Laboratory Animals

Rats play a crucial role in advancing scientific research and contributing to the understanding of various diseases. The striking structural similarity between the rat foot and the intricate anatomy of the human hand has drawn scientific interest to these animals. However, detailed morphometric and functional analyses of the adult Sprague–Dawley rat foot, including its dimensions, range of motion, and grip strength, remain limited in the literature. We aimed to examine the hind feet of 42 adult male Sprague–Dawley rats, each weighing 300–350 g. Using a multifaceted approach, we inspected hind paw morphology and performed descriptive statistical analyses of foot dimensions (a), measured unilateral hind foot grasping strength (b), assessed joint mobility (c), and examined the microscopic appearance of the hind foot third digit (d). One animal was additionally used to observe the hind limb skeletal architecture using microcomputed tomography. This study provides critical normative data regarding the structure and function of the hind foot in adult Sprague–Dawley rats, thereby facilitating future studies on musculoskeletal diseases.

This pilot study investigates whether trained behavioral tasks may serve as early, non-invasive indicators of distress in high-severity disease models in laboratory rats. Therefore, six Wistar rats in a diethylnitrosamine-induced hepatocellular carcinoma model, were trained to perform two tasks: lockbox solving and scale climbing. They were assessed over 18 weeks by body weight, hair corticosterone (hCORT), and Grimace Scale. Changes in task performance time and hCORT levels were observed and revealed a three-phase pattern. Initially, poor task performance (mean lockbox solving time 22 s) and elevated hCORT (mean 10.8 pg/mg) indicated a process of adaptation to the experiment. This was followed by improved performance (mean lockbox solving time 2 s) and reduced hCORT (mean 7.9 pg/mg), suggesting acclimatization. Later, performance declined again (mean lockbox solving time 9 s), accompanied by rising hCORT (mean 8.3 pg/mg), likely reflecting tumor progression and repeated anesthesia exposure. Lockbox solving appeared more variable than scale climbing. Acute stressors, such as room changes and prolonged anesthesia, had an impact on lockbox solving and scale climbing. After the initial housing room change lockbox solving time increased from 20 s to 49 s while scale climbing time rose from 12 s to 26 s. Case observations confirmed that task performance deficits coincided with illness or larger tumor burden. These findings support the use of trained behavior as a sensitive tool to detect distress. This approach could improve welfare monitoring in long-term or high-severity animal studies.

The chytrid fungus Batrachochytrium dendrobatidis (BD) poses a serious threat to amphibian species worldwide. BD infects keratinocytes of the skin and interferes with their physiological barrier function. The African clawed frog Xenopus laevis is a species with low susceptibility to BD. Nevertheless, any infection presents a challenge to the immune system and, when combined with other stressors, carries the risk of overwhelming the body’s defense. Infected animals may show greater variability in their scientific output than uninfected animals. This, in turn, means that more animals have to be used, which is not in line with the 3Rs principle. Moreover, releasing the fungus into the wastewater system should clearly be avoided to prevent infection of wild amphibians. Following confirmation of BD infection in the animals by Polymerase Chain Reaction (PCR), we decided to eradicate the fungus due to the reasons outlined above. This project involved not only the treatment of our frogs but also the timely coordinated disinfection of our entire permanently installed aquarium system. Itraconazole was applied as an immersion bath for the frogs and heat was used to disinfect the aquarium system. In this report, we outline our complex approach to a logistically demanding, labor-intensive project involving a large team of staff. We emphasize the need for straightforward planning, good communication and reporting on unexpected aspects and findings.

