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.

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.

Hepatic pathology and altered gene transcription in a murine model of acid ceramidase deficiency.

Farber disease (FD) is a rare lysosomal storage disorder (LSD) characterized by systemic ceramide accumulation caused by a deficiency in acid ceramidase (ACDase). In its classic form, FD manifests with painful lipogranulomatous nodules in extremities and joints, respiratory complications, and neurological involvement. Hepatosplenomegaly is commonly reported, and severe cases of FD cite liver failure as a cause of early death. Mice homozygous for an orthologous patient mutation in the ACDase gene (Asah1P361R/P361R) recapitulate the classical form of human FD. In this study, we demonstrate impaired liver function and elevation of various liver injury markers in Asah1P361R/P361R mice as early as 5 weeks of age. Histopathology analyses demonstrated significant formation and recruitment of foamy macrophages, invasion of neutrophils, progressive tissue fibrosis, increased cell proliferation and death, and significant storage pathology within various liver cell types. Lipidomic analyses revealed alterations to various lipid concentrations in both serum and liver tissue. A significant accumulation of ceramide and other sphingolipids in both liver and hepatocytes was noted. Sphingolipid acyl chains were also altered, with an increase in long acyl chain sphingolipids coinciding with a decrease in ultra-long acyl chains. Hepatocyte transcriptome analyses revealed significantly altered gene transcription. Molecular pathways related to inflammation were found activated, and molecular pathways involved in lipid metabolism were found deactivated. Altered gene transcription within the sphingolipid pathway itself was also observed. The data presented herein demonstrates that deficiency in ACDase results in liver pathology as well as sphingolipid and gene transcription profile changes that lead to impaired liver function.

Scientific and Regulatory Policy Committee Points to Consider*: The Toxicologic Pathologist's Role in the 3Rs.

Pathologists are trained medical professionals with special expertise in diagnostics, research, and pathophysiology. In these roles, pathologists are well qualified and positioned to engage in conversations about animal use replacement, reduction, and refinement (3Rs), thereby championing the guiding principles of the 3Rs. In particular, toxicology or nonclinical safety assessment is an important area where the discipline of toxicologic pathology can have a critical role in adopting 3Rs principles. As such, a working group of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee was formed to investigate and summarize some of the areas where veterinary pathologists working in the field of toxicology can increase involvement and impact on 3Rs. This "Points to Consider" publication provides an overview of areas within toxicology where the veterinary pathologist's perspective may maximize animal value, including refinement of study design, optimizing sample collection, the development of 3Rs focused regulatory policy, and humane end point determination.[Box: see text].

Electroencephalographic, physiologic and behavioural responses during cervical dislocation euthanasia in turkeys.

BACKGROUND: There is a critical need to develop appropriate on-farm euthanasia methods for poultry species. Euthanasia methods should affect the brain first causing insensibility, followed by cardiorespiratory arrest. Neck or cervical dislocation methods, either manual (CD) or mechanical (MCD), are reported to cause a prolonged time to loss of sensibility and death with inconsistent results upon application, especially MCD methods. However, there is limited information on cervical dislocation in turkeys. The overall objective of this study was to assess the welfare implications of CD and a newly developed MCD device for euthanasia of cull turkeys in comparison with intravenous (IV) pentobarbital sodium (1 mL/4.5 kg), the gold standard euthanasia method. Time to death using electroencephalographic (EEG) and behavioural responses were monitored in eight and eighteen week-old turkeys for five minutes after each euthanasia method application. Spectral analyses of EEG responses and onset of isoelectric EEGs were compared to baseline EEG recordings of birds under anesthesia and behavioural responses were studied among euthanasia treatments. A significant decrease in brain activity frequencies analysis and isoelectric EEG were recorded as time of brain death. RESULTS: All turkeys euthanized with IV pentobarbital sodium presented a rapid and irreversible decrease in the EEG activity at approximately 30s post-injection with minimal behavioural responses. CD and MCD methods caused EEG responses consistent with brain death at approximately 120 s and 300 s, respectively. Additionally, isoelectric EEGs resulted in all pentobarbital sodium and CD groups, but only in 54 and 88% of the eight and eighteen week-old turkeys in the MCD groups, respectively. There were few clear patterns of behavioural responses after CD and MCD application. However, cessation of body movement and time to isoelectric EEG after CD application were positively correlated. CONCLUSIONS: Use of CD and MCD resulted in a prolonged time to death in both age groups of turkeys. MCD application presents a number of welfare risks based on electroencephalographic and behavioural findings. Intravenous pentobarbital sodium induced rapid brain death, but possesses several on-farm limitations. To develop improvements in cervical dislocation methods, further investigations into combined or alternative methods are required to reduce the prolonged time to insensibility and death.

