How to see stress in chickens: On the way to a Stressed Chicken Scale.

For many species, scales are used to classify discomfort and stress (e.g., facial expression/pain scales). Although a significant number of vertebrates used for scientific purposes are chickens, a corresponding scale for birds has not yet been established. We developed a Stressed Chicken Scale (SCS) to investigate whether it is possible to assess discomfort in a chicken by its body posture. A selective review with additional handsearch was conducted to find suitable parameters for visual stress assessment. Seven potential body signals were identified: Tail and head position, eye closure, beak opening, leg and wing position, and plumage fullness (ruffled or fluffed up feathers). The SCS was evaluated for interobserver reliability with veterinary students (n = 20), using randomized pictures of stressed and unstressed chickens in lateral view (n = 80). Observers were able to identify the body signals on the pictures after a brief training session. Agreement scores for interobserver agreement ranged from κ = 0.31 (fair agreement) for eye closure to κ = 0.78 (substantial agreement) for beak opening. We found that the number of body signals displayed in a stressed expression had an impact on observers' overall assessment of the chickens, for example, chickens were more likely to be rated as stressed if more than 4 signals indicative of stress were present. We conclude that the 7 individual body signals can be used to identify discomfort in chickens.

Dog leukocyte antigen genotyping across class I and class II genes in beagle dogs as laboratory animals.

Dog leukocyte antigen (DLA) polymorphisms have been found to be associated with inter-individual variations in the risk, susceptibility, and severity of immune-related phenomena. While DLA class II genes have been extensively studied, less research has been performed on the polymorphisms of DLA class I genes, especially in beagle dogs commonly used as laboratory animals for safety evaluations in drug development. We genotyped four DLA class I genes and four DLA class II genes by locus-specific Sanger sequencing using 93 laboratory beagle dogs derived from two different strains: TOYO and Marshall. The results showed that, for DLA class I genes, 11, 4, 1, and 2 alleles, including a novel allele, were detected in DLA-88, DLA-12/88L, DLA-64, and DLA-79, while, for DLA class II genes, 1, 10, 6, and 7 alleles were detected in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. It was estimated that there were 14 DLA haplotypes, six of which had a frequency of ≥ 5%. Furthermore, when comparing the DLA diversity between TOYO and Marshall strains, the most common alleles and haplotypes differed between them. This is the first study to genotype all DLA loci and determine DLA haplotypes including all DLA class I and class II genes in dogs. Integrating information on the DLA diversity of laboratory beagle dogs should reinforce their benefit as an animal model for understanding various diseases associated with a specific DLA type.

Impact of quality and transparency in scientific writing on the reduction of animal usage in experimental protocols: a review based in pertinent literature.

The irreproducibility in scientific research has become a critical issue. Despite the essential role of rigorous methodology in constructing a scientific article, more than half of publications, on average, are considered non-reproducible. The implications of this irreproducibility extend to reliability problems, hindering progress in technological production and resulting in substantial financial losses. In the context of laboratory animal research, this work emphasizes the importance of choosing an appropriate experimental model within the 3R's principle (Refine, Reduce, Replace). This study specifically addresses a deficiency in data specification in scientific articles, revealing inadequacies in the description of crucial details, such as environmental conditions, diet, and experimental procedures. For this purpose, 124 articles from journals with relevant impact factors were analyzed, conducting a survey of data considered important for the reproducibility of studies. Important flaws in the presentation of data were identified in most of the articles evaluated. The results of this study highlight the need to improve the description of essential information, standardizing studies, and ensuring the reproducibility of experiments in areas such as metabolism, immunity, hormones, stress, among others, to enhance the reliability and reproduction of experimental results, aligning with international guidelines such as ARRIVE and PREPARE.

Dietary protein restriction regulates skeletal muscle fiber metabolic characteristics associated with the FGF21-ERK1/2 pathway.