Animals used in research and testing face physical and mental challenges, which may cause mild to severe pain and distress. Pain and distress are generally considered in the context of the analogy postulate and the precautionary principle. The analogy postulate refers to (patho-)physiological similarities between humans and vertebrates, assuming that manipulations causing pain and distress in humans do so equally in vertebrates. According to the precautionary principle, animals will be considered to have pain/distress in the absence of any scientific consensus as to the presence or absence of pain. Although those principles can be useful guides for humane treatment of laboratory animals, they do not address the subjectivity involved in recognising pain/distress. A reliable estimate of animal pain and distress can be derived from both behavioural and physiological indicators. Distress and pain are biological phenomena that motivate behaviours aimed at alleviating these states. If no behaviour achieves the expected outcome, the animal may switch to other modes, such as depression or learned helplessness. Pain is regarded as a biologically adaptive mechanism, alerting organisms to possible damage or injury. The effect of experimental procedures on perceived pain/distress may depend on physical and social environmental conditions. Observation schemes can be used to assess and monitor daily for clinical signs or problems. They can help reduce pain/distress and provide adequate care. Competent observation of animals is also basic to the correct implementation of humane endpoints. AI will transform research. Intelligent systems will generate data, enable early stress detection and improve animal welfare.

Although overeating is the root cause of many diseases and even premature death in laboratory animals, ad libitum feeding is still the prevailing practice in rodent husbandry. There is a need for practical methods of food restriction compatible with group housing. The diet board was developed as a mechanical solution to food restriction of group-housed rats. It tightly embeds food pellets in grooves cut into aspen boards, forcing the rats to gnaw wood to reach their food. In this two-year experiment, 146 nine-week-old outbred Hsd:Sprague Dawley^® rats were divided into four equal groups: diet board-fed males and females, and ad libitum-fed males and females. Blood samples for clinical chemistry were collected at regular intervals during the study, and times of spontaneous death or euthanasia were recorded. Triglyceride and cholesterol values were significantly lower in diet board males than ad libitum males. The restriction of food intake via a diet board significantly increased the 24-month survival rate for both female and male groups. The better observed survival rates amongst diet board-fed rats makes it possible to start long-term studies with far fewer rats than with ad libitum feeding. Thus, using a diet board appears to be a promising method for the reduction of animal numbers in long-term rat studies that is compatible with group housing.

Complete Freund’s adjuvant (CFA) is used in laboratory animals to stimulate strong immune responses. However, it can cause persistent local inflammation and tissue damage, raising welfare concerns. This scoping review aimed to (i) assess how frequently welfare-relevant outcomes are reported in CFA immunisation and antibody-production studies in mice, rats and rabbits; (ii) describe reported welfare-relevant outcomes and the methods used to assess them; (iii) chart CFA regimen details across studies; and (iv) summarise reported alternatives to CFA, including welfare comparisons where available. We followed a protocol registered in Systematic Reviews for Animals and Food and reported in accordance with PRISMA-ScR. We searched PubMed and Embase from 2015 onwards. Screening was conducted in two phases (title/abstract; full text) by one reviewer, with uncertainties resolved by a second reviewer. Data were charted on animal characteristics, CFA regimen details, welfare monitoring, reported local/systemic outcomes, analgesia/humane endpoints and comparator adjuvants. Of 1608 records screened, 689 were assessed at full-text level and 43 met inclusion criteria by reporting a welfare-relevant outcome (including ‘no adverse effects’): local outcomes were reported in 24/43 and systemic in 8/43; structured pain or behaviour monitoring occurred in 4/43 and formal scoring in 1/43 studies. Alternatives were evaluated in 32/43; in studies involving head-to-head comparisons with CFA (28/32), alternatives were more often better tolerated (22/28) than similar (6/28), with efficacy usually comparable or higher (31/32). Despite well-recognised welfare concerns with CFA, welfare-relevant outcomes are rarely reported. Standardised reporting of CFA regimens, welfare assessment methods and results is needed to support evidence-based refinement and comparison with alternatives.

