Development of a novel primate welfare assessment tool for research macaques.

Primates are important species for biomedical research and ensuring their good welfare is critical for research translatability and ethical responsibility. Systematic animal welfare assessments can support continuous programme improvements and build institutional awareness of areas requiring more attention. A multi-facility, collaborative project aimed to develop and implement a novel primate welfare assessment tool (PWAT) for use with research macaques. PWAT development involved: establishing an internal focus group of primate subject matter experts, identifying animal welfare categories and descriptors based on literature review, developing a preliminary tool, beta-testing the tool to ensure practicality and final consensus on descriptors, finalising the tool in a database with semi-automated data analysis, and delivering the tool to 13 sites across four countries. The tool uses input- and outcome-based measures from six categories: physical, behavioural, training, environmental, procedural, and culture of care. The final tool has 133 descriptors weighted based upon welfare impact, and is split into three forms for ease of use (room level, site level, and personnel interviews). The PWAT was trialled across facilities in March and September 2022 for benchmarking current macaque behavioural management programmes. The tool successfully distinguished strengths and challenges at the facility level and across sites. Following this benchmarking, the tool is being applied semi-annually to assess and monitor progress in behavioural management programmes. The development process of the PWAT demonstrates that evidence-based assessment tools can be developed through collaboration and consensus building, which are important for uptake and applicability, and ultimately for promoting global improvements in research macaque welfare.

Exploring rabbit as a nonrodent species for general toxicology studies.

To avoid adverse events in humans, toxicity studies in nonclinical species have been the foundation of safety evaluation in the pharmaceutical industry. However, it is recognized that working with animals in research is a privilege, and conscientious use should always respect the 3Rs: replacement, reduction, and refinement. In the wake of the shortages in routine nonrodent species and considering that nonanimal methods are not yet sufficiently mature, the value of the rabbit as a nonrodent species is worth exploring. Historically used in vaccine, cosmetic, and medical device testing, the rabbit is seldom used today as a second species in pharmaceutical development, except for embryo-fetal development studies, ophthalmic therapeutics, some medical devices and implants, and vaccines. Although several factors affect the decision of species selection, including pharmacological relevance, pharmacokinetics, and ADME considerations, there are no perfect animal models. In this forum article, we bring together experts from veterinary medicine, industry, contract research organizations, and government to explore the pros and cons, residual concerns, and data gaps regarding the use of the rabbit for general toxicity testing.

Implementation of an Alternative Training Method for Cardiac Blood Collection in Mice.

Training personnel to work with animals presents a variety of challenges, both logistically and with regard to animal welfare. These issues make training an ideal opportunity to evaluate practices and to implement the 3R principles (refinement, replacement, and reduction). Cardiac blood collection from mice is a procedure that can compromise the 3Rs by requiring repeated practice and animal euthanasia. The development of a non-animal training model would promote the 3R principles. Our goals for the development of a new training model for cardiac blood collection from mice were to reduce the number of mice needed to achieve competency, improve our culture of care, and refine the training approach by improving competency. The training model was developed using commonly available materials. The total cost of the model was less than $15 USD per model. Two training curricula were conducted concurrently over a 5-mo period: 1) a curriculum in which trainees used the model before progressing to live mice and 2) the traditional curriculum, which used euthanized mice throughout. The measured variables included the total number of mice used, proportions of trainees who reached competency, the time needed to reach competency, method comprehension, quality of skill performance, trainer and trainee feedback, and training costs. The alternative group used at least 10 fewer mice per technician as compared with the traditionally trained group. The alternative group had a higher competency rate, with 82% (9 of 11 trainees) reaching competency compared with 60% (3 of 5 trainees) in the traditional group. Skill comprehension and quality were superior in the alternative group, as evidenced by fewer gross lesions at necropsy. Overall, personnel in the alternative group provided positive feedback with regard to the use of fewer mice, acquisition of both skill and confidence, and benefits for compassion fatigue. The use of this model is now our standard approach for training personnel in cardiac blood collection in mice. Our results demonstrate that the use of models in training curricula can enhance skill development and reduce the use of mice.

