Laboratory Animals

Genetically engineered pigs are essential donors for xenotransplantation, requiring phenotypic stability and genetic definition. We evaluated the newly established GGTA1-knock-out “XENO” line, maintained as a closed herd for >10 generations, against Massachusetts General Hospital (MGH) miniature swine, commercial Landrace (LR), and Yorkshire × Landrace (Y × L) populations. One hundred and thirty-nine pigs were genotyped using an 80K SNP BeadChip. Morphometric monitoring at 18 months showed no significant differences in body weight, length, height, or heart girth between XENO and MGH pigs (all p > 0.05). Principal component and phylogenetic analyses separated the four groups into distinct genetic clusters, confirming the uniqueness of the XENO line. Chromosome-wide linkage disequilibrium was markedly higher in XENO (initial r² > 0.8; half-decay ≈ 50 kb) than in commercial lines, reflecting intensive inbreeding. Linkage disequilibrium-derived historical effective population size (N_e) in XENO was approximately 1.5-fold lower than in LR/Y × L, but comparable to MGH. ADMIXTURE analysis supported K = 3 ancestral components with <2% introgression into XENO. These findings demonstrate that closed-herd management preserves phenotypic uniformity while establishing a genetically homogeneous, independent donor line. XENO pigs exhibited overlapping growth trajectories with MGH animals, providing a genomically stable resource for preclinical xenotransplantation.

The design of many shelters for laboratory mice, particularly plastic shelters, is mainly driven by economic, ergonomic and hygiene considerations, with little focus on providing the animals with opportunities to express species-specific behaviours and control over their environment. Even in the presence of artificial shelters (plastic houses, tunnels), laboratory mice will readily burrow if given the opportunity. The main aim of this study was to gain a better understanding of the importance of access to sheltered space for laboratory mice. We pair-housed mice (BALB/c, SWISS and C57BL/6, male and female) in conventional laboratory cages that contained either (i) resources to build underground shelter (burrowing substrate) or (ii) artificial structures providing shelter (e.g. red plastic house). We assessed how mice used these resources, as well as their preferences when given a choice between different shelters. In addition, we assessed the insulating properties of different shelters. Mice spent most of their time underground in deep bedding and the least time inside the standard red plastic house, where they nested outside the house. They also showed a strong preference for the deep burrow cage, and the most relevant features of preferred shelters seem to be darkness and being underground. Nesting material alone was also less insulating, possibly contributing to an increased need for non-shivering thermogenesis.

Assessment of practical skills is a key component in ensuring competence in laboratory animal science (LAS), particularly for practitioners performing functions A, C and D as defined in the Directive 2010/63/EU, namely those who carry out procedures on animals, care for them and perform humane killing, respectively. To address the need for a harmonised, objective and transparent approach to assessing practical competence, Working Group 4 (WG4) of the Education and Training Platform for Laboratory Animal Science (ETPLAS) has developed a set of Direct Observation of Procedural Skills (DOPS) tools. These tools are tailored specifically to evaluate the intended learning outcomes of Modules 3.2, 6.2, 8 and 20–22 of the European Union (EU) Education and Training Framework. This report outlines the rationale, structure and guidance for implementing the DOPS tools. Based on Miller’s pyramid and established educational principles, DOPS tools are suitable for formative and summative assessments in simulated or real environments. They can be used both formatively to monitor progress during training and summatively to support end-of-training decisions about competence. Their implementation is crucial to supporting harmonised training, facilitating mutual recognition of competence and ultimately advancing both animal welfare and the quality of scientific research in the EU and beyond. The DOPS forms are available as supplemental material to this article and from the ETPLAS website.

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.

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.

