The perspectives of UK personnel towards current killing practices for laboratory rodents.

Rodents are the predominant species used for scientific research and must be humanely killed upon completion of the work. In the UK this is regulated by Schedule 1 of the Animals Scientific Procedures Act 1986, which lists permitted methodologies considered capable of humane killing, including overdose of an anaesthetic, exposure to carbon dioxide (CO2) gas, dislocation of the neck and concussion of the brain by striking the cranium. Although all are permitted, operator motivations behind method selection and individual operator preference remain unknown. The views of 219 laboratory animal personnel on institutional availability and use of Schedule 1 killing methods for laboratory rodents were obtained. Only 10% of participants reported that all four methods were available at their institution with 57.5% of respondents preferring cervical dislocation. For CO2, only 18.6% of participants reported using the recommended flow rate, while 45.5% did not know the flow rate employed. We highlight the urgent requirement for the development of quality-controlled training programmes, to improve knowledge and confidence in the selection and application of killing methods. We advocate for continuous review of killing practices to ensure best practice is reflected in legislation and achieve optimal protection of the welfare of laboratory rodents during killing.

Effects of Human Observer Presence on Pain Assessment Using Facial Expressions in Rabbits.

The goal of this study was to evaluate the effect of a human observer on Rabbit Grimace Scale (RbtGS) scores. The study scored video footage taken of 28 rabbits before and after orthopedic surgery, as follows: 24 h before surgery ( baseline), 1 h after surgery ( pain), 3 h after analgesia administration ( analgesia), and 24 h after surgery ( 24h) in the presence and absence of an observer. Videos were assessed twice in random order by 3 evaluators who were blind to the collection time and the presence or absence of an observer. Responses to pain and analgesia were evaluated by comparing the 4 time points using the Friedman test, followed by the Dunn test. The influence of the presence or absence of the observer at each time point was evaluated using the Wilcoxon test. Intra- and interrater reliabilities were estimated using the intraclass correlation coefficient. The scale was responsive to pain, as the scores increased after surgery and had decreased by 24 h after surgery. The presence of the observer reduced significantly the RbtGS scores (median and range) at pain (present, 0.75, 0 to 1.75; absent, 1, 0 to 2) and increased the scores at baseline (present, 0.2, 0 to 2; absent, 0, 0 to 2) and 24h after surgery (present, 0.33, 0 to 1.75; absent, 0.2, 0 to 1.5). The intrarater reliability was good (0.69) to very good (0.82) and interrater reliability was moderate (0.49) to good (0.67). Thus, the RbtGS appeared to detect pain when scored from video footage of rabbits before and after orthopedic surgery. In the presence of the observer, the pain scores were underestimated at the time considered to be associated with the greatest pain and overestimated at the times of little or no pain.

Pain Recognition in Rabbits.

Rabbits typically undergo at least one painful procedure during their lifetime and appropriate methods of assessment are essential to reduce or alleviate pain. Various methods of assessing pain in rabbits have been investigated, with the validity of spontaneous behavior and grimace scale scoring being the most studied to date. Assessment of pain is challenging, compounded by rabbits being a prey species that display freezing behavior in the presence of unfamiliar caregivers. Here we discuss some key changes in rabbits that can be used in the assessment of pain and provide some practical suggestions to ensure that the assessment can be carried out effectively.

Determining the preferred liquid reward in adult C57BL/6 mice.

Using food rewards to motivate mice is commonplace in behavioural research. Using a reward which is highly desirable is of benefit, as it can reduce the need to food restrict animals and can encourage higher levels of consumption, allowing for changes in levels of consumption following an intervention to be easily determined. Here, we aimed to determine the preferred milk reward for adult male C57Bl/6 mice. Thirty male C57Bl/6 mice were provided with soya milk, chocolate milk, almond milk or water for a four-hour period in a cross-over design. When planning studies that involve the use of a palatable liquid reward, soya milk is the preferred option for adult male C57BL/6 mice. Providing a liquid reward of high value will give increased levels of consumption, with little or no food restriction required.

Validation of the rabbit pain behaviour scale (RPBS) to assess acute postoperative pain in rabbits (Oryctolagus cuniculus).