Unmasking the Adverse Impacts of Sex Bias on Science and Research Animal Welfare.

Sex bias in biomedical and natural science research has been prevalent for decades. In many cases, the female estrous cycle was thought to be too complex an issue to model for, and it was thought to be simpler to only use males in studies. At times, particularly when studying efficacy and safety of new therapeutics, this sex bias has resulted in over- and under-medication with associated deleterious side effects in women. Many sex differences have been recognized that are unrelated to hormonal variation occurring during the estrous cycle. Sex bias also creates animal welfare challenges related to animal over-production and wastage, insufficient consideration of welfare (and scientific) impact related to differential housing of male vs female animals within research facilities, and a lack of understanding regarding differential requirements for pain recognition and alleviation in male versus female animals. Although many funding and government agencies require both sexes to be studied in biomedical research, many disparities remain in practice. This requires further enforcement of expectations by the Institutional Animal Care and Use Committee when reviewing protocols, research groups when writing grants, planning studies, and conducting research, and scientific journals and reviewers to ensure that sex bias policies are enforced.

Evaluation of a Behavioral Test for Sickness Behavior Associated with Fusarium Mycotoxin Ingestion in Female Beagle Dogs (Canis familiaris).

Animals exhibit behavioral changes during illness, including lethargy, anorexia, fever, adipsia, and anhedonia, which are believed to comprise an adaptive evolutionary strategy. Exploratory and social behaviors generally decrease during illness, but behavioral changes of dogs during illness have not been described. The objective of this study was to evaluate a novel canine behavior test during subclinical illness induced by dietary Fusarium mycotoxin. Twelve mature female beagle dogs received 3 treatment diets: a control diet (control), a diet formulated with grains contaminated with Fusarium mycotoxin (toxin), and the toxin diet together with a toxin binding agent (binder). All dogs received each diets for 14 d in a Latin square design with a 7-d washout period between diet trials. The test consisted of individually releasing dogs into the center aisle of the housing room for 4 min per day, during which interactions with familiar dogs in adjacent kennels were recorded by an observer outside the room who was blind to treatment groups. Total interactions, orientation, and attempted physical contact with other dogs were less frequent during the toxin and binder diet treatments. Conversely, frequencies of physical proximity and olfactory contact with familiar dogs in adjacent kennels were not associated with diet. In conclusion, induction of subclinical gastrointestinal illness influenced aspects of social interactions in beagle dogs. A clinical assessment sheet integrating these findings was developed to aid in early identification of subclinical illness in research dogs based on behavior.

Research Animal Behavioral Management Programs for the 21st Century.

Behavioral management programs have been developed commonly for research dogs and primates but rarely has program consideration been expanded to include all research species worked with. This is necessary to reduce animal stress and promote natural behaviors, which can promote good animal welfare and result in more robust and reproducible scientific data. We describe the evolution of consideration for research animal needs and define an umbrella-based model of research animal behavioral management programs, which may be used for all research species. In addition to developing a more comprehensive program, we emphasize the need for regular welfare assessments to determine whether the program is working cohesively and whether any aspects require modification.

Using Stakeholder Focus Groups to Refine the Care of Pigs Used in Research.

Research organizations should be proactive in regularly evaluating and refining their animal care and use programs in order to advance animal welfare and minimize distress. Pigs are often used in research, but few empirical studies have examined optimal husbandry and research use practices for pigs in a research environment. We developed the Pig Welfare Working Group (PWWG) to address the need for more formal guidelines on the management and use of pigs in research. The PWWG was a stakeholder focus group whose goal was to identify challenges and opportunities relevant to improving animal welfare through collaboration, knowledge sharing, and inclusive decision-making. Through consensus building, the PWWG developed 12 recommendations for behavioral management, housing, research procedures, transportation, and rehoming programs. The recommendations were rolled out across the contract research organization, business units, sites, and countries. Follow up will be conducted regularly to assess welfare, monitor progress toward implementing the recommendations, and recognize and reward participants making changes at their site.

