Carbon dioxide (CO 2 ) gradual-fill is commonly used to kill laboratory rats, but this use remains controversial due to a lack of agreement between studies. Inconsistencies may arise from differences in behaviors measured (e.g. active versus passive behaviors), in how rats cope with threats, or in rat sensitivity to CO 2 . The aims of the current study were to 1) describe active and passive responses during CO 2 forced exposure, 2) determine if these responses are consistent within individuals and across aversive stimuli, 3) assess individual differences in aversion to CO 2 in aversion-avoidance and approach-avoidance tests and 4) determine how responses in aversion tests relate to individual differences in behavior during forced exposure. Twelve Sprague Dawley female rats were exposed twice to three treatments: CO 2 , oxygen (O 2 ), and fox scent, and were exposed to CO 2 twice in each aversion test. The change in behavior from baseline was higher for rearing and locomotion when rats were exposed to CO 2 than when exposed to O 2 and fox scent. Responses varied among rats but were consistent across multiple tests within rats. For example, rearing was consistent within individuals between two exposures to CO 2 . Similarly, the strength of aversion was consistent within individuals across multiple exposures to CO 2 in aversion-avoidance and approach-avoidance testing. Latency to avoid CO 2 in aversion-avoidance tests was negatively correlated with rearing during CO 2 forced exposure. Collectively, these results indicate that rat responses to CO 2 vary between (but are consistent within) individuals, suggesting that rats vary in CO 2 sensitivity. However, even the less responsive rats avoided CO 2 concentrations far below those necessary to achieve unconsciousness, indicating that all rats likely experience negative states when euthanized with CO 2 .
Carbon dioxide (CO2) is commonly used to kill laboratory rats. Rats find CO2 aversive and aversion varies between individuals, indicating that rats vary in CO2 sensitivity. Healthy humans experience feelings of anxiety at concentrations similar to those avoided by rats, and these feelings are diminished by the administration of benzodiazepines. Our aim was to assess the effects of the benzodiazepine midazolam on individual thresholds of rat aversion to CO2. Six female Sprague Dawley rats were repeatedly exposed to CO2 gradual-fill in approach-avoidance testing. The first three exposures were to a control-treatment followed by three exposures to midazolam (0.375 mg/kg). Within each treatment aversion to CO2 was not affected by exposure number; however, tolerance increased from an average of 10.7% CO2 avoided during control sessions, to 15.5% CO2 avoided when treated with midazolam. These results indicate that rats experience anxiety when exposed to CO2, and that variation in rat CO2 sensitivity is driven by individual differences in the onset of these feelings of anxiety. No rat tolerated CO2 concentrations required to induce loss of consciousness.
The aim of this review is to summarize evidence regarding rat emotional experiences during carbon dioxide (CO 2) exposure. The studies reviewed show that CO 2 exposure is aversive to rats, and that rats respond to CO 2 exposure with active and passive defense behaviors. Plasma corticosterone and bradycardia increased in rats exposed to CO 2. As with anxiogenic drugs, responses to CO 2 are counteracted by the administration of anxiolytics, SRIs, and SSRI's. Human studies reviewed indicate that, when inhaling CO 2 , humans experience feelings of anxiety fear and panic, and that administration of benzodiazepines, serotonin precursors, and SSRIs ameliorate these feelings. In vivo and in vitro rat studies reviewed show that brain regions, ion channels, and neurotransmitters involved in negative emotional responses are activated by hypercapnia and acidosis associated with CO 2 exposure. On the basis of the behavioral, physiological, and neurobiological evidence reviewed, we conclude that CO 2 elicits negative emotions in rats.
Evidence indicates that carbon dioxide (CO2) 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 CO2 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 CO2 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. CO2 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 CO2, 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 CO2 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.
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