Considering the widespread use of rabbits in research that potentially causes pain and discomfort and the limited number of pain assessment validated tools in this species, we aimed to develop and validate a scale of acute postoperative pain in rabbits (RPBS). Footage of 58 rabbits from previous studies were used, recorded at 'baseline' (before orthopaedic and soft tissue surgeries), 'pain' (after surgery), 'analgesia' (after analgesic), and '24h post' (24 hours after surgery). The videos were randomised and assessed twice by four evaluators, within one-month interval between evaluations. After content validation, RBPS was further refined using the criteria from the validation. According to the principal component analysis, RPBS was considered unidimensional. The intra- and inter-observer reliability was excellent (ICC>0.80) for all evaluators. There was a high Spearman's correlation of the RPBS with unidimensional scales (>0.80) and a moderate correlation with the Rabbit Grimace Scale (0.68), confirming criterion validity. According to the mixed linear model, the scale was responsive, shown by the increase in pain scores after surgery. Construct validity was confirmed by known-group approach and internal relationships among items. Adequate item-total correlation (>0.3) was observed for all items, except for the attention to the affected area (0.04). The internal consistency was very good (Cronbach's α coefficient = 0.78; Mcdonald's ω coefficient = 0.83). The cut-off score for rescue analgesia was ≥3, with an area under the curve >0.95, demonstrating a high discriminatory capacity of the instrument. Scores 3 and 4 were within the uncertainty diagnostic zone. Specificity was 87% and sensitivity was 90%. It was concluded that the RPBS presented content, criterion, and construct validities, responsiveness, and reliability to assess acute pain in rabbits submitted to orthopaedic and soft tissue surgeries. The cut-off for rescue analgesia serves as a basis for the administration of analgesics to rabbits submitted to painful procedures.

Determining Candidate Hypobaric Hypoxia Profiles for Humane Killing of Laboratory Mice.

Millions of mice are used annually in scientific research and must be humanely killed. Despite significant welfare concerns, carbon dioxide exposure remains the most common killing method, primarily because there is no practical and humane alternative. We explored whether hypobaric hypoxia via gradual decompression could induce a non-recovery state in anesthetized male C57BL/6 and Balb/c laboratory mice. We aimed to determine if this was possible in a feasible timescale with minimal pathological consequences, as a proof-of-principle step. Systematic evaluation of two decompression rates (75, 150 ms-1) and three profile shapes (accelerated, linear, gradual) in a factorial design revealed that hypobaric hypoxia effectively induced a non-recovery state in anesthetized laboratory mice, irrespective of decompression rate and shape. Mice took longer to reach a non-recovery state with the 75 ms-1 decompression rate (75 ms-1: 257 ± 8.96 vs. 150 ms-1: 214 ± 7.26 s), with longer latencies in gradual and linear shaped profiles. Accelerated shaped profiles were least susceptible to meaningful refinement via rate. The only pathological changes of concern were moderate middle ear congestion and hemorrhage. These findings suggest that hypobaric hypoxia has potential, and subsequent work will evaluate the welfare consequences of gradual decompression in conscious mice, to identify decompression profiles that minimize welfare harms associated with ear barotrauma.

Evaluating Pain and Analgesia Effectiveness Following Routine Castration in Rabbits Using Behavior and Facial Expressions.

Prevention of pain in rabbits is a priority for both welfare and validity of scientific data. We aimed to determine if the rabbit grimace scale (RbtGS) could be used as a viable, rapid assessment tool in two breeds of rabbit, Dutch belted (DB) and New Zealand white (NZW), following orchidectomy, as an adjunct to behavioral analysis. All animals received analgesia. Rabbits were filmed and their behavior was recorded at multiple time points pre- and post-orchidectomy. Observers then scored specific pain associated behaviors for analysis. Time matched footage was also scored using the rabbit grimace scale (RbtGS). Following surgery, rabbits showed significant increases in the duration spent displaying key pain associated behaviors at 1 and 5 h post-surgery. DB rabbits that received low dose meloxicam (0.2 mg/kg) showed significantly more pain behaviors at 1 and 5 h post-surgery compared to those administered a combination of higher dose meloxicam (0.6 mg/kg) and a lidocaine/bupivacaine local infusion. DB rabbits showed an increase in RbtGS score at both 1 and 5 h post-surgery. In the NZW rabbits, an increase in RbtGS score was only observed at 1 h post-surgery. Using behavioral analysis as the gold standard for comparison, the RbtGS was an effective means of determining when rabbits are painful following orchidectomy. Higher dose meloxicam (0.6 mg/kg) combined with local anesthetic was a more effective method of reducing pain, compared to lower dose meloxicam (0.2 mg/kg) alone.

