A review of methods used to kill laboratory rodents: issues and opportunities

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

Determining candidate hypobaric hypoxia profiles for humane killing of laboratory mice

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

Welfare Risks of Repeated Application of On-Farm Killing Methods for Poultry

Council Regulation (EC) no. 1099/2009 on the protection of animals at the time of killing restricts the use of manual cervical dislocation in poultry on farms in the European Union (EU) to birds weighing up to 3 kg and 70 birds per person per day. However, few studies have examined whether repeated application of manual cervical dislocation has welfare implications and whether these are dependent on individual operator skill or susceptibility to fatigue. We investigated the effects of repeated application (100 birds at a fixed killing rate of 1 bird per 2 min) and multiple operators on two methods of killing of broilers, laying hens, and turkeys in commercial settings. We compared the efficacy and welfare impact of repeated application of cervical dislocation and a percussive killer (Cash Poultry Killer, CPK), using 12 male stockworkers on three farms (one farm per bird type). Both methods achieved over 96% kill success at the first attempt. The killing methods were equally effective for each bird type and there was no evidence of reduced performance with time and/or bird number. Both methods of killing caused a rapid loss of reflexes, indicating loss of brain function. There was more variation in reflex durations and post-mortem damage in birds killed by cervical dislocation than that found using CPK. High neck dislocation was associated with improved kill success and more rapid loss of reflexes. The CPK caused damage to multiple brain areas with little variation. Overall, the CPK was associated with faster abolition of reflexes, with fewer birds exhibiting them at all, suggestive of better welfare outcomes. However, technical difficulties with the CPK highlighted the advantages of cervical dislocation, which can be performed immediately with no equipment. At the killing rates tested, we did not find evidence to justify the current EU limit on the number of birds that one operator can kill on–farm by manual cervical dislocation

On Farm Evaluation of a Novel Mechanical Cervical Dislocation Device for Poultry

Urgent development of alternative on-farm killing methods for poultry is required following the number restrictions placed on the use of traditional manual cervical dislocation by European Legislation (EU 1099/2009). Alternatives must be proven to be humane and, crucially, practical in commercial settings with multiple users. We assessed the performance and reliability of a novel mechanical cervical dislocation device (NMCD) compared to the traditional manual cervical dislocation (MCD) method. NMCD was based on a novel device consisting of a thin supportive glove and two moveable metal finger inserts designed to aid the twisting motion of cervical dislocation. We employed a 2 × 2 factorial design, with a total of eight stockworkers from broiler and layer units (four per farm) each killing 70 birds per method. A successful kill performance was defined as immediate absence of rhythmic breathing and nictitating membrane reflex; a detectable gap in the vertebrae and only one kill attempt (i.e., one stretch and twist motion). The mean stockworker kill performance was significantly higher for MCD (98.4 ± 0.5%) compared to NMCD (81.6 ± 1.8%). However, the MCD technique normally used by the stockworkers (based previous in-house training received) affected the performance of NMCD and was confounded by unit type (broilers), with the majority of broiler stockworkers trained in a non-standard technique, making adaption to the NMCD more difficult. The consistency of trauma induced by the killing methods (based on several post-mortem parameters) was higher with NMCD demonstrated by “gold standard” trauma achieved in 30.2% of birds, compared to 11.4% for MCD (e.g., dislocation higher up the cervical region of the spine i.e., between vertebrae C0–C1, ≥1 carotid arteries severed), suggesting it has the potential to improve welfare at killing. However, the results also suggest that the NMCD method requires further refinement and training optimization in order for it to be acceptable as an alternative across poultry industry, irrespective of previous MCD technique and training

Welfare assessment of novel on-farm killing methods for poultry

<p>Diagrammatic representation of the location of two drill sites either side of the sagittal suture into the skull over the forebrain (a); and placement of bi-polar electrode wires (through the two drill sites) to rest, one on each of the dorsal surfaces of the right and left telencephalon and reference electrode placed between the skin and the skull (b).</p

Effect of an immune challenge and two feed supplements on broiler chicken individual breast muscle protein synthesis rate

Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single 2H2O dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SIGNIFICANCE: The present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.</p