Breast cancer is the most common cancer in women worldwide, underscoring the need for ethical and effective preclinical models to advance disease management. This study aimed to highlight the importance of humane endpoints in a rat model of mammary carcinogenesis undergoing ladder resistance training to evaluate and ensure the absence of animal suffering. Twenty-eight female Wistar rats were assigned randomly to four groups of seven animals per group: sedentary, sedentary induced (with N-methyl-N-nitrosourea (MNU)), exercised and exercised MNU-induced. At 7 weeks of age, the animals in the induced groups were injected intraperitoneally (i.p.) with the carcinogenic agent (N-methyl-N-nitrosourea; i.p; 50 mg/kg). The animals’ welfare was monitored using a set of biological characteristics. The resistance exercise training, started at 9 weeks old and comprised climbing a ladder three times per week for 18 weeks, gradually increasing the load throughout the protocol. Of 14 MNU-induced rats, 9 (64%) developed mammary cancer. Exercised rats tended to show delayed tumor ulceration despite larger tumor volumes. No significant differences in bodyweight were observed, although weight loss appeared earlier in sedentary-induced animals. Exercised rats exhibited more stress signs, including reduced grooming and one case of chromodachryorrhea, yet had lower tumor-related humane endpoint scores. Three rats were euthanised early due to tumor-related endpoints. Minor tail wounds occurred but did not affect exercise or welfare. These results indicate that, with careful endpoint monitoring, the exercise protocol is well tolerated and may improve performance in tumour-bearing rats, supporting ethical standards while allowing accurate investigation of resistance training effects.

The use of animals as a model for humans has a long history. This paper describes in a bird’s eye view the main developments from the ancient period until recent times. Although animal models have been used since the early Greek and Roman civilizations, the main increase in their use dates from the end of the Second World War. Not only did the number of animals used in experiments increase dramatically in this period, but also the variety of species, as well as the purposes for which animals were used. However, from the 1970s onwards, the number of animal experiments has decreased. The reasons for this decrease are briefly explored.

The implementation of the 3R principles (Replace, Reduce, Refine) requires a comprehensive understanding of the various levels at which they operate, as well as the factors that may hinder or support their advancement. Central to this process is incorporating the perspectives of stakeholders in biomedical research to develop practical and relevant strategies for action. In a qualitative study, we conducted 35 semi-structured interviews with Swiss researchers working on the 3Rs or animal experimentation. Interviews were analysed following a reflexive thematic analysis, and we identified two categories of facilitation: originative facilitation, which concerns epistemic aspects of the 3Rs, and executive facilitation, which concerns interventionist aspects of the 3Rs. In this article, we report on the former dimension, which can be further divided into the conceptualisation of the 3Rs on the one hand, and the education of researchers on the other hand. Although grounded in the Swiss context, these findings reveal key insights for researchers, policymakers, and institutional stakeholders internationally. As efforts to align animal research practices with ethical standards continue to grow globally, understanding how the 3Rs are conceptualised and taught is essential for creating coherent, cross-border strategies that foster responsible scientific practice. Clarifying definitions and reinforcing researcher education are critical steps in ensuring the consistent and meaningful implementation of the 3Rs across national boundaries.

Temporomandibular disorders affect about 12% of the industrialized population, and their complex management often results in recurrence or treatment failure. Among animal models, the pig is particularly relevant owing to its anatomical similarity to humans, although specific differences in the zygomatic arch and mandibular fossa require adapted surgical approaches. We describe and validate a standardized surgical approach to the porcine temporomandibular joint (TMJ) suitable for translational research. Eleven cadavers of Sus scrofa domestica were dissected to identify key anatomical structures. A high condylectomy was performed to establish a reproducible surgical pathway, and the caudal intra-articular injection technique was tested using methylene blue to assess its feasibility. The pig’s TMJ area consists of superficial muscles such as the platysma and parotidoauricularis, with deeper structures such as the parotid gland, facial nerve and major vessels, while the bony anatomy includes a large condylar head and a superficial zygomatic arch. The proposed surgical approach to the pig TMJ involves a retromandibular incision with preauricular extension, subperiostal dissection to expose the zygomatic arch and articular capsule, followed by an incision to access the condyle, disc and mandibular fossa, allowing for condylectomy with careful preservation of the facial nerve and surrounding structures. This study highlights the anatomical differences with humans, such as the presence of the parotidoauricularis muscle, which lies superficially to the facial nerve and serves as a useful landmark for a safe dissection. The described approach provides a reliable and reproducible technique for future TMJ surgical studies in the pig model.