Harmonisation of welfare indicators for macaques and marmosets used or bred for research.

Background: Accurate assessment of the welfare of non-human primates (NHPs) used and bred for scientific purposes is essential for effective implementation of obligations to optimise their well-being, for validation of refinement techniques and novel welfare indicators, and for ensuring the highest quality data is obtained from these animals. Despite the importance of welfare assessment in NHP research, there is little consensus on what should be measured. Greater harmonisation of welfare indicators between facilities would enable greater collaboration and data sharing to address welfare-related questions in the management and use of NHPs. Methods: A Delphi consultation was used to survey attendees of the 2019 NC3Rs Primate Welfare Meeting (73 respondents) to build consensus on which welfare indicators for macaques and marmosets are reliable, valid, and practicable, and how these can be measured. Results: Self-harm behaviour, social enrichment, cage dimensions, body weight, a health monitoring programme, appetite, staff training, and positive reinforcement training were considered valid, reliable, and practicable indicators for macaques (≥70% consensus) within a hypothetical scenario context involving 500 animals. Indicators ranked important for assessing marmoset welfare were body weight, NHP induced and environmentally induced injuries, cage furniture, huddled posture, mortality, blood in excreta, and physical enrichment. Participants working with macaques in infectious disease and breeding identified a greater range of indicators as valid and reliable than did those working in neuroscience and toxicology, where animal-based indicators were considered the most important. The findings for macaques were compared with a previous Delphi consultation, and the expert-defined consensus from the two surveys used to develop a prototype protocol for assessing macaque welfare in research settings. Conclusions: Together the Delphi results and proto-protocol enable those working with research NHPs to more effectively assess the welfare of the animals in their care and to collaborate to advance refinement of NHP management and use.

The identification of effective welfare indicators for laboratory-housed macaques using a Delphi consultation process.

Despite the importance for both animal welfare and scientific integrity of effective welfare assessment in non-human primates, there has been little or no consensus as what should be assessed. A Delphi consultation process was undertaken to identify the animal- and environment-based measures of welfare for laboratory-housed macaques and to determine their relative importance in on-site welfare assessments. One-hundred fifteen potential indictors were identified through a comprehensive literature search, followed by a two-round iterative electronic survey process to collect expert opinion. Stable group response and consensus about the validity, reliability, and feasibility of the proposed indicators (67.5% agreement) was achieved by the completion of Round Two. A substantially higher proportion of environment-based measures (72%: n = 44/61) were considered as valid, reliable, and feasible compared to the animal-based measures (22%: n = 12/54). The indicators that ranked most highly for assessing welfare were the presence of self-harm behaviours and the provision of social enrichment. This study provides an empirical basis upon which these indicators can be validated and then integrated into assessment tools developed for macaques and emphasises the need to include both animal- and environment-based indicators for accurate welfare monitoring.

Postoperative pain behaviours in rabbits following orthopaedic surgery and effect of observer presence.