Under conditions of dietary amino acid balance, decreasing the dietary crude protein (CP) level in pigs has a beneficial effect on meat quality. To further elucidate the mechanism, we explored the alteration of muscle fiber characteristics and key regulators related to myogenesis in the skeletal muscle of pigs fed a protein restricted diet. Compared to pigs fed a normal protein diet, dietary protein restriction significantly increased the slow-twitch muscle fiber proportion in skeletal muscle, succinic dehydrogenase (SDH) activity, the concentrations of ascorbate, biotin, palmitoleic acid, and the ratio of s-adenosylhomocysteine (SAM) to s-adenosylhomocysteine (SAH), but the fast-twitch muscle fiber proportion, lactate dehydrogenase (LDH) activity, the concentrations of ATP, glucose-6-phosphate, SAM, and SAH in skeletal muscle, and the ratio of serum triiodothyronine (T3) to tetraiodothyronine (T4) were decreased. In conclusion, we demonstrated that dietary protein restriction induced skeletal muscle fiber remodeling association the regulation of FGF21-ERK1/2-mTORC1 signaling in weaned piglets.

Effective in vitro evaluation of the risk of histamine release related to valemetostat tosylate using MRGPRX2-expressing cells.

Mas-related G-protein-coupled receptor X2 (MRGPRX2), expressed on mast cells, is associated with drug-induced pseudo-allergic reactions. Although it is well known that there are differences of sensitivity between species in the pseudo-allergic reactions, no platform for evaluating a human risk of the pseudo-allergic reactions observed in nonclinical studies has been established. Valemetostat tosylate, developed as an anti-cancer drug, induced histamine release in a nonclinical study with dogs. The purpose of the current study was to identify the mechanism and assess the human risk of valemetostat-tosylate-induced histamine release using dog and human MRGPRX2-expressing cells. In an experiment with human or dog MRGPRX2-expressing cells, valemetostat tosylate caused activation of human and dog MRGPRX2. Importantly, the EC50 for dog MRGPRX2 was consistent with the Cmax value at which histamine release was observed in dogs. Furthermore, the EC50 for human MRGPRX2 was ca. 27-fold higher than that for dog MRGPRX2, indicating a species difference in histamine-releasing activity. In a clinical trial, histamine release was not observed in patients receiving valemetostat tosylate. In conclusion, an in vitro assay using human and animal MRGPRX2-expressing cells would be an effective platform to investigate the mechanism and predict the human risk of histamine release observed in nonclinical studies.

EPC1/2 regulate hematopoietic stem and progenitor cell proliferation by modulating H3 acetylation and DLST.

Enhancers of polycomb 1 (EPC1) and 2 (EPC2) are involved in multiple biological processes as components of histone acetyltransferases/deacetylase complexes and transcriptional cofactors, and their dysfunction was associated with developmental defects and diseases. However, it remains unknown how their dysfunction induces hematopoietic stem and progenitor cell (HSPC) defects. Here, we show that depletion of EPC1/2 significantly reduced the number of hematopoietic stem and progenitor cells (HSPCs) in the aorta-gonad mesonephros and caudal hematopoietic tissue regions by impairing HSPC proliferation, and consistently downregulated the expression of HSPC genes in K562 cells. This study demonstrates the functions of EPC1/2 in regulating histone H3 acetylation, and in regulating DLST (dihydrolipoamide S-succinyltransferase) via H3 acetylation and cooperating with transcription factors serum response factor and FOXR2 together, and in the subsequent HSPC emergence and proliferation. Our results demonstrate the essential roles of EPC1/2 in regulating H3 acetylation, and DLST as a linkage between EPC1 and EPC2 with mitochondria metabolism, in HSPC emergence and proliferation.

Differential effects of four laboratory animal control diets on gut microbiota in mice.

BACKGROUND: The gut microbiota is intricate and susceptible to multiple factors, with diet being a major contributor. The present study aimed to investigate the impact of four commonly used laboratory animal control diets, namely Keao Xieli's maintenance diet (KX), HFK's 1025 (HF), Research Diets' D12450B (RD), and Lab Diet's 5CC4 (LD), on the gut microbiota of mice. RESULTS: A total of 40 mice were randomly assigned to four groups, and each group was fed one of the four diets for a duration of 8 weeks. The assessment of gut microbiota was conducted using 16S rRNA sequencing both at the beginning of the study (week 0) and the end (week 8), which served as the baseline and endpoint samples, respectively. Following the 8-week feeding period, no significant differences were observed in physiological parameters, including body weight, visceral weight, and blood biochemical indices, across the four groups. Nonetheless, relative to the baseline, discernible alterations in the gut microbiota were observed in all groups, encompassing shifts in beta-diversity, hierarchical clustering, and key genera. Among the four diets, HF diet exhibited a significant influence on alpha-diversity, RD diet brought about notable changes in microbial composition at the phylum level, and LD diet demonstrated an interconnected co-occurrence network. Mantel analysis indicated no significant correlation between physiological parameters and gut microbiota in the four groups. CONCLUSION: Overall, our study demonstrated that the four control diets had a minimal impact on physiological parameters, while exerting a distinct influence on the gut microbiota after 8 weeks. © 2024 Society of Chemical Industry.

