy industry?

The contentious issue of cow-calf separation at birth is incongruent with many views on acceptable farming practices, and carries the risk of eroding public trust in the dairy industry if it is not addressed. The available evidence provides little support for the practice, but research on best practices for maintaining cow-calf contact in a way that enhances animal welfare while preserving farm profitability is nascent. In this article, the authors address the research questions that require answers to better inform producers and facilitate their decision-making and prepare the dairy industry to take another evolutionary step forward.

Social housing improves dairy calves' performance in a competition test.

On most dairy farms, calves are housed individually until weaning. However, depriving calves of an early social environment impairs behavioral development. We studied the effect of early-life social housing on calves' competitive skills. In this study, Holstein heifers were pseudorandomly assigned to either individual housing (n = 9) or pair housing (with a nonfocal companion, n = 9) at the age of 11 d. After 14 d of housing treatment, calves underwent a competition test for milk access against a group-reared calf; consisting of 2 test sessions per day for 5 d (session duration: 74.42 ± 2.29 s; mean ± standard error). Pair-housed calves performed better than individually housed calves: throughout the competition days, individually housed calves increased their latency to approach the milk bottle and decreased their time spent drinking in contrast to pair-housed calves, which exhibited stable latencies to reach the milk bottle and increased their time drinking. To control for the influence of personality on their competitive abilities, all calves were subjected to personality tests assessing boldness before being exposed to the housing treatment. Our findings indicate that calves assessed as bolder during the pretreatment personality test tended to approach the milk bottle faster. Our results provide additional evidence of the beneficial effects of social housing on dairy calves' behavioral development.

Review: Using animal welfare to frame discussion on dairy farm technology.

The use of technology on dairy farms has increased dramatically over the last half-century. The ways that scientists describe the potential benefits and risk of technology are likely to affect if it is accepted for use on farms. The aim of our study was to identify papers that describe a linkage between technologies used on dairy farms and the welfare of dairy cattle. Our systematic review identified 380 papers, of which 28 met our inclusion criteria and were used to describe the technologies examined, the welfare-relevant measures used, and the ways in which authors framed welfare benefits and risks associated with the technologies. The large majority (27 of 28 papers) used positive frames, considering how the technology could improve welfare. Some authors carefully articulated the logic linking the specific measures to specific welfare-related outcomes (such as the use of accelerometer data to draw inferences about changes in lying times), but others made more general inferences (about health and welfare) that were not (and perhaps could not) be assessed. We conclude that much of the framing focused on animal welfare is biased toward welfare benefits and that future work should strive to address both potential benefits and harms; more balanced coverage may better inform solutions to the problems encountered by the people and animals affected by the technology. Welfare is a complex and multifaced concept, and it is unlikely that any one technology (or perhaps even a combination of technologies) can adequately capture this complexity. Thus, we encourage authors to restrict their claims to specific, welfare-relevant measures that can be assessed independently and thus validated. More general claims about welfare should be treated with skepticism.

To Treat or Not to Treat: Public Attitudes on the Therapeutic Use of Antibiotics in the Dairy Industry-A Qualitative Study.

This paper describes the views of 779 U.S. residents on questions related to therapeutic antibiotic use in dairy cattle. An online survey was conducted with qualitative (open-ended) questions. Respondents were offered one of three scenarios with varying degrees of information describing a farmer with a sick cow that would benefit from antibiotic therapy. The text replies to the open-ended questions were analyzed by grouping responses with similar comments and identifying patterns or themes. Content analysis showed that many of the participants in this study provided farmers with the social license to treat sick cows with antibiotics; however, some participants commented on the social license not necessarily extending to antibiotic use for growth promotion or prophylactic use. Our findings are not generalizable, but may provide some insight that should be considered when developing policies and practices regarding the use of antibiotics on dairy farms that may promote improved alignment with societal values.

The effects of pain following disbudding on calf memory.

Studies have found evidence of pain in the hours following hot-iron disbudding, but little is known about longer-lasting pain following this procedure. Work on humans and rats has shown that lasting pain can have negative effects on the formation and recall of memories. The objective of this study was to assess whether lasting pain following disbudding affects learning and memory in calves. A modified hole-board apparatus was used to assess how quickly calves were able to learn the locations of 4 bottles containing milk dispersed among 11 locations with empty bottles. At 14 d of age and after 6 d of training on this task, calves (n = 30) were randomly assigned to 3 treatments: disbudding with analgesic on the day of the procedure, disbudding with analgesic throughout the study, and sham disbudding. All calves were sedated, given a lidocaine cornual local block and a single injection of an nonsteroidal anti-inflammatory drugs. Starting on the day after their disbudding treatment, calves were tested daily using the modified hole-board apparatus. After 12 d of testing, the locations of the 4 milk-containing bottles were switched, and calves then relearned the locations of the rewarded bottles over the next 6 daily test sessions. We found general working memory (i.e., short-term memory) and reference memory (i.e., long-term memory) increased over the 12 d of testing, declined when locations were switched on d 13, and then again increased over the final 6 d of testing. We did not find an effect of treatment on any measure, perhaps because there was no lasting pain or because effects were too minor to detect using this test of spatial memory.

Graduate Student Literature Review: Sociability, fearfulness, and coping style-Impacts on individual variation in the social behavior of dairy cattle.

Dairy cattle typically live in groups, but individuals within these groups vary in their social behavior. An improved understanding of factors affecting the expression of social behavior may help refine management practices on farms to better accommodate the needs of all individuals within the herd. In this paper, we review (1) some examples of how social behavior is expressed in cattle, (2) commonly assessed personality traits in this species (i.e., sociability and fearfulness) as well as coping style, and (3) how these can affect the expression of social behavior of dairy cattle and in turn their welfare. We also identify understudied social behaviors that personality might influence (social learning, social stress, and social buffering of negative emotions), and that could inform how to improve the welfare of intensively housed dairy cattle.