Professionals engaged in animal experimentation within laboratory animal facilities face a range of practical hardships, including emotional distress, professional stress, ethical conflicts, and work environment-related challenges. To address these issues, it is crucial to systematically identify the psychological and emotional burdens, as well as the specific stressors, experienced by individuals in this field. This study examines the ethical conflicts and compassion fatigue encountered by personnel working with laboratory animals in Korea. An online survey was conducted with 165 participants, including animal researchers (laboratory technicians, postdoctoral researchers, and graduate students), principal investigators, animal facility managers, and attending veterinarians. The results indicated that animal researchers experienced lower levels of stress and fewer ethical dilemmas than other occupational groups, including principal investigators, attending veterinarians, and animal facility managers. Female respondents reported a higher frequency of ethical dilemmas than male respondents, regardless of occupational role. Participants with less than six years of work experience reported fewer ethical dilemmas than those with longer tenure, suggesting that ethical sensitivity might increase with prolonged exposure to animal experimentation. Euthanasia and animal pain were identified as the primary sources of ethical stress. Multivariate logistic regression analysis revealed that experiences of ethical dilemmas, years of employment, and occupational role were significantly associated with stress levels. These findings underscore the need for organizational-level interventions to mitigate ethical stress and promote a healthier research environment. Strengthening emotional resilience and supporting the overall well-being of personnel working with laboratory animals are essential goals of such efforts.

Successfully implementing the 3R principles relies on a range of interdependent factors operating across multiple levels. As such, any effort to advance the 3Rs must engage with this complexity and incorporate the lived experiences of stakeholders to inform policy advancements. In an interview-based study, we gathered insights from 35 Swiss researchers on the implementation of the 3Rs in Switzerland. Using reflexive thematic analysis, we identified two overarching categories of facilitation: originative facilitation, referring to the collective meaning-making of the 3Rs, and executive facilitation, encompassing politico-institutional conditions that support their implementation. The present paper focusses on the latter domain, in which key themes include political support fostering a pro-3R research environment, the cultivation of a 3R culture within research institutions, clearer 3R guidance from cantonal commissions on animal experimentation, and strategic use of the Swiss 3R Competence Centre as a leading national 3R intermediary. These themes detail major institutional stakeholders that could further promote the implementation of the 3Rs from the perspective of researchers. Accordingly, the results showed that advancing the 3Rs is not just a matter of niche innovations but should also be orchestrated from a broader institutional perspective and with specific objectives to achieve. While these findings are specific to the Swiss context, they provide valuable information for countries with similar regulations on animal experimentation or a similar political structure.

Transparent reporting of animal research is both a scientific and ethical imperative and is linked to both research quality and the principles of Replacement, Reduction, and Refinement (3Rs). Persistent concerns about poor reproducibility, limited translation and research waste have driven reforms in reporting standards and data-sharing requirements. However, despite considerable progress over the past 60 years, substantial gaps remain. Many published studies still lack essential methodological detail, such as animal characteristics, welfare measures, bias minimization methods (such as randomization and blinding), and sample size justification. Non-reporting of negative or null results further distorts the evidence base. A major barrier to change has been inadequate training of researchers in best practices for experimental design and statistical analysis. We describe how standardized reporting frameworks can promote research quality and highlight tools available to assist the researcher in implementing best practices in study design, reporting, and data accessibility. Over the next decade, emerging applications such as automated compliance tools and AI-assisted screening will offer scalable approaches to adoption of reporting standards. Preclinical science itself will be profoundly altered by advances in alternative models, open data, and linked machine-readable research outputs that will shift the research culture from isolated studies to an integrated cumulative evidence ecosystem. However, an expanded evidence base will be of value only if methodological quality is prioritized over novelty. Embedding best practices at study inception coupled with transparency will enhance reproducibility, reduce waste, and strengthen the reliability and translational value of animal research.