Euthanasia of meat rabbits with carbon dioxide: Behavioral and physiologic responses to gas chamber gradual- and fast-fill rates.

The use of CO2 inhalation with different gas chamber fill rates has not been evaluated for euthanasia in commercial meat rabbits. Our objectives were to evaluate the behavioral and physiologic responses of rabbits (pre-weaned to adult) when exposed to gradual- and fast-fill rates of CO2, and to determine the time to onset of insensibility and death. Cull rabbits (n = 81) were randomly assigned to either a gradual-fill chamber displacement rate of 28% volume change/min (n = 42) or a fast-fill chamber displacement rate of 58% volume change/min (n = 39). The fast-fill rate resulted in a more rapid loss of sensibility at a lower CO2 chamber concentration and in a faster death than for gradual-fill. There were minimal differences in behavioral responses between fill rates with no clear signs of distress. These findings indicate that CO2 at the studied displacement rates is suitable for commercial meat rabbit euthanasia.

Euthanasie des lapins de boucherie au gaz carbonique : comportement et réponses physiologiques aux taux de remplissage graduels et rapides. L'usage de l'inhalation du CO2 avec différents taux de remplissage de la chambre à gaz n'a pas été évalué pour l'euthanasie des lapins de boucherie commerciaux. Nos objectifs consistaient à évaluer les réponses comportementales et physiologiques des lapins (des lapins pré-sevrés à des lapins adultes) lors de l'exposition à des taux de remplissage graduels et rapides de CO2, et à déterminer le moment de l'insensibilité et de la mort. Les lapins de réforme (n = 81) ont été assignés au hasard soit à un taux de déplacement graduel du volume de la chambre de 28 % changement/min. (n = 42) ou à un taux de déplacement rapide du volume de la chambre de 58 % changement/min (n = 39). Le taux de remplacement rapide a produit une perte de sensibilité plus rapide à une concentration inférieure de CO2 dans la chambre et une mort plus rapide que le remplissage graduel. Il y avait des différentes minimales dans les réponses comportementales entre les taux de remplissage sans signes clairs de détresse. Ces résultats indiquent que le CO2 aux taux de déplacement étudiés est approprié pour l'euthanasie des lapins de boucherie commerciaux.(Traduit par Isabelle Vallières).

Efficacy of buprenorphine for management of surgical castration pain in piglets.

BACKGROUND: Surgical castration is a painful procedure, performed routinely on commercial pig farms to prevent boar taint and reduce aggression. The objectives of this study were to assess the efficacy of 0.04 mg/kg buprenorphine (BUP) in reducing pain in castrated piglets, using behavioral indicators and vocalization analysis. This study also sought to further validate the Piglet Grimace Scale (PGS) as a pain assessment tool. A pilot study first assessed the safety of BUP or 0.2 mg/kg butorphanol administration to piglets (n = 4 per treatment). When no side effects were noted with BUP, ten litters of 5-day old piglets (n = 60 total, 15 per treatment group) were used, and randomly assigned to one of four possible treatments: BUP (castrated or uncastrated), saline, or sham. Treatments were administered as an intramuscular injection 20 min prior to surgical castration. Piglets were video recorded 1 h pre-procedure, post-castration for 8 h and for another hour, 24 h post-procedure. Behaviors were scored continuously for the first 15 min of each hour and 511 still-images of piglet faces were scored using the PGS. Vocalizations were recorded from each piglet at three points in the study: at initial handling, injection, and castration. RESULTS: Butorphanol caused some piglets to become groggy and vomit and was not further evaluated. BUP-castrated piglets demonstrated significantly fewer pain behaviors and less facial grimacing compared to saline-treated pigs (P < 0.0001 and P = 0.0073, respectively). There was no difference between the pain behaviours displayed by BUP-castrated piglets compared to BUP-uncastrated and sham piglets (P = 0.9986 and P = 0.7484). There was also no difference in PGS score between BUP-castrated and BUP-uncastrated piglets (P = 0.9376). Piglets in the BUP-castrated group produced vocalizations of similar frequency, amplitude, power, and energy to saline-treated piglets. CONCLUSIONS: Buprenorphine was highly effective in alleviating castration-associated pain behaviors and facial grimacing in piglets, without causing any obvious side effects. Its administration did not reduce piglet vocalizations at the time of castration. The PGS corresponded well to piglet pain behaviors and has utility as a pain assessment tool.