Exploring the effect of pain on response to reward loss in calves.

Negative emotional states are known to interact, potentially aggravating one another. In this study, we used a well validated paradigm (successive negative contrast, SNC) to determine if pain from a common procedure (disbudding) influences responses to a reward downshift. Holstein calves (n = 30) were trained to approach a 0.5 L milk reward. Latency to approach, number of vocalisations and pressure applied on the bottle were recorded during training. To assess how pain affected responses to reward downshift, calves were randomly assigned to one of three treatments before the downshift. Two groups were disbudded and provided the 'gold standard' of pain mitigation: intraoperative local anesthesia and analgesia. One of these disbudded groups was then provided supplemental analgesic before testing. The third group was sham disbudded. All calves were then subjected to the reward downshift by reducing the milk reward to just 0.1 L. Interactions were detected between test session and daily trials on pressure applied for the Disbudded group (estimate ± SEM: 0.08 ± 0.05), and on vocalisations for the Sham (0.3 ± 0.1) and Disbudding + Analgesia (0.4 ± 0.1) groups. Our results indicate that SNC is a promising paradigm for measuring negative affect in calves and suggests that pain potentially affects the response to a reward downshift.

Social acceptance of genetic engineering technology.

Genetic engineering of animals has been proposed to address societal problems, but public acceptance of the use of this technology is unclear. Previous work has shown that the source of information proposing the technology (e.g. companies, universities), the term used to describe the technology (e.g. genome editing, genetic modification), and the genetic engineering application (e.g. different food products) affects technology acceptance. We conducted three mixed-method surveys and used a causal trust-acceptability model to understand social acceptance of genetic engineering (GE) by investigating 1) the source of information proposing the technology, 2) the term used to describe the technology, and 3) the GE application for farm animals proposed. Further, participants expressed their understanding of technology using a range of terms interchangeably, all describing technology used to change an organism's DNA. We used structural equation modelling and confirmed model fit for each survey. In each survey, perceptions of benefit had the greatest effect on acceptance. Following our hypothesized model, social trust had an indirect influence on acceptance through similar effects of perceived benefit and perceived risk. Additional quantitative analysis showed that the source of information and technology term had little to no effect on acceptance. Applications involving animals were perceived as less beneficial than a plant application, and an application for increased cattle muscle growth was perceived as more risky than a plant application. When assessing the acceptability of applications participants considered impacts on plants, animals, and people, trust in actors and technologies, and weighed benefits and drawbacks of GE. Future work should consider how to best measure acceptability of GE for animals, consider contextual factors and consider the use of inductive frameworks.

Crowd sourcing remote comparative lameness assessments for dairy cattle.

Lameness assessments are rarely conducted routinely on dairy farms and when completed typically underestimate lameness prevalence, hampering early diagnosis and treatment. A well-known feature of many perceptual tasks is that relative assessments are more accurate than absolute assessments, suggesting that creating methods that allow for the relative scoring of which cow is more lame will allow for reliable lameness assessments. Here we developed and tested a remote comparative lameness assessment method: we recruited nonexperienced crowd workers via an online platform and asked them to watch 2 videos side-by-side, each showing a cow walking, and to identify which cow was more lame and by how much (on a scale of -3 to 3). We created 11 tasks, each with 10 video pairs for comparison, and recruited 50 workers per task. All tasks were also completed by 5 experienced cattle lameness assessors. We evaluated data filtering and clustering methods based on worker responses and determined the agreement among workers, among experienced assessors, and between these groups. A moderate to high interobserver reliability was observed (intraclass correlation coefficient, ICC = 0.46 to 0.77) for crowd workers and agreement was high among the experienced assessors (ICC = 0.87). Average crowd-worker responses showed excellent agreement with the average of experienced assessor responses (ICC = 0.89 to 0.91), regardless of data processing method. To investigate if we could use fewer workers per task while still retaining high agreement with experienced assessors, we randomly subsampled 2 to 43 (1 less than the minimum number of workers retained per task after data cleaning) workers from each task. The agreement with experienced assessors increased substantially as we increased the number of workers from 2 to 10, but little increase was observed after 10 or more workers were used (ICC > 0.80). The proposed method provides a fast and cost-effective way to assess lameness in commercial herds. In addition, this method allows for large-scale data collection useful for training computer vision algorithms that could be used to automate lameness assessments on farm.

Calves peak-end memory of pain.

In humans, the 'peak-end' rule states that recollection of an experience is most often influenced by the peak (the most intense moment) and end of the experience. We investigated whether calves followed the peak-end rule in their memory of a painful procedure: disbudding. As proxies for retrospective and 'real-time' reports of pain, we used conditioned place aversion, and reflex pain behaviours. In two separate trials, calves were subjected to two disbudding conditioning sessions (one horn per treatment), acting as their own control. In the first trial, calves (n = 22) were disbudded and remained in a pen for 4 h, and disbudded and left in another pen for 4 h with an additional 2 h following an analgesic treatment. In the second trial, calves (n = 22) were disbudded and left in pens for 6 h during both treatments, receiving the analgesic at either 2 h or 4 h after disbudding. Calves were then tested for place aversion. For both trials we did not observe a preference for the pens where calves received analgesic treatment towards the end of the session. We did not find an association between aversion and the sum, peak or end of pain behaviours. Results are not consistent with a peak-end effect in calves' memory of pain.