Development and validation of a cynomolgus macaque grimace scale for acute pain assessment.

Cynomolgus macaques may undergo surgical procedures for scientific and veterinary purposes. Recognition and assessment of pain using validated tools is a necessary first step for adequately managing pain in these primates. Grimace scales are one means of assessing the occurance of acute pain using action units such as facial expressions and posture. The aim of this study was to create and validate a Cynomolgus Macaque Grimace Scale (CMGS). Cynomolgus macaques (n = 43) were video recorded before and after a surgical procedure. Images were extracted from videos at timepoints at which breakthrough pain might be expected based on analgesic pharmacokinetics. Using the CMGS images were scored by 12 observers blinded to animal identification, times, and conditions. To validate the tool, detailed behavioral analyses emphasizing changes to baseline activity ethograms were compared to grimace scores. Four action units were identified related to potential pain including orbital tightening, brow lowering, cheek tightening, and hunched posture. The CMGS tool was found to have moderate inter- (ICCaverage action unit mean ± SD: 0.67 ± 0.28) and good intra- (ICCsingle mean ± SD: 0.79 ± 0.14) observer reliability. Grimace scores increased significantly (p < 0.0001) in the first four post-operative timepoints compared to baseline, correlating with behavioral findings (rho range = 0.22-0.35, p < 0.001). An analgesic intervention threshold was determined and should be considered when providing additional pain relief. The CMGS was shown to be a reliable and valid tool; however, more research is needed to confirm external validity. This tool will be highly valuable for refining analgesic protocols and acute peri-procedural care for cynomolgus macaques.

Use of nonaversive handling and training procedures for laboratory mice and rats: Attitudes of American and Canadian laboratory animal professionals.

Nonaversive or low stress handling techniques can reduce fear and stress in research rodents, ultimately improving study data quality. Uptake of low stress handling has been slow in the USA and Canada. In this study we explored the understanding, experience, and attitudes toward low stress handling of rats and mice in laboratory animal professionals from the USA (US) and Canada (CA). Participants (n = 40) were recruited for a standardized interview and job categories were divided into veterinary/PhD level roles (doctoral level; DL) and non-veterinary/non-PhD level roles (non-doctoral level, NDL) (US: 23, DL: 9, NDL: 14; CA: 17, DL: 8, and NDL: 9). Interviews were transcribed and analyzed using NVIVO. Two research assistants independently coded themes for each question and consolidated responses based on commonality. Laboratory animal professionals understood the benefits of low stress handling and training techniques with rats and mice, stating reduced stress, better data, and improved welfare, with CA participants more likely to mention animal welfare as a benefit, and DL more likely to mention improved research data and reduced stress. Participants across demographic groups indicated improved job satisfaction and decreased stress as the positive impacts low stress handling would have on their positions. The primary perceived barriers to low stress handling implementation were researcher attitudes, the time needed to implement and use these techniques, and training personnel to use the techniques properly and consistently. To promote refinement of handling of rats and mice, more educational opportunities on the benefits and implementation of low stress handling techniques need to be provided to laboratory animal professionals, as well as to researchers.

Refining restraint techniques for research pigs through habituation.

