The EU Directive 2010/63 on the protection of animals used for scientific purposes, as well as the Swiss Animal Welfare Legislation, demand monitoring and documentation of specific aspects of an animal experiment, including welfare-related issues and the (retrospective) assessment of the severity of the procedures that the animals underwent. A score sheet is an efficient tool for the evaluation of the burden of an animal during an experiment and, if properly designed and used, helps adhere to the 3Rs principle. It must be adapted to the specifics of each experiment and explicitly conceived for it. Several score sheet examples have been published; however, some contain fundamental flaws or are designed for specific settings only, requiring modifications to fit other experimental designs. This paper suggests an eight-step procedure to design a score sheet that can be adapted to any animal species and experimental conditions.

The use of Vascular Access Buttons™ (VABs) is gaining traction in animal research as a less invasive alternative to traditional venipuncture methods and a superior option compared with existing alternatives, such as vascular access ports (VAPs). By enabling repeated blood collection and intravenous drug administration with reduced stress and discomfort for the animals, VABs contribute to improved data quality and enhanced animal welfare in animal studies. While increasingly used in rodents, their application in non-rodent species remains underexplored. This study evaluated the feasibility and reliability of the VAB system in two Göttingen Minipigs, comparing its performance with the conventional vena cava cranialis puncture. Pharmacokinetic assessments and clinical pathology analyses revealed consistent results across both techniques, demonstrating the VAB system's ability to generate reproducible, high-quality data. Additionally, its durability and ease of use highlight its potential as a practical and ethical alternative to both traditional venipunctures and VAPs in pharmacokinetic and long-term studies in minipigs. These findings support the integration of the VAB system in toxicological and pharmacokinetic research, particularly in studies requiring repeated blood collection or chronic intravenous dosing.

Laboratory animal welfare has received increasing attention in recent years as housing protocols move toward favoring environments that allow natural behaviors. Within this study, the effects of housing male and female Sprague Dawley rats in standard cages versus taller cages with an upper shelf were investigated. To determine differences in behavior and physiology based upon cage type, home-cage assessment of ultrasonic vocalizations and analysis of fecal corticosterone metabolites, as well as various behavioral tests, were performed. Rats in shelved cages produced significantly less 50 kHz calls, demonstrated better working memory in the spontaneous alternation task and had higher concentrations of fecal corticosterone metabolites. No differences were observed in the open field, elevated plus maze or light-dark box. While no significant treatment differences were found in the ultrasonic vocalization playback paradigm, results confirmed previous evidence of approach behavior upon 50 kHz call playback. The observed differences in behavior and physiology as a consequence of housing conditions inevitably have implications for experimental reproducibility as comparing studies across laboratories may be difficult if different housing parameters are utilized. The results of this study can also be used in guiding future animal welfare protocols given that certain cage modifications such as increased vertical space and/or the presence of a shelf might improve welfare. Investigation of additional parameters and strains of rodents will enhance our understanding of optimal laboratory animal housing conditions.

The aim of this experimental, descriptive study was to evaluate feasibility, safety and side effects of alfaxalone and midazolam by intranasal instillation for anaesthesia induction in rabbits. We included 26, healthy, female New Zealand White Rabbits undergoing general anaesthesia in context of a study to test different coatings for stifle joint endoprosthesis. Midazolam (0.1 mg/kg) and alfaxalone 3 mg/kg (group 1) or 4 mg/kg (group 2) were mixed and administered intranasally. The number of sneezes, swallows and evasive attempts were recorded. Time to lateral recumbency, presence of salivation, nystagmus, induction and intubation qualities were scored. If intubation was not possible, a top-up of 1 mg/kg alfaxalone was administered intranasally. If still not sufficient, anaesthesia was induced by mask-insufflation of isoflurane. Data were analysed using IBM SPSS Statistics 30.0.0.0 and non-parametric data compared using either a Mann-Whitney test or a chi-square test. Overall, 10 animals assigned to group 1 and 16 animals to group 2 were included in the study. In 24/26 rabbits (92.3%) no significant complications were noted. One rabbit showed 20 s of apnoea after induction and one rabbit died during induction. Top-up dosages of alfaxalone were necessary in three cases and in two of these three, isoflurane administration was also required to complete anaesthetic induction. The median time to lateral recumbency was 32.5 s in group 1 and 15 s in group 2. By intranasal application of midazolam with alfaxalone at both dosages, the anaesthetic state was induced shortly after application.