This study compared the macroscopic adhesion scoring performance of large language models (LLMs: ChatGPT-o3, ChatGPT-5, Gemini-2.5 Pro) with that of novice veterinary surgeons, using expert consensus as the reference. Eighty standardized postoperative laparotomy cases in Wistar rats were photographed and scored using the Nair 0–4 adhesion scale. Two novice surgeons and three LLMs independently evaluated each case; the expert reference was defined by a surgeon and a pathologist. Group differences were analyzed using the Kruskal–Wallis test with Dunn–Bonferroni post hoc comparisons, correlations by Bonferroni-adjusted Spearman coefficients, human interobserver reliability by intraclass correlation coefficient (ICC) (A,1), and agreement with the expert by quadratic-weighted Cohen’s κ and exact-match accuracy. Overall differences were significant. ChatGPT-o3, ChatGPT-5, Gemini-2.5 Pro, and Novice 1 assigned lower scores, while Novice 2 assigned higher scores. Correlations with the expert were significant for Novice 1 (ρ = 0.706), Novice 2 (ρ = 0.593), and ChatGPT-o3 (ρ = 0.617), but not for ChatGPT-5 or Gemini-2.5 Pro. Inter-observer reliability among human raters was moderate (ICC = 0.55). Importantly, absolute exact-match accuracies were modest across all evaluators, with the highest accuracy observed for Novice 1 (33.8%) and ⩽26.3% for the LLMs. While novices outperformed the models, these findings highlight the intrinsic difficulty of fine-grained Nair 0–4 adhesion scoring on two-dimensional intraoperative images and indicate that current LLMs are better suited as calibrated decision-support tools rather than stand-alone raters.

The involvement of non-scientific staff in discussions about animal welfare and scientific quality is essential for biomedical research progress. In this study, we developed a survey to collect the self-perception of animal care staff (ACS) and laboratory technicians about their involvement in scientific planning and conduct. Participants were contacted to complete an anonymous online questionnaire. We obtained 850 responses, mainly from Europe: 564 from ACS and 286 from laboratory technicians. Job satisfaction was assessed as positive by ACS and laboratory technicians despite the low frequency of culture of care activities and mental health meetings. Both groups expressed their desire to be trained in research planning and conduct; however, regular training was not reported. In addition, the inability to act on animal welfare concerns owing to experimental reasons was reported by both groups. Over half of the participants felt valued and appreciated by the lead scientists or animal facility manager; however, it is not clear how they are acknowledged, as their names on the authors list or in the manuscript acknowledgments are barely included. Our results indicated that involvement of ACS and laboratory technicians in planning and conducting studies would improve their understanding of how experiments are done, and therefore communication processes, work satisfaction, animal welfare, and scientific quality. Finally, we provided recommendations to improve the engagement of ACS and laboratory technicians in discussions about animal research planning and conduct.

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.

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.

The 18th National Congress of the Spanish Society for Laboratory Animal Science (SECAL), held in Bilbao from 18 to 21 November 2025, brought together approximately 400 professionals from across the laboratory animal science community. The meeting featured a comprehensive and high-quality scientific programme addressing key topics such as biosafety, occupational health, the use of aquatic models, technological innovation and the implementation of the 3Rs (Replacement, Reduction and Refinement) principles. The congress combined plenary sessions, workshops, oral and poster presentations and round-table discussions, fostering knowledge exchange and professional interaction. A dedicated session on the translation of animal research to clinical applications provided an interdisciplinary perspective involving scientists, welfare experts and patient representatives. The event highlighted the continued commitment of the community to scientific excellence, ethical responsibility and innovation, reinforcing the role of the SECAL Congress as a leading national forum in laboratory animal science.

Live cephalopods used in scientific research are protected under European Directive 2010/63/EU due to growing evidence that adult cephalopods can experience pain. This protection also extends to paralarval Octopus vulgaris, despite the absence of evidence that these early life stages are capable of nociception or pain. Consequently, the question arises whether welfare protection should be applied as a precaution when evidence of sentience is lacking, or only once it has been empirically demonstrated. Ethical reasoning under such uncertainty is influenced by views about sentience, scientific necessity, and societal relevance, and prior work suggests these views differ between scientists and laypeople. However, it remains unclear how these considerations combine when evidence for sentience is incomplete. This study investigated what influences ethical reasoning about the use of O. vulgaris paralarvae in research. An online survey (N = 205) presented cephalopod biologists, other biologists, and non-biologists with one of three scenarios describing a gene expression experiment on O. vulgaris paralarvae in which nociception-related genes were expressed, not expressed, or not yet investigated. The provided scientific information did not significantly affect ethical judgments, but beliefs about pain capacity, disciplinary background, and gender did. These findings suggest that ethical evaluations in the context of uncertain biological evidence are driven more by prior beliefs, disciplinary background, and gender than by scientific information and familiarity with research ethics. This underscores the need for more accessible science communication, improved ethical engagement, and more objective evaluation of ethics education to support well-informed and inclusive decision-making in animal research governance.

