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Isoflurane anesthesia prior to carbon dioxide euthanasia is recognized as a refinement by many guidelines. Facilities lacking access to a vaporizer can use the "drop" method, whereby liquid anesthetic is introduced into an induction chamber. Knowing the least aversive concentration of isoflurane is critical. Previous work has demonstrated that isoflurane administered with the drop method at a concentration of 5% is aversive to mice. Other work has shown that lower concentrations (1.7% to 3.7%) of isoflurane can be used to anesthetize mice with the drop method, but aversion to these concentrations has not been tested. We assessed aversion to these lower isoflurane concentrations administered with the drop method, using a conditioned place aversion (CPA) paradigm. Female C57BL/6J (OT-1) mice ( n  = 28) were randomly allocated to one of three isoflurane concentrations: 1.7%, 2.7%, and 3.7%. Mice were acclimated to a light-dark apparatus. Prior to and following dark (+ isoflurane) and light chamber conditioning sessions, mice underwent an initial and final preference assessment; the change in the duration spent within the dark chamber between the initial and final preference tests was used to calculate a CPA score. Aversion increased with increasing isoflurane concentration: from 1.7% to 2.7% to 3.7% isoflurane, mean ± SE CPA score decreased from 19.6 ± 20.1 s to -25.6 ± 23.2 s, to -116.9 ± 30.6 s ( F 1,54  = 15.4, p  < 0.001). Our results suggest that, when using the drop method to administer isoflurane, concentrations between 1.7% and 2.7% can be used to minimize female mouse aversion to induction., Competing Interests: Declaration of conflicting interestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: IJM is employed by the Animal Welfare Institute, which funded MJB’s salary during the conduct of this study. In her role as adjunct professor in the UBC Animal Welfare Program, IJM had limited advisory input on practical aspects of the study design and execution. After this study was completed, MJB began graduate work on a different thesis topic with IJM as co-supervisor. In her role as M.Sc. co-supervisor, IJM also assumed a supervisory role of MJB’s non-thesis research, and provided input on the data analysis, interpretation, and drafting of this manuscript.

Anesthesia with isoflurane prior to carbon dioxide euthanasia is recommended as a refinement, but vaporizer access can be limited. An alternative to vaporizers is the 'drop' method, introducing a fixed volume of isoflurane into the induction chamber. Previous work suggests that isoflurane administered at a concentration of 5% via the drop method is effective but aversive to mice; lower concentrations have not been tested. We assessed mouse behavior and insensibility with induction using the drop method for isoflurane concentrations below 5%. Male Crl:CD-1 (ICR) mice ( n  = 27) were randomly allocated to one of three isoflurane concentrations: 1.7%, 2.7%, and 3.7%. During induction, measures of insensibility and stress-related behaviors were recorded. All mice reached a surgical plane of anesthesia, and mice exposed to higher concentrations did so more quickly; as concentrations increased from 1.7 to 2.7 and 3.7%, the time to recumbency (Least squares means ± SE: 120.5 s ± 8.1, 97.9 s ± 8.1, and 82.8 s ± 8.1, respectively), loss of righting reflex (149.1 s ± 8.5, 127.7 s ± 8.5, and 100.7 s ± 8.5, respectively), and loss of pedal withdrawal reflex (214.5 s ± 8.3, 172.2 s ± 8.3, and 146.4 s ± 8.3, respectively) all declined. Rearing was the most frequently performed stress-related behavior, and was most pronounced immediately following isoflurane administration for all treatments. Our results indicate that the drop method can be used to effectively anesthetize mice with isoflurane concentrations as low as 1.7%; future work should assess mouse aversion., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Here we document how rats separate their living space into different functional regions. Five groups of four female Sprague Dawley rats were housed in caging systems that consisted of two standard cages connected by a tube. Both cages were provided with the same amount of bedding and nesting materials, but only one contained food and water. Nesting cover and weight of each cage were measured once a week for five weeks during cage cleaning. We found that the cages with food and water had 9% less nesting material coverage but had gained 90% more weight when compared with cages where food and water were absent. These results indicate that, when provided with separate spaces, rats move nesting materials away from the cage containing food and water sources, but preferentially excrete in the cage with water and food.

Evidence indicates that carbon dioxide (CO 2 ) induces negative affective states (including anxiety, fear and distress) in laboratory rodents, but many countries still accept it for euthanasia. Alternative methods (e.g. inhalant anaesthetic) may represent a refinement over CO 2 but are not widely adopted. We conducted an online survey of Canadian and European laboratory animal professionals and researchers ( n  = 592) to assess their attitudes towards the use of CO 2 and alternative methods for rodent euthanasia using quantitative 7-point scale (from 1 (= strongly oppose) to 7 (= strongly favour) and qualitative (open-ended text) responses. CO 2 was identified as the most common method used to kill rodents, and attitudes towards this method were variable and on average ambivalent (mean ± SD score on our 7-point scale was 4.4 ± 1.46). Qualitative analysis revealed four themes relating to participant attitude: (a) the animal's experience during gas exposure; (b) practical considerations for humans; (c) compromise between the animal's experience and practical considerations; and (d) technical description of the procedure or policies. Many participants (51%) felt that there were alternatives available that could be considered an improvement over CO 2 , but perceived barriers to implementing these refinements. Qualitative analysis of these responses revealed five themes: (a) financial constraints; (b) institutional culture; (c) regulatory constraints; (d) research constraints; and (e) safety concerns. In conclusion, concerns regarding the use of CO 2 often focused on the animal's experience, but barriers to alternatives related to operational limitations. New research is now required on to how best to overcome these barriers.