Comparison of the fecal microbiota of domestic commercial meat, laboratory, companion, and shelter rabbits (Oryctolagus cuniculi).

BACKGROUND: Rabbits are cecotrophic, hindgut-fermenters that rely heavily on their gastrointestinal microbiota for optimal digestion of plant-based diets. Dysbiosis, caused by disruption of the gastrointestinal microbiota, is known to predispose rabbits to rabbit enteritis complex (REC), a major cause of morbidity and mortality. The objectives of this study were to describe the fecal microbiota of domestic rabbits from a variety of settings (commercial meat, companion, laboratory, and shelter) and to identify how factors such as age, season, and routine antimicrobial use affect the fecal microbiota composition. RESULTS: A total of 86 pooled commercial meat, 54 companion, 14 pooled laboratory, and 14 shelter rabbit fecal samples were evaluated using 16S rRNA gene sequencing of the V4 region. In all sample types, the predominant bacterial phylum was Firmicutes. Other commonly identified phyla (composing ≥ 1% of the total microbiota composition) were Verrucomicrobia, Proteobacteria, and Bacteroidetes. Significant differences in composition were noted between commercial, companion, laboratory, and shelter rabbit samples for proportions of Verrucomicrobia (P < 0.01), Proteobacteria (P < 0.01), and Lentisphaerae (P = 0.01) within the total microbiota. Within the commercial meat rabbit samples, significant differences between the microbiota composition of growers (n = 42) and does (n = 44) were limited to one unclassified Firmicutes (P = 0.03) and no differences were identified at the phylum level. Significant differences were present between fecal samples taken from rabbits during the summer (n = 44) compared to the winter (n = 42), with Firmicutes (P = 0.04), Verrucomicrobia (P = 0.03), Proteobacteria (P = 0.02), Deinococcus-Thermus (P = 0.04), Armatimonadates (P = 0.003), and Actinobacteria (P = 0.03) forming significantly different proportions of the microbiota. The only significant difference in composition between those farms that routinely reported antimicrobial use and those that did not was in one unclassified Bacteroidetes (P < 0.05) and no differences were identified at the phylum level. CONCLUSIONS: Rabbit husbandry and diet, in addition to season, significantly influence the fecal microbiota composition of domestic rabbits, while age of the rabbit post-weaning has minimal impact.

Biosecurity practices and causes of enteritis on Ontario meat rabbit farms.

Infectious enterocolitis is a significant cause of mortality in meat rabbits. Disease risk is enhanced by intensive rearing practices and poor on-farm biosecurity. This investigation was undertaken in farmed meat rabbits during an Ontario-wide outbreak of enteritis with high mortality to determine the prevalence of causative agents. A survey evaluating on-farm biosecurity practices was also conducted to identify potential means of pathogen contamination and zoonotic risks. Gross and microscopic pathology evaluations combined with microbiologic testing were conducted on 95 rabbits over spring and winter months. Escherichia coli and Clostridium spiroforme were most commonly associated with enteritis in rabbits regardless of age or season and lesions were significantly more severe in mature does (P < 0.0001). The survey results demonstrated a lack of consistent on-farm biosecurity practices. The infectious nature of enteric disease of rabbits combined with poor biosecurity practices may contribute to disease transmission within and between farms.

Pratiques de biosécurité et causes d'entérite dans des fermes d'élevage de lapins de l'Ontario. L'entérocolite infectieuse est une cause importante de mortalité chez les lapins d'élevage. Le risque de maladie est accru par des pratiques d'élevage intensives et une mauvaise biosécurité à la ferme. Cette enquête a été entreprise chez des lapins d'élevage durant une éclosion d'entérite à l'échelle de l'Ontario qui présentait un taux de mortalité élevé afin de déterminer la prévalence des agents étiologiques. On a aussi réalisé un sondage évaluant les pratiques de biosécurité à la ferme afin d'identifier les modes potentiels de contamination des agents pathogènes et les risques zoonotiques. Des évaluations pathologiques macroscopiques et microscopiques combinées à des tests microbiologiques ont été réalisés sur 95 lapins au cours des mois d'été et d'hiver. Escherichia coli et Clostridium spiroforme étaient le plus communément associés à l'entérite chez les lapins sans égard à l'âge ou à la saison et les lésions étaient significativement plus graves chez les lapines adultes (P < 0,0001). Les résultats du sondage ont démontré l'absence de pratiques de biosécurité uniformes à la ferme. La nature infectieuse de la maladie entérique des lapins et de mauvaises pratiques de biosécurité peuvent contribuer à la transmission de la maladie dans les fermes et entre ces dernières.(Traduit par Isabelle Vallières).