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.

Various studies have documented psychological strain in laboratory animal professionals (LAPs). However, reliable data on relevant stressors remain limited. This study aimed to assess potential stressors experienced by LAPs in German-speaking countries and analyse their impact on psychological strain. An online survey was conducted in summer 2023, assessing the participants’ mental strain using the General Health Questionnaire (GHQ-12), common general stressors using the Copenhagen Psychosocial Questionnaire (COPSOQ), as well as potential job-specific stressors. The final sample included 932 anonymous LAP participants. Data were analysed descriptively and using hierarchical regression analysis. Tasks like the euthanasia of surplus animals or being unable to alleviate animal distress due to scientific needs emerged as job-specific stressors. However, the results of the hierarchical regression indicated that general stressors, such as work–privacy conflict, physical and emotional demands, the need to hide emotions, and the degree of one’s own influence at work, explained at least as much of the variance in psychological strain as the specific stressors. In addition, work experience, function, and gender significantly influenced the GHQ score. The results from this study could contribute to fostering a culture of care by developing general protection means for the psychological hazards in the workplaces of LAPs, thus reducing their adverse effects on personnel, laboratory animals and research results.

Magnesium regulates myocardial depolarisation, vascular tone, neuromuscular function and provides analgesia. This study examined its analgesic and haemodynamic effects, comparing the effects of intravenous saline 0.9% (NaCl; control n = 8) and magnesium (n = 7) infusions in minipigs undergoing thoracotomy. Preoperative single-housed activity was recorded for 1 h. Intraoperatively, arterial blood pressure (BP) and heart rate (HR) were recorded once telemetry was established; data from the final 60 min of anaesthesia were compared between groups. Postoperative activity, HR and BP were recorded continuously using in-pen CCTV and telemetry, respectively. Indicators of pain, including HR and BP were sampled after tracheal extubation (T_TE) at +0.5, +2.5, +4.5, +6, +8.5, +15.5, +17.5, +19.5, +20, +23, +25.5, +26 and +29 h. Changes in HR and BP before and after analgesia at T_TE +6, +17.5, +23 and +29 h, and pre- and post-pain assessments at T_TE +2.5, +6, +17.5, +20, +23, +26 and +29 h were compared using a paired t-test. During anaesthesia, no electrocardiography derangements were noted. There was no difference between groups in the area under the curve for any cardiovascular variable. Magnesium-treated animals had an increased incidence of hypotension during anaesthesia. Postoperatively, activity decreased significantly for both groups for duration (all time points) and frequency (T_TE +0.5, +4.5, +8.5, +15.5 h), but no treatment effect was observed. Pain scores were comparable between groups. Following pain assessment and analgesia administration at T_TE +17.5, Group NaCl had significant increases in HR and mean arterial pressure The magnesium dose evaluated produced no harm nor obvious benefit.

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.

Recommendations for safe anaesthesia are common in medical practice, have been formulated for traditional laboratory species, i.e. rodents, but do not exist for laboratory pigs, sheep, goats and cattle. The guidelines presented here were commissioned by the Federation of European Laboratory Animal Science Associations (FELASA) and serve to underscore EU Directive 2010/63/EU (Article 14) which require that, ‘procedures are carried out with general or local anaesthesia and analgesia or another appropriate method to ensure pain, suffering and distress are kept to a minimum’. The guidelines are based on a review of: (i) medical and veterinary medical guidelines promoting safe anaesthetic practice; (ii) scientific literature pertaining to anaesthesia and analgesia in pigs, sheep, goats and cattle; and (iii) a consideration of the ethical, legal and scientific requirements when anaesthetizing laboratory animals. The guidelines aim to make recommendations for the provision of safe, practical and effective anaesthesia and analgesia to laboratory pigs and ruminants. Recommended standards for sedation and restraint (I), general principles of anaesthesia (II), monitoring anaesthesia (III) and pain assessment (IV) in the same species have been described by this working group (WG) and are set out in four parts in this document.

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.

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.