An approximately 7.5-month-old female Sprague Dawley rat () housed in a research facility presented for skin lesions including multifocal crusting and hypotrichosis. On presentation the research rat was mildly underconditioned with a distended abdomen and later developed small-bowel diarrhea. Abdominal ultrasound demonstrated diffusely thickened intestinal segments, and veterinary staff palpated a linear intra-abdominal mass. The rat was unresponsive to supportive care, and the rat was euthanized and necropsied. An elongate pink-tan mesenteric mass and severe, diffuse dilation and thickening of the small intestine were observed. Histopathology revealed mild to severe inflammation in most tissues with an abundance of eosinophils and eosinophilic granulomas in the abdominal lymph nodes. Clinical and histopathological findings are similar to hypereosinophilic syndromes (HES) described in a variety of species, including humans. Previous reports of HES in rats are limited to descriptions in rat strain Matsumoto Eosinophilia Shinshu (MES), which has been used as a model of human HES. Unlike MES rats, the rat in this report had diarrhea as well as eosinophilic infiltrates in the skin and heart, all of which are commonly described in human cases of HES.

Eight male Dorper sheep were anesthetized with isoflurane to evaluate the effects of anesthesia on cardiopulmonary parameters, echocardiographic variables and cardiac output (CO) using echocardiography methods compared with thermodilution. Heart rate (HR), respiratory rate ( R), systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and mean arterial pressure (MAP) were recorded. CO was obtained by thermodilution using a pulmonary artery catheter (CO), Teichholz method (CO) and pulmonary artery Doppler (CO). Echocardiographic variables (interventricular septum (IVSs and IVSd), left ventricular chamber (LVIDs and LVIDd) and left ventricular free wall (LVFWs and LVFWd) in systole and diastole, respectively; ejection fraction (EF%); fractional shortening (FS%); pulmonary artery pressure gradient (PG), peak flow velocity (V) and velocity-time integral of the flow (VTI) were recorded. All parameters were measured at baseline and every 15 min during the transanesthetic and recovery periods (T15, T30, T45, T60, T15, T30, T45, T60). Significant reductions in SAP, MAP, DAP and CO were observed during the transanesthetic period. Bland-Altman analysis comparing CO and CO showed mean biases of 0.49 l min (limits of agreement (LOA), -2.12 to 3.12 l min), 0.56 l min (-1.82 to 2.96 l min) and 0.75 l min (-2.04 to 3.56 l min) at baseline, during anesthesia and in recovery, respectively. For CO and CO, mean biases were -0.53 l min (-2.90 to 1.90 l min), -0.66 l min (-4.72 to 3.39 l min) and -0.94 l min (-4.62 to 2.73 l min) at baseline, during anesthesia and in recovery, respectively. Isoflurane decreases blood pressure and CO without affecting echocardiographic parameters. CO values obtained by thermodilution and echocardiography methods are not interchangeable.

To provide an alternative to oral gavage for treatments in laboratory mice, we have recently developed and introduced the micropipette-guided drug administration (MDA) method. This procedure is based on the presentation of a palatable solution consisting of sweetened condensed milk diluted with water, encouraging voluntary consumption of the vehicle and drug formulations. In this study, we compared the MDA method in male and female C57BL/6N and BALB/c mice, two inbred strains widely used in basic and preclinical research. Administering a diluted condensed milk solution daily over a period of 30 days, we observed that male C57BL/6N mice reached the fully voluntary drinking stage within five days or less, requiring the least amount of time to drink from the micropipette. Compared with males, female C57BL/6N mice showed increased consumption times during the initial administration period, yet they all managed to reach a fully voluntary stage within seven days or less. By contrast, BALB/c mice in general, and female BALB/c mice in particular, consistently required more time to consume the diluted condensed milk solution, whether administered voluntarily (no restraint) or semi-voluntarily (with mild tail restraint). Notably, a substantial portion of BALB/c mice (12.5% of males and 62.5% of females) failed to achieve fully voluntary consumption by MDA, despite their ability and willingness to drink the condensed milk solution when using a mild tail restraint. Taken together, the present study identified significant strain and sex differences in the MDA method as applied to laboratory mice.

