Mice are the most widely used model species for drug discovery and scientific research. Consequently, it is important to refine laboratory procedures and practices to ensure high standards of welfare and scientific data quality. Recent studies have identified that the standard practice of handling laboratory mice by their tails increases behaviours indicative of anxiety, which can be overcome by handling mice using a tunnel. However, despite clear negative effects on mice’s behaviour, tunnel handling has yet to be widely implemented. In this study, we provide the first evidence that tail handling also reduces mice’s responses to reward. Anhedonia is a core symptom of clinical depression, and is measured in rodents by assessing how they consume a sucrose solution: depressed mice consume less sucrose and the size of their licking bouts when drinking (their ‘lick cluster sizes’) also tend to be smaller. We found that tail handled mice showed more anhedonic responses in both measures compared to tunnel handled mice, indicative of a decreased responsiveness to reward and potentially a more depressive-like state. Our findings have significant implications for the welfare of laboratory mice as well as the design and interpretation of scientific studies, particularly those investigating or involving reward.

Rodents are the most widely used species for scientific purposes. A critical pre-requisite of their use, based on utilitarian ethical reasoning, is the provision of a humane death when necessary for scientific or welfare grounds. Focussing on the welfare challenges presented by current methods, we critically evaluate the literature, consider emerging methodologies that may have potential for refinement and highlight knowledge gaps for future research. The evidence supports the conclusion that scientists and laboratory personnel should seek to avoid killing laboratory rodents by exposing them to carbon dioxide (CO₂), unless exploiting its high-throughput advantage. We suggest that stakeholders and policymakers should advocate for the removal of CO₂ from existing guidelines, instead making its use conditionally acceptable with justification for additional rationale for its application. With regards to physical methods such as cervical dislocation, decapitation and concussion, major welfare concerns are based on potential inaccuracy in application and their susceptibility to high failure rates. There is a need for independent quality-controlled training programmes to facilitate optimal success rates and the development of specialist tools to improve outcomes and reliability. Furthermore, we highlight questions surrounding the inconsistent inclusion criteria and acceptability of physical methods in international regulation and/or guidance, demonstrating a lack of cohesion across countries and lack of a comprehensive ‘gold standard’ methodology. We encourage better review of new data and championing of open access scientific resources to advocate for best practice and enable significant changes to policy and legislation to improve the welfare of laboratory rodents at killing.

Whether and to what extent animals experience emotions is crucial for understanding their decisions and behaviour, and underpins a range of scientific fields, including animal behaviour, neuroscience, evolutionary biology and animal welfare science. However, research has predominantly focused on alleviating negative emotions in animals, with the expression of positive emotions left largely unexplored. Therefore, little is known about positive emotions in animals and how their expression is mediated. We used tail handling to induce a negative mood in laboratory mice and found that while being more anxious and depressed increased their expression of a discrete negative emotion (disappointment), meaning that they were less resilient to negative events, their capacity to express a discrete positive emotion (elation) was unaffected relative to control mice. Therefore, we show not only that mice have discrete positive emotions, but that they do so regardless of their current mood state. Our findings are the first to suggest that the expression of discrete positive and negative emotions in animals is not equally affected by long-term mood state. Our results also demonstrate that repeated negative events can have a cumulative effect to reduce resilience in laboratory animals, which has significant implications for animal welfare.

Millions of mice are used annually in scientific research and must be humanely killed. Despite significant welfare concerns, carbon dioxide exposure remains the most common killing method, primarily because there is no practical and humane alternative. We explored whether hypobaric hypoxia via gradual decompression could induce a non-recovery state in anesthetized male C57BL/6 and Balb/c laboratory mice. We aimed to determine if this was possible in a feasible timescale with minimal pathological consequences, as a proof-of-principle step. Systematic evaluation of two decompression rates (75, 150 ms⁻¹) and three profile shapes (accelerated, linear, gradual) in a factorial design revealed that hypobaric hypoxia effectively induced a non-recovery state in anesthetized laboratory mice, irrespective of decompression rate and shape. Mice took longer to reach a non-recovery state with the 75 ms⁻¹ decompression rate (75 ms⁻¹: 257 ± 8.96 vs. 150 ms⁻¹: 214 ± 7.26 s), with longer latencies in gradual and linear shaped profiles. Accelerated shaped profiles were least susceptible to meaningful refinement via rate. The only pathological changes of concern were moderate middle ear congestion and hemorrhage. These findings suggest that hypobaric hypoxia has potential, and subsequent work will evaluate the welfare consequences of gradual decompression in conscious mice, to identify decompression profiles that minimize welfare harms associated with ear barotrauma.

Millions of poultry are farmed inten-sively every year across the United Kingdom (UK) to produce both meat and eggs. There are inevitable situa-tions that require birds to be emergency killed on farm to alleviate pain and suffering. In Europe and the UK, emergency methods are regulated by the European Council Regulation (EC) No. 1099/2009 and The Wel-fare of Animals at the Time of Killing Regulations (Eng-land 2015; Scotland 2012; Wales and Northern Ireland 2014). Cervical dislocation has been reported to be the most widely used method prior to these legislative changes which took place from 1 January 2013. Based on limited scientific evidence and concern for bird wel-fare, these legislative changes incorporated restrictions based on bird weight for both manual (<= 3 kg) and mechanical (<= 5 kg) cervical dislocation, and introduced an upper limit in the number of applications for manual cervical dislocation (up to 70 birds per person per day). Furthermore, it removed methods which showed evi-dence of crushing injury to the neck. However, since legal reform new scientific evidence surrounding the wel-fare consequences of cervical dislocation and the devel-opment of novel methods for killing poultry in small numbers on farm have become available. Whether the UK poultry industry have adopted these novel methods, and whether legislative reform resulted in a change in the use of cervical dislocation in the UK remains unknown. Responses from 215 respondents working across the UK poultry industry were obtained. Despite legal reform, manual cervical dislocation remains the most prevalent method used across the UK for killing poultry on farm (used by 100% of farms) and remains the preferred method amongst respondents (81.9%). The use of alternative methods such as Livetec Nex (R) and captive bolt guns were available to less than half of individuals and were not frequently employed for broilers and laying hens. Our data suggests there is a lack of a clear alternative to manual cervical dislocation for individuals working with larger species and a lack of gold standard methodology. This risks bird welfare at killing and contributes to inconsistency across the indus-try. We suggest providing stakeholders with practical alternatives prior to imposing legislative changes and effective knowledge transfer between the scientific com-munity and stakeholders to promote positive change and protect bird welfare.

Killing is often an unavoidable and necessary procedure for laboratory mice involved in scientific research, and providing a humane death is vital for public acceptance. Exposure to carbon dioxide (CO ₂ ) gas is the most widely used methodology despite well proven welfare concerns. Consequently, the continued use of CO ₂ and its globally permitted status in legislation and guidelines presents an ethical dilemma for users. We investigated whether killing with hypobaric hypoxia via gradual decompression was associated with better welfare outcomes for killing laboratory mice. We compared the spontaneous behaviour of mice exposed to CO ₂ , decompression or sham conditions, and used analgesic or anxiolytic interventions to determine their relative welfare impact. Gradual decompression resulted in longer times to unconsciousness and death and the pharmacological interventions support the notion of a minimally negative animal experience, while providing further evidence for pain and anxiety associated with exposure to CO ₂ . Decompression resulted in moderate ear haemorrhage, but our welfare assessment suggests this may happen when mice are unconscious. Hence, gradual decompression could be the basis of significant refinement for killing laboratory mice. Future work should corroborate behaviour with neurobiological markers of loss of consciousness to verify the conscious phase of concern for animal welfare.