Reply: Mouse euthanasia by isoflurane inhalation: a controversial method for the animal welfare and a negative impact on oocyte quality

We thank Dr Golledge for his comments and valuable remarks on our study and we agree with him on several points.

The aim of our work was to assess the impact of two methods of euthanasia (cervical dislocation versus inhalation of an overdose of isoflurane) on the quality of mouse oocytes. Euthanize many animals simultaneously is a prerequisite for conducting studies with large cohorts, especially for researches on female gametes. A major benefit of euthanasia by isoflurane inhalation is the ability to sacrifice several mice in the same time compared with the commonly used method by cervical dislocation. Even if several studies have demonstrated that isoflurane is aversive to rodents, this aversion is weaker than aversion to other agents like carbon dioxide or other inhalant anaesthetics. ¹ So, we agree with Dr Golledge on the fact that isoflurane is the ‘least worst’ option of inhalation method of euthanasia, and like him, we do not think that euthanasia by inhalation is more ‘humane’ than the physical method. Use of a lethal dose of volatile anaesthetics to euthanize laboratory animals may be motivated by an anthropomorphism in order to reduce pain and stress through the simultaneous anaesthesia. In any case, euthanasia with isoflurane can be considered as a more humane method, and we agree with the opinion of Dr Golledge on the fact that euthanasia by physical methods may offer a higher animal welfare even if it would be an unpleasant technique for operators.

Unlike the Canadian guidelines, the National Centre for Scientific Research in France considers isoflurane inhalation as one of the most appropriate method of euthanasia for small rodents (http://www.cnrs.fr/infoslabos/reglementation/euthanasie2.htm). The physicochemical characteristics of the molecule explain that the induction of unconsciousness occurs rapidly. Administration of isoflurane requires adequate equipment with an effective ventilation system to avoid environmental pollution (the main risk to experimenters). In our study, we used a home-made inhalation closed chamber of small capacity designed so that the effective gas concentration is reached there within seconds. Isoflurane concentration in the closed chamber did not fall below 5% in order to maintain a concentration at least three times greater than the minimal alveolar concentration (generally at 1.3 vol%, depending on the species). We misspoke concerning the time taken to kill mice. Indeed, we mentioned in our article the approximate time taken for unconsciousness. Before beginning the dissection, confirmation of the animal death was checked by the absence of any respiratory movement with stop heartbeat. Unfortunately, we did not measure accurately the mean time to the last visually detectable respiratory movement, as assessed by Dr Golledge.

The use of anaesthetic agents to perform euthanasia may interfere with the phenomena studied and possibly affect experimental results. In our study, we observed that isoflurane had a negative impact on mouse oocyte quality, with ovary dissection hindered by the occurrence of micro-haemorrhagic events. Similarly, several studies on rodents recently showed that isoflurane decreased the sperm motility parameters. ^2,3 Since mice are the most common animal model for scientific purposes, researchers should be aware of these iatrogenic experimental variables before interpreting their data.

Anyway, it is important that scientific research continues in respect of ethical rules to achieve euthanasia in the least painful and stressful way possible. A reduction in the duration of stress or distress during euthanasia of mice may result from a prior induction of anaesthesia with volatile anaesthetics, as demonstrated by Dr Golledge. ⁴