Rabbits are widely used in studies focusing on pain. However, pain is undertreated in this species and one possible factor to explain this is the lack of evaluation methods. The objective of this study was to identify behaviours related to orthopaedic pain in rabbits and to evaluate the influence of the presence of an observer on these behaviours. Twenty-eight rabbits undergoing orthopaedic surgery and filmed 24 hours before surgery, and 1 hour (before rescue analgesia), 4 hours (3 hours after rescue analgesia), and 24 hours post-recovery were observed in the presence and absence of an observer. The frequency and/or duration of behaviours were compared over time and between the presence and absence of the observer using the Friedman and Wilcoxon tests respectively. Data are expressed as median and interquartile range and a significant difference was considered when p<0.05. At 1 hour post-recovery, the rabbits showed reduced activity, hopping, change posture, position in the cage, explore, and open eyes in both the presence and absence of the observer. In the absence of the observer, quadrupedal posture, interact with pinecone, and eat carrot also decreased, while wince behaviour increased. In the presence of the observer, before surgery, the rabbits were less active (Presence-280; 162-300, Absence-300; 300-300) and presented a lower duration of explore (Presence-3; 0-32, Absence-40; 4-63). Post-recovery the rabbits flinched less (Presence-0; 0-0, Absence-0; 0-1) and suspended the affected limb less (Presence-0; 0-0, Absence-0; 0-65). After rescue analgesia the rabbits put weight on and raised the affected limb less (Presence-0; 0-0, Absence-0; 0-2) and licked the affected area less (Presence-0; 0-0, Absence-0; 0-2). These findings demonstrate that the presence of the observer inhibited pain-free behaviours in the rabbits, leading to a false impression of pain, and after the surgery the rabbits masked some pain signs related to the affected area.

Negative mood affects the expression of negative but not positive emotions in mice.

Whether and to what extent animals experience emotions is crucial for understanding their decisions and behaviour, and underpins a range of scientific fields, including animal behaviour, neuroscience, evolutionary biology and animal welfare science. However, research has predominantly focused on alleviating negative emotions in animals, with the expression of positive emotions left largely unexplored. Therefore, little is known about positive emotions in animals and how their expression is mediated. We used tail handling to induce a negative mood in laboratory mice and found that while being more anxious and depressed increased their expression of a discrete negative emotion (disappointment), meaning that they were less resilient to negative events, their capacity to express a discrete positive emotion (elation) was unaffected relative to control mice. Therefore, we show not only that mice have discrete positive emotions, but that they do so regardless of their current mood state. Our findings are the first to suggest that the expression of discrete positive and negative emotions in animals is not equally affected by long-term mood state. Our results also demonstrate that repeated negative events can have a cumulative effect to reduce resilience in laboratory animals, which has significant implications for animal welfare.

Influence of the Competition Context on Arousal in Agility Dogs.

To determine whether participation in dog agility has an impact on canine arousal and welfare, this study aimed firstly to identify the effect of the competition context on arousal changes experienced by dogs, as distinct from purely physical participation in agility, and secondly to assess the handlers' ability to recognize this. Behaviors indicative of changes in arousal were recorded for twenty dogs immediately before completion of both a competition and a training run, whilst the accuracy of handlers' observations of their dogs' behavior was examined via questionnaire. Whilst a moderate number of behaviors presented with greater frequency or duration in competition, the total number of different arousal behaviors performed was higher for dogs in competition (p < 0.01). Context had a relatively modest effect on the level of arousal of agility dogs, with a greater number of behaviors indicating increased arousal in competition. Such increased arousal may adversely influence the success of dog-handler partnerships in competition.  In both contexts, handlers observed fewer behaviors than their dogs performed and this finding may have implications for dog welfare.

Opportunities for refinement in neuroscience: Indicators of wellness and post-operative pain in laboratory macaques.

Being able to assess pain in nonhuman primates undergoing biomedical procedures is important for preventing and alleviating pain, and for developing better guidelines to minimise the impacts of research on welfare in line with the 3Rs principle of Refinement. Nonhuman primates are routinely used biomedical models however it remains challenging to recognise negative states, including pain, in these animals. This study aimed to identify behavioural and facial changes that could be used as pain or general wellness indicators in the rhesus macaque (Macaca mulatta). Thirty-six macaques scheduled for planned neuroscience procedures were opportunistically monitored at four times: Pre-Operative (PreOp), Post-Operative (PostOp) once the effects of anaesthesia had dissipated, Pre-Analgesia (PreAn) on the subsequent morning prior to repeating routine analgesic treatment, and Post-Analgesia (PostAn) following administration of analgesia. Pain states were expected to be absent in PreOp, moderate in PreAn, and mild or absent in PostOp and PostAn when analgesia had been administered. Three potential pain indicators were identified: lip tightening and chewing, which were most likely to occur in PreAn, and running which was least likely in PreAn. Arboreal behaviour indicated general wellness, while half-closed eyes, leaning of the head or body shaking indicated the opposite. Despite considerable individual variation, behaviour and facial expressions could offer important indicators of pain and wellness and should be routinely quantified, and appropriate interventions applied to prevent or alleviate pain, and promote positive welfare.