Pigs are common research models and are strong animals that can be difficult to restrain. Improper restraint can put pigs and research personnel at risk for injury and induce stress, which can affect research outcomes. This study aimed to refine restraint techniques for research pigs using habituation and operant conditioning. Forty-four (22 males, 22 females; 4 months old, ~8.1 kg) Göttingen minipigs were randomly assigned to a control (C: no interventions) or a treatment group (T). Pigs in the T group received 3 min training sessions 3 days/week for the first 14 d after arrival. Training sessions included human socialization and habituation to a hammock sling for blood collection. Blood collection occurred on day 13 for all pigs by novel technicians. Pigs were placed in the sling, blood was collected from the radial vein, and serum cortisol levels were determined (ug/dL). Pig behavior was recorded and scored for duration of time spent struggling (s) and vocalizing (s). Novel human approach tests occurred on day 12, before blood collection, and day 14, after blood collection. Pigs were scored on latency to touch the human (s) and duration of time spent in contact with the human (s). Pig weight was taken upon arrival and on day 15. Separate linear models were fitted for response variables struggle duration in sling, serum cortisol, latency to touch human, time spent in contact with human, and body weight. Fixed effects were treatment and sex. Prior to blood collection, there was no difference in response to a novel human (P > 0.05) but after blood collection, T pigs were quicker to approach (estimate: -5.352, SE: 1.72, P = 0.003) and spent more time in contact with the novel human (estimate: 3.091, SE: 1.448, P = 0.039). T pigs also had lower cortisol levels during blood collection (estimate: -2.36, SE: 0.657, P = 0.001). There was no difference in behavior while in the sling (P > 0.05). The results of the study suggest that even small investments in habituation and training pigs to study procedures is beneficial in reducing stress and improving human-animal relationships, but more time would be beneficial to promote calmer behavior in the sling.

Compassion Fatigue and Coping Mechanisms of Laboratory Animal Professionals from Europe, China, and Japan.

Laboratory animal professionals (LAP) may experience situations that contribute to compassion fatigue (CF). The goal of this research was to better understand CF in LAP in and across employment categories. Surveys were distributed through LAP organizational listservs in the European Union (EU), China, and Japan, and results were analyzed to identify CF prevalence, personal and work-related factors, coping mechanisms, and beneficial work-support programs. Independent χ²-tests compared personal and work-related factors and feelings of CF. Feelings of CF and coping mechanisms were compared with personality scores using independent sample t tests. There were 302 respondents from the EU, 39 from China, and 77 from Japan. Over half of respondents from the EU (52%) and China (56%) reported experiencing CF (52%), with fewer (32%) reporting CF in Japan. No major differences were found based on employer type. Personality scores were significantly related to feelings of CF and preferred coping mechanisms. Work-related factors that contributed to feelings of CF in over half of respondents included staffing levels, workplace relationships, and availability of programs geared to address CF. Across regions, talking to someone, physical activity, getting away from work, and self-care were effective coping mechanisms in over 50% of respondents. Fewer than 30% of respondents indicated that their place of employment had CF support programs, and even fewer (8% to 28%) indicated that these programs were helpful. The study results suggest that to be effective, employer CF programs for LAP should consider providing quiet places at work and programs for self-care, promoting physical and mental health and social support systems, and establishing opportunities to memorialize animals.

Challenges with Assessing and Treating Pain in Research Primates: A Focused Survey and Literature Review.

Research primates may undergo surgical procedures making effective pain management essential to ensure good animal welfare and unbiased scientific data. Adequate pain mitigation is dependent on whether veterinarians, technicians, researchers, and caregivers can recognize and assess pain, as well as the availability of efficacious therapeutics. A survey was conducted to evaluate primate veterinary approaches to pain assessment and alleviation, as well as expressed challenges for adequately managing primate pain. The survey (n = 93 respondents) collected information regarding institutional policies and procedures for pain recognition, methods used for pain relief, and perceived levels of confidence in primate pain assessment. Results indicated that 71% (n = 60) of respondents worked at institutions that were without formal experimental pain assessment policies. Pain assessment methods were consistent across respondents with the majority evaluating pain based on changes in general activity levels (100%, n = 86) and food consumption (97%, n = 84). Self-reported confidence in recognizing and managing pain ranged from slightly confident to highly confident, and there was a commonly expressed concern about the lack of objective pain assessment tools and science-based evidence regarding therapeutic recommendations of analgesics for research primates. These opinions correspond with significant gaps in the primate pain management literature, including limited specific pharmacokinetic data and efficacy testing for commonly used analgesics in research primate species as well as limited research on objective and specific measures of pain in research primates. These results demonstrate that there are inconsistencies in institutional policies and procedures surrounding pain management in research primates and a lack of objective pain assessment methods. Demonstrating the gaps and challenges in primate pain management can inform guideline development and suggest areas for future research.