The grimace scale: a useful tool for assessing pain in laboratory animals.

Accurately and promptly assessing pain in experimental animals is extremely important to avoid unnecessary suffering of the animals and to enhance the reproducibility of experiments. This is a key concern for veterinarians, animal caretakers, and researchers from the perspectives of veterinary care and animal welfare. Various methods including ethology, immunohistochemistry, electrophysiology, and molecular biology are used for pain assessment. However, the grimace scale, which was developed by taking cues from interpreting pain through facial expressions of non-verbal infants, has become recognized as a very simple and practical method for objectively evaluating pain levels by scoring changes in an animal's expressions. This method, which was first implemented with mice approximately 10 years ago, is now being applied to various experimental animals and is widely used in research settings. This review focuses on the usability of the grimace scale from the "cage-side" perspective, aiming to make it a more user-friendly tool for those involved in animal experiments. Differences in facial expressions in response to pain in various animals, examples of applying the grimace scale, current automated analytical methods, and future prospects are discussed.

High-mortality epizootic Mycobacterium ulcerans ecovar Liflandii in a colony of Zaire Dwarf Clawed Frogs (Hymenochirus boettgeri).

Mycobacterium ulcerans ecovar Liflandii (MuLiflandii) was identified as the causative agent of mycobacteriosis in a research colony of Zaire dwarf clawed frogs (Hymenochirus boettgeri) at the University of Michigan. Clinical presentation included lethargy, generalized septicemia, cutaneous granulomas, coelomic effusion, and acute mortality. Identification of the mycobacterial species was based on molecular, microbiological, and histopathologic characteristics. These findings indicate that MuLiflandii is a primary cause of morbidity and mortality in Zaire dwarf clawed frogs and should be considered in the differential diagnosis of sepsis and coelomic effusion in amphibians. Mycobacterial speciation is important given the variability in pathogenesis within the family Mycobacteriaceae and the implications for both animal and human health as potential zoonoses. The Zaire dwarf clawed frog is a species common in the pet trade, and these findings provide consideration for this pathogen as a potentially important public health concern. This is the first report of MuLiflandii infection in the genus Hymenochirus and illustrates the diagnostic challenges of differentiating among both mycolactone-producing mycobacteria and Mycobacterium marinum. Furthermore, we demonstrate the utility of environmental sampling for this pathogen within the tank system, suggesting this mode of sampling could replace the need for direct frog surveillance.

Necropsy in Mouse Research.

Necropsy is the postmortem examination of an animal's body. Experiments on laboratory animals are performed to gather scientific data relevant to basic and, even more often, translational research, mainly in the field of translational medicine. A necropsy conducted on laboratory animals subjected to experimental research provides an opportunity to exhaustively explore pathological processes that took place during life. In order to achieve that goal, procedures of biomaterial collection should be performed timely, bearing in mind the inevitable process of postmortem tissue decay, and precisely, to avoid mechanical tissue damage.In this chapter, procedures of collecting organs and tissue in a way that ensures the preservation of the organ structure, tissue organization, morphological characteristics of cells, and last but not least, intracellular protein and nucleic acid content and chromosome organization are going to be described step by step.

Exploring the Use of Animal Models in Craniofacial Regenerative Medicine: A Narrative Review.