Staphylococcus aureus nasal carriage is considered a risk factor for infections, and the development of nasal decolonization strategies is highly relevant. Despite they are not naturally colonized by Staphylococcus, mice are a good model for S. aureus nasal colonization. Murine models are easy to manipulate, and inter-laboratory reproducibility makes them suitable for nasal colonization studies. Strategies using bioluminescent bacteria allow for the monitoring of infection over time without the need to sacrifice animals for bacterial quantification. In this study, we evaluated S. aureus nasal colonization in three mouse strains (BALB/c, C57BL/6, and Swiss Webster) using a bioluminescent strain (SAP231). In vitro, a visible Bioluminescent Signal Emission (BLSE) was observed until 10⁶ bacteria and detected by IVIS® imaging system up to 10⁴ cells. Animals were inoculated with one or two doses of approximately 10⁹ colony-forming units (CFU) of SAP231. Swiss Webster mice showed the longest colonization time, with some animals presenting BLSE for up to 140 h. In addition, BLSE was higher in this strain. BALB/c and C57BL/6 strains showed consistent BLSE results for 48 h. BLSE intensity was higher in Swiss Webster inoculated with both doses. Three different positions for image capture were evaluated, with better results for the lateral and ventrodorsal positions. After the loss of BLSE, bacterial quantification was performed, and Swiss Webster mice presented more bacteria in the nasal cavity (approximately 10⁵ CFU) than the other strains. Our results demonstrate that bioluminescent S. aureus allow monitoring of nasal colonization and estimation of the bacterial burden present in live animals until 48 h.

Health monitoring is essential for ensuring animal health and reliable research results. Each animal facility should establish adequate health monitoring methods, and microbiological quality control should be implemented through regular health surveillance. Recently, specific pathogen free (SPF) mice have been housed in individually ventilated cage (IVC) racks in the majority of mouse facilities globally, and health monitoring is implemented using a soiled bedding sentinel (SBS). Even though SBS monitoring is a standard method, it has a limitation in that some pathogens are not sufficiently transmitted to the sentinel housed in the IVC. The exhaust air dust polymerase chain reaction (EAD PCR) method has been reported to be a reliable complementary method to SBS monitoring based on research findings. In Korea, health monitoring programs using EAD PCR have not yet been applied to laboratory animal facilities. The microbiological status of mouse colonies housed in the two IVC racks was compared using SBS and EAD PCR monitoring in our SPF mouse facility. Except for Helicobacter spp. and Staphylococcus aureus, the detection of 16 pathogens did not differ between the two methods. In the detection of Helicobacter spp., EAD PCR was found to be more sensitive than SBS. Helicobacter spp. were not found by SBS, whereas four S. aureus positive samples were detected by either SBS or EAD PCR test. According to our findings, EAD PCR can be used as a supplement to SBS monitoring. Moreover, EAD PCR can reduce the number of animals used, making it a 3R (Replacement, Reduction, Refinement)-consistent method.

An integrated rearing system for Cantareus aspersus under environmental conditions in an experimental orchard is proposed. In this study, the natural behaviour, circadian rhythms and suitable rearing conditions of the edible snail were optimised to produce homogeneous growth and low variability. The growth was standardised, and growth pattern fit was assessed with various models. One thousand fry were cultured in the orchard, and a random sample of 100 snails were measured weekly for 23 weeks. The rearing system had the following characteristics: snails and earthworms were included in the experimental rearing orchard; a homogeneous group of juvenile snails of the same age and size and high rearing density (500 snails/m²) was used; snails were fed with layers mash ad libitum; and mixed and fringes vegetation was planted in the orchard. A commercial size of 60% of snails was achieved in 21 weeks and 95% in 23 weeks. The different models showed a good fit, and the quadratic model obtained the best fit. This experimental snail orchard proposal can be extended to other areas, although it must be corrected according to different environmental conditions and fit to other species of interest. This experimental model could constitute a viable alternative to traditional models of animal experimentation with mammals, and given its high adaptability, it could be applied in different fields of science.