Effect of 8-oxoguanine glycosylase deficiency on aflatoxin B1 tumourigenicity in mice.

The mycotoxin aflatoxin B1 (AFB1) may initiate cancer by causing oxidatively damaged DNA, specifically by causing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesions. Base excision repair removes these lesions, with 8-oxoguanine glycosylase (OGG1) being the rate-limiting enzyme. The aim of this study was to determine the effect of ogg1 deficiency on AFB1-induced oxidatively damaged DNA and tumourigenesis. Female wild-type, heterozygous and homozygous ogg1 null mice were given a single dose of 50mg/kg AFB1 or 40 µl dimethyl sulfoxide (DMSO) ip. Neither ogg1 genotype nor AFB1 treatment affected levels of oxidised guanine in lung or liver 2h post-treatment. AFB1-treated ogg1 null mice showed exacerbated weight loss and mortality relative to DMSO-treated ogg1 null mice, but AFB1 treatment did not significantly increase lung or liver tumour incidence compared with controls, regardless of ogg1 genotype. Suspect lung masses from three of the AFB1-treated mice were adenomas, and masses from two of the mice were osteosarcomas. No osteosarcomas were observed in DMSO-treated mice. All liver masses from AFB1-treated mice were adenomas, and one also contained a hepatocellular carcinoma. In DNA from the lung tumours, the K-ras mutation pattern was inconsistent with initiation by AFB1. In conclusion, ogg1 status did not have a significant effect on AFB1-induced oxidatively damaged DNA or tumourigenesis, but deletion of one or both alleles of ogg1 did increase susceptibility to other aspects of AFB1 toxicity.

Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development.

The highly conserved ANP32 proteins are proposed to function in a broad array of physiological activities through molecular mechanisms as diverse as phosphatase inhibition, chromatin regulation, caspase activation, and intracellular transport. On the basis of previous analyses of mice bearing targeted mutations of Anp32a or Anp32e, there has been speculation that all ANP32 proteins play redundant roles and are dispensable for normal development. However, more recent work has suggested that ANP32B may in fact have functions that are not shared by other ANP32 family members. Here we report that ANP32B expression is associated with a poor prognosis in human breast cancer, consistent with the increased levels of Anp32b mRNA present in proliferating wild-type (WT) murine embryonic fibroblasts and stimulated WT B and T lymphocytes. Moreover, we show that, contrary to previous assumptions, Anp32b is very important for murine embryogenesis. In a mixed genetic background, ANP32B-deficient mice displayed a partially penetrant perinatal lethality that became fully penetrant in a pure C57BL/6 background. Surviving ANP32B-deficient mice showed reduced viability due to variable defects in various organ systems. Study of compound mutants lacking ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles for ANP32A in mouse development that became apparent only in the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b being the most critical for normal development.

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.

The PepT1-transportable soy tripeptide VPY reduces intestinal inflammation.

BACKGROUND: Inflammatory bowel disease (IBD) is a chronic inflammation of the gastrointestinal tract. The peptide transporter PepT1 is responsible for the intestinal uptake of dietary peptides, and its expression in the gastrointestinal tract is up-regulated during intestinal inflammation, indicating that PepT1 may be a promising target for IBD therapeutics. METHODS: The transport of soy-derived di- and tripeptides across Caco-2 intestinal epithelial cells was examined, and the anti-inflammatory effects of the transported peptide VPY were evaluated in vitro in Caco-2 and THP-1 macrophages, and in vivo in a mouse model of DSS-induced colitis. RESULTS: VPY inhibited the secretion of IL-8 and TNF-α, respectively, from Caco-2 and THP-1 cells. VPY transport and anti-inflammatory activity in Caco-2 cells was reduced in the presence of Gly-Sar, indicating this activity was mediated by PepT1. In mice, VPY treatment reduced DSS-induced colitis symptoms and weight loss, improved colon histology, reduced MPO activity, and decreased gene expression of the pro-inflammatory cytokines TNF-α, IL-6, IL-1ß, IFN-γ and IL-17 in the colon. CONCLUSIONS AND GENERAL SIGNIFICANCE: VPY is a novel PepT1 substrate that can inhibit the production of pro-inflammatory mediators in vitro in intestinal epithelial and immune cells, and reduce the severity of colitis in mice by down-regulating the expression of pro-inflammatory cytokines in the colon, suggesting that VPY may be promising for the treatment of IBD.

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.

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.

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.

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.

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.

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.