Good education and training for scientists undertaking animal experiments is important for providing understanding of key issues in experimental design (ED) and for alleviating continuing concerns about the conduct of animal and research studies. We present here outputs from the FELASA Experimental Design Working Group, set up to consider the current provision of ED teaching and how it might be improved and harmonised across the laboratory animal science community. It is hoped that these outputs will provide practical help to ED teachers who wish to enhance the effectiveness of their teaching; they include• A list of learning outcomes (LOs) that should be achieved by learners, principally aimed at early career researchers;• An example of an (adaptable) template of how these LOs could be addressed in 16 h (12 h tuition plus breaks), ideally as a 2-day workshop. If circumstances make 12 h tuition impossible to achieve, key LOs for a shorter course are identified;• Guidance and recommendations for running ED courses, including some ideas for achieving effective learning, the ideal skill set for tutors, some teaching scenarios, and the amount of statistics to have in a basic experimental design course;• A glossary of relevant terms (in supplemental material);• A description of how the 2-day course format ran on two trial occasions, with results of informal assessment of participants as well as their feedback, both immediate and a year afterwards, indicating it was effective;• A programme for a potential 2-day, training-the-trainers style, workshop, describing its key elements and the results of trialling this with a range of ED tutors.

The 3Rs strongly shape the practice of laboratory animal use, as well as related policies worldwide. This success should not obscure the fact that implementing the 3Rs comes with challenges. A major problem is that it is fundamentally unclear under which conditions the 3Rs may be considered fulfilled in specific contexts. We argue that this lack of clarity is largely a result of the fact that the normative nature of the 3Rs has so far been disregarded. Hence, this paper seeks to answer the following research question: how is the normative nature of the 3Rs to be understood, and how can this understanding transparently guide their implementation? Based on a distinction between different types of norms, we show that the 3Rs, which have been called 'principles' since their origin, are indeed to be understood as principles in a substantive (norm-theoretical) sense. That is, they are norms that command the highest possible realization of their content. This understanding of the normative nature of the 3Rs has a significant effect on their implementation in practical contexts. As we will argue, it turns the orthodox idea of implementation strategies upside down. Building on this theoretical claim, we propose an ethics tool designed to help applicants, review committee members and authorities to apply the 3Rs transparently and, above all, in accordance with a reflected understanding of the relevant EU Directive's intention (Directive 2010/63/EU) and of the work of Russell and Burch, the pioneers of this milestone in the promotion of animal welfare in research.

Cervical dislocation (CD) is a widely used method worldwide for humanely killing adult laboratory mice in accordance with national legislation, such as the Animals (Scientific Procedures) Act 1986 in the UK. However, concerns have been raised regarding the limitations and potential risks associated with CD, including a reported failure rate of up to 20% and the risk of injury to the thoracic or lumbar spine region. To address these concerns, we have adopted a CD method that avoids the use of tension on the tail or any additional tools. In this study, we detail our process of validation through self-reporting and direct observation leading up to present our implementation of computerised tomography and three-dimensional imaging software to evaluate the effectiveness and efficiency of this tail-force free CD method. Our findings reveal a 100% success rate in achieving accurate cervical dislocation without causing damage to other vertebrae, thereby providing an improved and more reliable approach to humane killing for both male and female adult laboratory mice.

Murine aggression has profound implications on animal welfare and husbandry. This report examines how three distinct combinations of environmental enrichment - wheel, igloo and tunnel; wheel, igloo, and tunnel with nesting; and tunnel with nesting - affect aggressive behaviour in CD-1 male mice. We found that combining wheel/igloo/tunnel enrichment with nesting or replacing the wheel/igloo with two tunnels while maintaining the nesting enrichment reduced aggression. These findings not only suggest how enrichment can improve the welfare of aggressive male mice but also emphasise the need for further research to determine the optimal combination of enrichment.

Inflammatory bowel diseases (IBDs) are a major health burden, and incidence as well as prevalence have increased over the last decades. Colitis animal models are used to explore the underlying pathogenesis of and therapeutic options for IBD. Since the reporting of specific aspects of colitis studies using mice has been criticized in the past, we performed a scoping review based on the PRISMA guidelines to assess putative improvements in the quality of reporting. A defined search for dextran sulfate sodium (DSS) murine colitis models was performed in three literature databases (PubMed, PubMed Central®, and Embase) for two time periods: 2007/2008 and 2017. Data were extracted from 122 articles published in 2007/2008 and 236 publications from 2017. We checked the articles for the reporting of details of the colitis model (DSS properties, manufacturer, concentration, duration of application, mouse strain, sex, source), measures to reduce allocation, performance and detection bias (randomization and blinding), and information on clinical assessment, refinements, and humane endpoints. Our results showed that there was significant improvement over time in the scores for refinement and, based on this, also the completeness score. However, the other aspects were poorly reported, suggesting that this research field may need to adopt reporting guidelines such as ARRIVE, the Gold Standard Publication Checklist, or the colitis methods checklist when writing and reviewing publications.