The craniofacial region contains skin, bones, cartilage, the temporomandibular joint (TMJ), teeth, periodontal tissues, mucosa, salivary glands, muscles, nerves, and blood vessels. Applying tissue engineering therapeutically helps replace lost tissues after trauma or cancer. Despite recent advances, it remains essential to standardize and validate the most appropriate animal models to effectively translate preclinical data to clinical situations. Therefore, this review focused on applying various animal models in craniofacial tissue engineering and regeneration. This research was based on PubMed, Scopus, and Google Scholar data available until January 2023. This study included only English-language publications describing animal models' application in craniofacial tissue engineering (in vivo and review studies). Study selection was based on evaluating titles, abstracts, and full texts. The total number of initial studies was 6454. Following the screening process, 295 articles remained on the final list. Numerous in vivo studies have shown that small and large animal models can benefit clinical conditions by assessing the efficacy and safety of new therapeutic interventions, devices, and biomaterials in animals with similar diseases/defects to humans. Different species' anatomical, physiologic, and biological features must be considered in developing innovative, reproducible, and discriminative experimental models to select an appropriate animal model for a specific tissue defect. As a result, understanding the parallels between human and veterinary medicine can benefit both fields.

A survey of the impact of COVID-19 on the management of animal experiments and laboratory animal facilities in Korea.

The pandemic has affected the lives of people all over the world. The effects of the pandemic on laboratory animal facilities and their operations through this unusual global event are poorly understood. Here, we have applied a methodological framework of qualitative approach including semi-structured interviews to investigate laboratory animal operations in Korea and how it has shaped the on-going management and laboratory operations of such facilities. A total of fifty-two individuals, including members and administrators of the Institutional Animal Care and Use Committee (IACUC), researchers, and animal facility managers and staff, were surveyed through purposeful sampling. Survey questions explored how the pandemic impacted the IACUC and the functioning of animal facilities, and what steps to take in preparation of a future pandemic-like crisis. Our survey found evidence of an increase in animal experiments in Korea during the pandemic that correlated with increases in research funding during that period, such as for vaccine development. Also, operational challenges due to pandemic-related health issues in personnel were resolved through overtime, rather than by reducing facility operations. Moreover, a refinement of post-approval monitoring (PAM) practices was also discussed by respondents. Taken together, our study offers insights into animal facility operations during the pandemic and outlines recommendations for safeguarding operations in such future scenarios.

Pain management in zebrafish : Report from a FELASA Working Group.

Empirical evidence suggests fishes meet the criteria for experiencing pain beyond a reasonable doubt and zebrafish are being increasingly used in studies of pain and nociception. Zebrafish are adopted across a wide range of experimental fields and their use is growing particularly in biomedical studies. Many laboratory procedures in zebrafish involve tissue damage and this may give rise to pain. Therefore, this FELASA Working Group reviewed the evidence for pain in zebrafish, the indicators used to assess pain and the impact of a range of drugs with pain-relieving properties. We report that there are several behavioural indicators that can be used to determine pain, including reduced activity, space use and distance travelled. Pain-relieving drugs prevent these responses, and we highlight the dose and administration route. To minimise or avoid pain, several refinements are suggested for common laboratory procedures. Finally, practical suggestions are made for the management and alleviation of pain in laboratory zebrafish, including recommendations for analgesia. Pain management is an important refinement in experimental animal use and so our report has the potential to improve zebrafish welfare during and after invasive procedures in laboratories across the globe.

Regular medical checkup program (in K-MEDI hub) to enhance the welfare of laboratory dogs and pigs.

BACKGROUND: The importance of animal welfare is being recognized worldwide. Recently, the increasing demand for enhanced laboratory animal welfare has led to clinically featured transformations of animal research institutes. This study aims to describe the process and findings of veterinary medical check-ups and its influence on laboratory dogs and pigs welfare. Regular medical checkups were conducted by the attending veterinarian twice a year to ensure the health and welfare of dogs and pigs in our animal research institute. Based on the findings from the medical checkup, we assessed the current health of dogs and pigs,providing reasonable treatments to prevent the risk of complications. RESULTS: Blood tests and physical examinations revealed clinically relevant findings. Some of these findings were due to insufficient postoperative care after invasive surgical experiments and the remaining were predictable side effects after surgical experiments. However, one finding involved severe gum bleeding due to retained deciduous teeth. This animal was euthanized because it was judged to reach the humane endpoint. Majority of the dogs and pigs at our animal research institute were considered to be healthy, based on the comprehensive results of the medical checkups. CONCLUSIONS: Regular medical checkups by the attending veterinarian established enhanced animal welfare, ensuring the accuracy and reproducibility of animal studies. This pioneering veterinary animal care program can serve as a potential advanced guideline for animal research institutes to improve dogs and pigs welfare.