Mental well-being and diversity, equity, and inclusiveness in the veterinary profession: Pathways to a more resilient profession.

Mental well-being (MWB) and diversity, equity, and inclusiveness (DEI) continue to be critical within the veterinary profession but there is less information regarding how professional associations around the world tackle these issues. A mixed-method study including an international online survey in English (n = 137 responses via snowball sampling), fourteen interviews, and two webinars was used to identify the availability and impact of MWB and DEI support programs for veterinarians. Survey results showed that more veterinary organizations designated MWB and DEI challenges (54%, n = 43/79 and 58%, n = 45/78, respectively) as a key priority area than veterinary clinics (26%, n = 15/57 and 33%, n = 19/57, respectively). Whereas, MWB support programs were available in a moderate number of mainly English-speaking countries, DEI support programs were available in only a few countries and focused primarily on specific groups, with an unknown impact due to their recent implementation. Universally, survey respondents believed activities for specific groups, such as MWB webinars, training, and awareness campaigns, as well as MWB/DEI helplines and DEI peer-to-peer support programs had a high impact (median 3.5-4/5) yet were underemployed by both veterinary organization and veterinary clinics. Further feedback from respondents during focused interviews indicated that requiring initial and continuing training as well as tailored group activities would be most beneficial to improve MWB/DEI throughout the veterinary professional career. There are many areas of the intersection between MWB and DEI that remain to be elucidated in the future studies. Having a sufficient sample size, improving accessibility, and addressing varying cultural perceptions are the main challenges, as seen in our study. To truly address MWB and DEI disparities, change is also needed in veterinary workplace culture and environment. In conclusion, raising awareness for an inclusive profession, including increasing openness and acceptance to enhance DEI and destigmatizing MWB challenges, is needed to ensure a thriving, modern veterinary profession.

Applied Animal Ethics in Industrial Food Animal Production: Exploring the Role of the Veterinarian.

Industrial food animal production practices are efficient for producing large quantities of milk, meat, and eggs for a growing global population, but often result in the need to alter animals to fit a more restricted environment, as well as creating new animal welfare and health problems related to animal confinement in high densities. These practices and methods have become normalized, to the extent that veterinarians and others embedded in these industries rarely question the ethical challenges associated with raising animals in this fashion. Moral 'lock-in' is common with those working in food animal industries, as is the feeling that it is impossible to effect meaningful change. Animal welfare issues associated with the industrialization of food animal production are 'wicked problems' that require a multi- and transdisciplinary approach. We argue that veterinarians, as expert animal health and welfare advocates, should be critical stakeholders and leaders in discussions with producers and the food animal sector, to look for innovative solutions and technology that will address current and future global sustainability and food security needs. Solutions will necessarily be different in different countries and regions, but ethical issues associated with industrial food animal production practices are universal.

Developing Recommendations for Cumulative Endpoints and Lifetime Use for Research Animals.