Students and course providers have found online options for laboratory animal science (LAS) courses helpful because the content is accessible with flexible timing options. This study aimed to explore whether a blended LAS course with video lectures would be preferred and perceived effective, accounting for students’ educational levels as well as prior experiences and future expectations in the use of animals. Data were collected by a feedback survey including three 5-Likert type scales and open-ended questions created by the authors. Of the 134 course students from various programmes, 101 consented to the use of their responses in this study. The analyses indicated that the respondents were generally satisfied with the blended course, especially the hands-on components, even though some found video lectures challenging due to their own ineffective time and study management skills. Plans to use animals in the future increased satisfaction with the course significantly, while the education level or previous experience with animals did not affect the outcome. Background variables did not affect views about video lectures significantly. Conclusively, the blended structure seemed to provide sustainable LAS course experiences for normal and unpredictable times.

Effective multimodal analgesia techniques are required when pigs are used as models in orthopedic human research. Regional anesthesia is a widely used technique to provide perioperative analgesia in animals undergoing orthopedic surgery. The brachial plexus (BP) block is indicated to desensitize the forelimb in many species but has not been yet described in pigs. The main aims of this study were to develop an ultrasound-guided axillary approach for the BP and to evaluate injectate spread and nerve staining in pig cadavers. Eight fresh F1 cross breed German Large White and German Landrace male pig cadavers were enrolled. Two cadavers were used for anatomical dissection of the axillary space and to determine the disposition of the BP. Six cadavers were used to perform a bilateral axillary ultrasound-guided approach for the BP, and after injecting 0.3 ml/kg of a solution of 2% lidocaine and new methylene blue (L-NMB), these were dissected to determine injectate spread and nerve staining. Upon dissection, the BP was observed in all the cases surrounded by the axillary sheath and in close proximity to the axillary artery. Ultrasonographic scanning and guidance for the approach to the BP was feasible in all cadavers and upon dissection, all the nerves forming the BP were stained in all their quadrants and in all the cases. In conclusion, the injection of 0.3 ml/kg of L-NMB through an ultrasound-guided axillary approach to the BP was feasible and adequate to entirely stain the BP in all pig cadavers. Further clinical studies are required to evaluate the effectiveness of this technique in live animals.

The term ‘culture of care’ in the context of using animals for scientific purpose describes the culture in organisations that provides support to staff to strive for continuous improvement in:

• animal care and welfare;

• support and recognition of staff involved in the animal care and use programme;

• scientific quality;

• openness and transparency.

We developed a systematic process for reporting observations and events that have the potential to help with continuous learning, improving animal welfare and supporting staff. The process took learning from the safety, health and environment arena on accident prevention. The two key aspects were (a) the systematic logging of observations and events; and (b) the learning approach to following up on observations. Underpinning our systematic process is the ‘Learning from Observations and Events Log’. Reported observations and events can relate to positive practices, general observations as well as near misses.

We created an environment to promote continuous improvement for both animals and staff by recognising, rewarding and sharing good practice, as well as where near misses are openly reported and learnt from. Supporting animal welfare, staff welfare, improving scientific quality and transparency are the four key pillars of a positive culture of care.

We recognised early on that using a system and learning approach to follow up on observations and events rather than a people and blame approach was key to developing open reporting and a positive culture. In the systems approach, errors are consequences rather than causes, having their origins in systemic factors.