Animal bite anaphylaxis, rarely diagnosed but underappreciated.

BACKGROUND: Animal allergies are common, with reactions ranging from rhinoconjunctivitis from respiratory exposure to anaphylaxis, usually from animal bites. Since animal bites are also common, this raises the question of how often anaphylaxis occurs following a bite. MATERIALS AND METHODS: A PubMed, Google, and Google Scholar literature review was performed using keywords such as animal bite anaphylaxis. An inquiry was made to the Anaphylaxis Registry ANAPHYLAXIE.net to see if additional cases were contained in the registry. RESULTS: Approximately 40 cases of animal bite anaphylaxis are described in the literature, mostly from rodent bites (mice, rats, hamsters, and guinea pigs). A survey of laboratory animal bite anaphylaxis in the U.S. identified previously unreported cases, suggesting that most cases are not reported. CONCLUSION: Anaphylaxis from animal bites is rarely reported, but occurs more frequently than suggested by case reports and should be considered in a symptomatic patient following a bite.

Determination of critical-sized defect of mandible in a rabbit model: Micro-computed tomography, and histological evaluation.

Objective: To evaluate a rabbit model of mandibular box-shaped defects created through an intraoral approach and determine the minimum size defect that would not spontaneously heal during the rabbit's natural life (or critical-sized defect, CSD). Methods: Forty-five 6-month-old rabbits were randomly divided into five defect size groups (nine each). Mandibular box-shaped defects of different sizes (4, 5, 6, 8, and 10 mm) were created in each hemimandible, with the same width and depth (3 and 2 mm, respectively). Four, 8, and 12 weeks post-surgery, three animals per group were euthanized. New bone formation was assessed using micro-computed tomography (MCT) and histomorphometric analyses. Results: Box-shaped defects were successfully created in the buccal region between the incisor area and the anterior part of the mental foramen in rabbit mandibles. Twelve weeks post-surgery, MCT analysis showed that the defects in the 4, 5, and 6 mm groups were filled with new bone, while those in the 8 and 10 mm groups remained underfilled. Quantitative analysis revealed that the bone mass recovery percentage in the 8 and 10 mm groups was significantly lower than that in the other groups (p < 0.05). There was no significant difference in the bone mass recovery percentage between the 8 and 10 mm groups (p > 0.05). Histomorphometric analysis indicated that the area of new bone formation in the 8 and 10 mm groups was significantly lower than that in the remaining groups (p < 0.05). There was no significant difference in the new bone area between the 8 and 10 mm groups (p > 0.05). Conclusions: The dimensions of box-shaped CSD created in the rabbit mandible through an intraoral approach were 8 mm × 3 mm × 2 mm. This model may provide a clinically relevant base for future tissue engineering efforts in the mandible.

The curative effect of ozonated bidistilled water on Escherichia coli-induced endometritis in rats.

BACKGROUND: Endometritis is a prevalent and challenging condition that affects livestock, and its effective treatment is the use of intrauterine antibiotics. Antibiotic use may lead to negative consequences, including residue in tissues and the emergence of antibiotic resistance. OBJECTIVES: The aim of this study was to assess the therapeutic potential of ozonated bidistilled water in the treatment of Escherichia coli-induced endometritis in rat models, comparing it to conventional antibiotic treatment. METHODS: A total of 21 female Wistar Albino rats were used for this study. E. coli from the uterus of cows with endometritis at a concentration of 1.0 × 109 cfu/mL inoculated into rat uteruses. After inoculation, it was confirmed macroscopically that experimental endometritis was induced in all individuals. And, the rats with endometritis were randomly divided into three groups treated with intrauterine bidistilled water enriched with ozone, antibiotics and 0.9% NaCl. The uterine tissues were examined histopathologically. RESULTS: In the semiquantitative analyses, significantly lower histopathological scores were observed for both the bidistilled water enriched with ozone and antibiotic-treated endometritis groups compared to the control group (p < 0.05). CONCLUSIONS: Ozone-enriched bidistilled water can be used in the treatment of experimentally induced endometritis in rats, and can provide effective microbiological and histopathological improvement. Further studies involving larger populations of different species are needed to determine the medical suitability of this new treatment.