As commissioned by the Federation of European Laboratory Animal Science Associations, these working group recommendations define the requirements to achieve the humane killing of fish, compare methods of killing, recommend methods of killing depending on context, and detail protocols leading to good practice. With a review of current practices in a fish laboratory and available literature to guide the recommendations, the concept of ideal euthanasia is discussed, and the dilemma of a prompt but stressful death versus a slow but stress-free experience is introduced. Noticeably, the context of fish killing varies widely whether to satisfy European Directive requirements, efficacy for species and/or developmental stages, scientific needs, health and safety, or animal welfare. Examples in the recommendations are based on the most common laboratory fish species, such as zebrafish , and the most commonly used methods of killing, such as overdose of anaesthesia, hypothermic shock, electrical stunning, and concussion - percussive blow to the head. Practical applications of completion of death, refinements, and protocols for good practice are proposed for all developmental stages and depending on the potential fate of the carcass as a scientific sample.

The streptozotocin (STZ)-induced hyperglycaemic rat model is widely used in diabetes research, particularly for investigating diabetic retinopathy; however, animal welfare concerns are often underreported. This study evaluated welfare outcomes in two commonly used rat lines, Brown Norway (BN/Crl) and Sprague Dawley (RjHan:SD). Data were collected from a series of diabetic retinopathy studies, in which a total of 183 BN/Crl and 76 RjHan:SD male rats received 40-65 mg/kg STZ via intraperitoneal injection and were monitored for 5-12 weeks. Welfare parameters, including body weight development and urologic complications (notably paraphimosis), were recorded and compared. Both lines achieved hyperglycaemia (≥16 mmol/l) within three weeks. However, BN/Crl rats exhibited a high incidence (82.5%) and severity of paraphimosis, along with marked weight loss, resulting in 13.7% of the animals reaching humane endpoint criteria for euthanasia. Weight loss positively correlated with STZ doses, with the highest dose (65 mg/kg) leading to 17.2% humane endpoint rate. In contrast, RjHan:SD rats exhibited significantly fewer urologic complications and maintained better weight gain, with none reaching humane endpoint. Our findings suggest that, while BN/Crl rats may offer advantages for ocular research owing to their pigmented eyes, their susceptibility to severe welfare issues raises concerns regarding their routine use. Furthermore, standardised supportive treatments, such as insulin supplementation, are worth considering for the model. This study highlights that careful selection of model animals, disease induction protocols and supportive treatments can optimise research outcomes and avoid loss of experimental animals, while adhering to the 3Rs.

The amphibian is an alternative animal model for developmental biology and toxicology. The Frog Embryo Teratogenesis Assay- (FETAX) stands as a validated test for ecotoxicology and chemical hazard characterization. Conventionally, fertilized eggs are obtained through adult hormonal injection. In adherence to the 3R principles, our proposed method offers the opportunity to obtain embryos through natural amplexus, rearing adults in controlled conditions that replicate the most favourable environmental parameters.

Animals used in science and education should be used by competent laboratory animal science (LAS) staff, both for reasons of reproducibility and to safeguard animal welfare. In this article, we propose entrustable professional activities (EPAs) as a structure to support and assess development of competence and valid entrustment decisions of persons working with laboratory animals in practice following, or in combination with, basic training. We propose the creation of a consensus framework and provide concepts that would encourage harmonisation in competence-based development. We anticipate that these will ensure that supervisors, trainers and competence assessors can more reliably establish the operator's competence in procedures on animals which are used in scientific research or education professionally.