Research animals are important for scientific advancement, and therefore, their long-term welfare needs to be monitored to not only minimize suffering, but to provide positive affective states and experiences. Currently, there is limited guidance in countries around the world on cumulative and experimental endpoints. This paper aims to explore current opinions and institutional strategies regarding cumulative use and endpoints through a scoping survey and review of current regulations and welfare assessment tools, and ultimately to provide recommendations for assessment of cumulative and lifetime use of research animals. The survey found that only 36% of respondents indicated that their institution had cumulative use endpoint policies in place, but these policies may be informal and/or vary by species. Most respondents supported more specific guidelines but expressed concerns about formal policies that may limit their ability to make case-by-case decisions. The wide diversity in how research animals are used makes it difficult for specific policies to be implemented. Endpoint decisions should be made in an objective manner using standardized welfare assessment tools. Future research should focus on robust, efficient welfare assessment tools that can be used to support planning and recommendations for cumulative endpoints and lifetime use of research and teaching animals.

Recommendations for Ensuring Good Welfare of Horses Used for Industrial Blood, Serum, or Urine Production.

Various pharmaceutical products have been derived from horse blood and urine for over a century. Production of biologics and therapeutics from these samples is a niche industry and often occurs in regions with little regulation or veterinary oversight. To ensure good welfare of horses maintained for these purposes, guidance has been developed to support the industry.

Pain Management in Farm Animals: Focus on Cattle, Sheep and Pigs.

Pain causes behavioral, autonomic, and neuroendocrine changes and is a common cause of animal welfare compromise in farm animals. Current societal and ethical concerns demand better agricultural practices and improved welfare for food animals. These guidelines focus on cattle, sheep, and pigs, and present the implications of pain in terms of animal welfare and ethical perspectives, and its challenges and misconceptions. We provide an overview of pain management including assessment and treatment applied to the most common husbandry procedures, and recommendations to improve animal welfare in these species. A cost-benefit analysis of pain mitigation is discussed for food animals as well as the use of pain scoring systems for pain assessment in these species. Several recommendations are provided related to husbandry practices that could mitigate pain and improve farm animal welfare. This includes pain assessment as one of the indicators of animal welfare, the use of artificial intelligence for automated methods and research, and the need for better/appropriate legislation, regulations, and recommendations for pain relief during routine and husbandry procedures.

Moving Beyond the Absence of Pain and Distress: Focusing on Positive Animal Welfare.

For many years, researchers, veterinarians, animal ethics committees, and regulators have focused on minimizing pain and distress as a primary goal of refinement when working with animals in science. More recent publications as well as a shift in animal ethics and public opinion have emphasized promotion of positive affective states, culminating in the concept of positive animal welfare. Robust measures are required to know when positive animal welfare states are occurring, and a number of measures are proposed and discussed. Regardless of whether there are newer methods available that focus exclusively on measuring positive affective states, consistent consideration of research animal behavioral programs, refinement, and adopting periodic stand-alone animal welfare assessments for all species involved will help to push the care and practices of research animals towards an increased focus on positive animal welfare.

Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians.

OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.

Using Paper Nest Pucks to Prevent Barbering in C57BL/6 Mice.

Little research has been conducted to examine the influence of various methods of providing nest materials-such as dispersing them, providing them as single units, or clustering them-on the behavior and welfare of group-housed mice. In this study, 6 wk-old C57BL/6NCrl mice were housed 3 per cage and randomized into 1 of 3 nest-material groups: 1) one facial tissue per cage (control; female mice, 3 cages; male mice, 3 cages); 2) an 8-g 'puck' of compressed nesting material and a facial tissue (females, 3 cages; males, 3 cages); or 3) 8 g of dispersed paper strips and a facial tissue (females, 3 cages; males, 3 cages). Mouse behavior (agonistic, stereotypic, nesting), physical examination data, and nest scores were evaluated over 16 d. The results showed that mice in the puck and control groups spent more time manipulating nest materials after cage changes than did mice in the paper-strip group. Average nest scores were highest in the paper-strip group compared with controls and puck cages. Female cages with pucks showed no barbering, whereas all other female mice cages demonstrated barbering. Overall, nest pucks may provide a time-consuming activity for mice and may help protect female C57BL/6 mice from barbering. However, more research is needed to replicate and expand these study results.