Can grimace scales estimate the pain status in horses and mice? A statistical approach to identify a classifier.

Pain recognition is fundamental for safeguarding animal welfare. Facial expressions have been investigated in several species and grimace scales have been developed as pain assessment tool in many species including horses (HGS) and mice (MGS). This study is intended to progress the validation of grimace scales, by proposing a statistical approach to identify a classifier that can estimate the pain status of the animal based on Facial Action Units (FAUs) included in HGS and MGS. To achieve this aim, through a validity study, the relation between FAUs included in HGS and MGS and the real pain condition was investigated. A specific statistical approach (Cumulative Link Mixed Model, Inter-rater reliability, Multiple Correspondence Analysis, Linear Discriminant Analysis and Support Vector Machines) was applied to two datasets. Our results confirm the reliability of both scales and show that individual FAU scores of HGS and MGS are related to the pain state of the animal. Finally, we identified the optimal weights of the FAU scores that can be used to best classify animals in pain with an accuracy greater than 70%. For the first time, this study describes a statistical approach to develop a classifier, based on HGS and MGS, for estimating the pain status of animals. The classifier proposed is the starting point to develop a computer-based image analysis for the automatic recognition of pain in horses and mice.

Handling method alters the hedonic value of reward in laboratory mice.

Mice are the most widely used model species for drug discovery and scientific research. Consequently, it is important to refine laboratory procedures and practices to ensure high standards of welfare and scientific data quality. Recent studies have identified that the standard practice of handling laboratory mice by their tails increases behaviours indicative of anxiety, which can be overcome by handling mice using a tunnel. However, despite clear negative effects on mice's behaviour, tunnel handling has yet to be widely implemented. In this study, we provide the first evidence that tail handling also reduces mice's responses to reward. Anhedonia is a core symptom of clinical depression, and is measured in rodents by assessing how they consume a sucrose solution: depressed mice consume less sucrose and the size of their licking bouts when drinking (their 'lick cluster sizes') also tend to be smaller. We found that tail handled mice showed more anhedonic responses in both measures compared to tunnel handled mice, indicative of a decreased responsiveness to reward and potentially a more depressive-like state. Our findings have significant implications for the welfare of laboratory mice as well as the design and interpretation of scientific studies, particularly those investigating or involving reward.

Rhesus macaque personality, dominance, behavior, and health.

Previous studies of nonhuman primates have found relationships between health and individual differences in personality, behavior, and social status. However, despite knowing these factors are intercorrelated, many studies focus only on a single measure, for example, rank. Consequently, it is difficult to determine the degree to which these individual differences are independently associated with health. The present study sought to untangle the associations between health and these individual differences in rhesus macaques (Macaca mulatta). We studied 85 socially housed macaques at the Oregon and California National Primate Research Centers, and used veterinary records to determine the number of injuries and illnesses for each macaque. We measured personality using 12 items from a well-established primate personality questionnaire, performed focal observations of behaviors, and calculated dominance status from directional supplant data. All twelve personality questionnaire items were reliable and were used to represent five of the six personality dimensions identified in rhesus macaques-Dominance, Confidence, Openness, Anxiety, and Friendliness (also known as Sociability). Following this, we fit generalized linear mixed effects models to understand how these factors were associated with an animal's history of injury and history of illness. In the models, age was an offset, facility was a random effect, and the five personality dimensions, behavior, sex, and dominance status were fixed effects. Number of injuries and illnesses were each best represented by a negative binomial distribution. For the injury models, including the effects did improve model fit. This model revealed that more confident and more anxious macaques experienced fewer injuries. For the illness models, including the fixed effects did not significantly improve model fit over a model without the fixed effects. Future studies may seek to assess mechanisms underlying these associations.