Swine are a common neurobehavioral model. Visual event related potentials (VERPs) are an electroencephalogram (EEG) technique that assesses visual processing and can inform brain function and sensory changes after trauma or disease. We hypothesized that piglet visual EEG processing for 2D conspecific (CS) images would produce more cortical activity than a simple white square stimulus. We measured VERPs in healthy piglets presented with a 2D CS piglet image (5) and compared these results with animals presented with a simple white square (WS, 5). EEG waveforms were input into a source localization model of the brain to estimate cortical activity. N1 and P2 amplitudes and latencies and current density were extracted for each animal. Visual processing of CS produced longer N1 and P2 latencies than WS in the visual processing regions, suggesting that pigs may require longer processing times for more detailed images. Contrary to our hypothesis, CS had lower P2 amplitudes (frontal and left temporal) and current density (right temporal and occipital), which suggests that CS requires less processing power. Magnitudes may be related to the brightness of the stimuli presented (a feature that was not controlled for) with WS having on average a higher lux (112) than CS (98). Regardless, latency differences between CS and WS demonstrate that visual processing is sensitive to subtle stimulus features which can inform future studies on pig behavior and attention. Finally, these data serve as a healthy reference to compare VERPs in experimental cohorts subject to brain injury or other neurological diseases affecting visual processing.

The use of zebrafish as an animal model for biomedical and toxicological research has increased dramatically over the past decade, alongside a growing need to adopt the 3Rs principles to ensure ethically acceptable animal experimentation. Currently, one of the main challenges concerns 'surplus' animals that are unavoidably generated as part of an experimental procedure and are unsuitable for experimental analysis because they do not have the desired genotype, are too old or have the wrong sex. However, justifying the sacrifice of animals for these reasons is morally debatable and current ethics legislation in some countries insists they should nevertheless be maintained and left to die of natural causes. It is therefore imperative to develop strategies which can identify unwanted animals at a sufficiently early, non-sentient developmental stage so that they can then be sacrificed in an ethically more acceptable manner. In this manuscript we present a reliable medium-throughput method for non-invasive genotyping of zebrafish at developmental stages when sacrifice is considered ethically acceptable. This method is based on the use of low frequency shaking to induce the detachment of a limited number of cells from the embryos. These cells are then analysed by polymerase chain reaction-based genotyping approaches.

Researchers often need to justify their choice of sample size, particularly in fields such as animal and clinical research, where there are obvious ethical concerns about relying on too many or too few study subjects. The common approach is still to depend on statistical power calculations, typically carried out using simple formulas and default values. Over-reliance on power, however, not only carries the baggage of statistical hypothesis tests that have been criticized for decades, but also blocks an opportunity to strengthen the research in the design phase by learning about challenges in interpretation before the study is carried out. We recommend constructing a 'quantitative backdrop' in the planning stage of a study, which means explicitly connecting ranges of possible research outcomes to their expected real-life implications. Such a backdrop can facilitate considerations of how potential results, for example represented by intervals, will ultimately be interpreted. It can also serve, in principle, to help select single values of interest for use in traditional power analyses, or, better, inform sample size investigations based on the goal of achieving an interval width narrow enough to distinguish values deemed practically or clinically important from those not representing practically meaningful effects. The latter bases calculations on a desired precision, rather than desired power. Sample size justification should not be seen as an automatic math exercise with a right answer, but as a nuanced investigation of measurement, design, analysis and interpretation challenges. Construction of the quantitative backdrop provides a tangible starting place for such an investigative process.

In this study we aimed to determine the optimal epidural dose and concentration of diphenhydramine (DPH) for prolonged analgesia while preserving motor function, comparing its effects with lidocaine. A single-blind, randomized-controlled trial was conducted with 32 healthy rabbits assigned to two experiments (2.5 ± 0.5 kg). Experiment 1 ( = 25) assessed optimal DPH doses (4, 8, 12, 18, 24 mg kg), while Experiment 2 ( = 15) compared DPH concentrations (5%, 10%, 15%, 20%) at 12 mg kg. Antinociceptive effects were measured using pinprick tests on the perineal and digital areas, and motor blockade was evaluated using a modified 4-point grading system for limb paralysis. The results showed that epidural administration of 10% DPH at 12 mg kg provided profound analgesia, with hindlimb analgesia lasting 201 ± 2.5 min and motor blockade persisting for 157.2 ± 3.4 min. In contrast, 5% DPH resulted in 96 ± 15 min of analgesia and 5.2 ± 2.2 min of motor block. Lidocaine failed to provide effective digital analgesia. No significant difference was observed between 10% and 15% DPH in analgesic and motor effects. The motor blockade duration was consistently shorter than the antinociceptive effect. DPH demonstrated superior potency, and a prolonged, sensory-selective epidural block compared with lidocaine, minimizing motor blockade and the risk of akinesia-like analgesia. DPH (preferably >5%) offered effective and safe analgesia, making it a promising option for hindlimb analgesia in rabbits, reducing animal suffering and providing better outcomes than lidocaine.