CIRS-LAS - a novel approach to increase transparency in laboratory animal science for improving animal welfare by reducing laboratory animal distress.

The 3Rs principle is highly topical in animal-based research. These include, above all, new scientific methods for conducting experiments without an animal model, by using non-animal models (Replace), reducing the number of laboratory animals (Reduction) or taking measures to keep the stress on the laboratory animal as low as possible (Refinement). Despite numerous modern alternative approaches, the complete replacement of animal experiments is not yet possible. The exchange in the team about the daily work with laboratory animals, about open questions and problems, contributes to a reflection of one's own work and to a better understanding of the work of the others. CIRS-LAS (Critical Incident Reporting System in Laboratory Animal Science) represents a reporting system for incidents in laboratory animal science. It is urgently needed because the lack of transparency about incidents leads to the repetition of failed experiments. Negative experiences from animal-based experiments are often not mentioned in publications, and the fear of hostility is still very high. Therefore, a constructive approach to errors is not a matter of course. To overcome this barrier, CIRS-LAS was created as a web-based database. It addresses the areas of reduction and refinement of the 3Rs principle by providing a platform to collect and analyze incidents. CIRS-LAS is open to all individuals working with laboratory animals worldwide and currently exists with 303 registered members, 52 reports, and an average of 71 visitors per month. The development of CIRS-LAS shows, that an open and constructive error culture is difficult to establish. Nevertheless, the upload of a case report or the search in the database leads to an active reflection of critical occurrences. Thus, it is an important step towards more transparency in laboratory animal science. As expected, the collected events in the database concern different categories and animal species and are primarily reported by persons involved in an experiment. However, reliable conclusions about observed effects require further analysis and continuous collection of case reports. Looking at the development of CIRS-LAS, its high potential is shown in considering the 3Rs principle in daily scientific work.

Future of TABST and LABST in the Indian Pharmacopoeia Monographs A Humane Society International/India Workshop Report.

Humane Society International India (HSI India) organized and facilitated a workshop on the 'Future of Target Animal Batch Safety Test (TABST) and Laboratory Animal Batch Safety Test (LABST) in the Indian Pharmacopoeia (IP) Monographs'. The workshop hosted key Indian regulators from the Indian Pharmacopoeia Commission (IPC) and the Central Drugs Standard Control Organization (CDSCO), industry representatives from the Indian Federation of Animal Health Companies (INFAH), Asian Animal Health Association (AAHA), and international experts representing the European Directorate for the Quality of Medicines (EDQM), the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH), and multinational veterinary products manufacturers. The workshop was organized to encourage a bidirectional flow of information and to discuss the deletion of TABST and LABST from the veterinary vaccine monographs in the IP. This workshop was built from the symposium held by Humane Society International on the 'Global Harmonization of Vaccine Testing Requirements' held in 2019. This report details the outcomes of the workshop with proposed activities to be taken up as part of the next steps for the elimination or waiving of these tests.

Education in laboratory animal science and the 3Rs.

While laboratory animal research continues to be crucial for scientific and medical advancement, it still raises relevant ethical considerations. In order to foster public trust and support, all animal use must be relevant, responsible, competent and humane, and education and training of scientists in laboratory animal science (LAS) is vital to achieve these goals. However, education must be effective in generating meaningful learning and promoting a culture of competence, professionalism, accountability and transparency. With the ongoing technological and pedagogical revolution in education, LAS educators are adopting innovative educational practices, including e-learning modules, interactive simulations and virtual reality tools, to create and deliver inspirational educational experiences that are immersive, interactive, learner-centric and effective. Drawing from their expertise and experience, the authors of the articles included in this special edition bring forward new technologies and approaches, as well as novel perspectives to well-established concepts and methodologies, hopefully valuable contributions for better engagement and improved learning on LAS and the 3Rs.