The composition and initial evaluation of a grimace scale in ferrets after surgical implantation of a telemetry probe.

Reliable recognition of pain is difficult in ferrets as many currently available parameters are non-specific, inconsistent and/or impractical. Grimace scales have successfully been applied to assess pain in different animal species and might also be applicable to ferrets. To compose a Ferret Grimace Scale (FGS), we studied the facial musculature of ferrets and compared lateral photographs of 19 ferret faces at six time points before and after intraperitoneal telemetry probe implantation. We identified the Action Units (AUs) orbital tightening, nose bulging, cheek bulging, ear changes and whisker retraction as potential indicators of pain in ferrets. To evaluate whether these AUs could reliably be used to identify photographs taken before and after surgery, the photographs were scored 0, 1 or 2 (not, moderately or obviously present) by 11 observers that were blinded to the treatment and timing of the photographs. All AU-scores assigned to the photographs taken five hours after surgery were significantly higher compared to their time-matched baseline scores. Further analysis using the weights that were obtained using a Linear Discriminant Analysis revealed that scoring orbital tightening alone was sufficient to make this distinction with high sensitivity, specificity and accuracy. Including weighted scores for nose bulging, cheek bulging and ear change did not change this. As these AUs had more missing values than orbital tightening, their descriptions should be re-evaluated. Including whisker retraction, which had a negative weight, resulted in lower accuracy and should therefore in its current form be left out of the FGS. Overall, the results of this study suggest that the FGS and the AU orbital tightening in particular could be useful in a multifactorial pain assessment protocol for ferrets. However, before applying the FGS in practice, it should be further validated by incorporating more time points before and after applying (different) painful stimuli, and different levels of analgesia.

Docking piglet tails: How much does it hurt and for how long?

Tail docking in pigs has the potential for evoking short- as well as long-term physiological and behavioural changes indicative of pain. Nonetheless, the existing scientific literature has thus far provided somewhat inconsistent data on the intensity and the duration of pain based on varying assessment methodologies and different post-procedural observation times. In this report we describe three response stages (immediate, short- and long-term) through the application of vocalisation, behavioural and nociceptive assessments in order to identify changes indicative of potential pain experienced by the piglets. Furthermore, we evaluated the following procedural differences: (1) cautery vs. non-cautery docking; (2) length of tail removal. Sound parameters showed a significantly greater call energy and intensity exhibited by docked vs. sham-docked piglets (P<0.05). Observations of general activity of the animals in a test situation failed to detect a difference among treatments (P>0.05) up to 48h post-tail docking. Similarly, no difference in mechanical nociceptive thresholds indicative of long term pain was observed at 17weeks following neonatal tail docking (P>0.05). The present results highlight the potential for the use of measures of vocalisation to detect peri-procedural changes possibly associated with evoked pain. Nonetheless, activity and nociceptive measures failed to identify post-docking anomalies, suggesting that alternative methodologies need to be implemented to clarify whether tail docking is associated with short- and long-term changes attributable to pain experienced by the piglets.

Characterization of short- and long-term mechanical sensitisation following surgical tail amputation in pigs.

Commercial pigs are frequently exposed to tail mutilations in the form of preventive husbandry procedures (tail docking) or as a result of abnormal behaviour (tail biting). Although tissue and nerve injuries are well-described causes of pain hypersensitivity in humans and in rodent animal models, there is no information on the changes in local pain sensitivity induced by tail injuries in pigs. To determine the temporal profile of sensitisation, pigs were exposed to surgical tail resections and mechanical nociceptive thresholds (MNT) were measured in the acute (one week post-operatively) and in the long-term (either eight or sixteen weeks post-surgery) phase of recovery. The influence of the degree of amputation on MNTs was also evaluated by comparing three different tail-resection treatments (intact, 'short tail', 'long tail'). A significant reduction in MNTs one week following surgery suggests the occurrence of acute sensitisation. Long-term hypersensitivity was also observed in tail-resected pigs at either two or four months following surgery. Tail amputation in pigs appears to evoke acute and sustained changes in peripheral mechanical sensitivity, which resemble features of neuropathic pain reported in humans and other species and provides new information on implications for the welfare of animals subjected to this type of injury.