Dr Huw Golledge and colleagues, from Newcastle University, have used an NC3Rs strategic grant to help identify the most humane methods of euthanasia for laboratory rodents.
Principal Investigator: Huw Golledge, Senior Research Associate
Organisation: Newcastle University
Award: £295,595, in 2009, over 36 months
Title: Assessing the humaneness of gas euthanasia techniques for laboratory rodents
Read more about Dr Golledge's research
There is a lack of consensus on which inhalational euthanasia methods are most humane
Worldwide, an estimated 100 million mice and rats are used every year in scientific research. The majority are killed via inhalation euthanasia, with carbon dioxide (CO2) being the most commonly used agent. A number of studies, however, suggest that CO2 is aversive to rodents, and may even cause acute pain at high concentrations. In mice CO2 can directly stimulate the amygdala and induce fear-like behaviours at around 10% concentration. Since rodents must be exposed to approximately 30% CO2 before consciousness is lost this raises serious welfare concerns.
Whether there is an alternative to CO2 which is both more humane and practical is unclear. Volatile anaesthetics, such as isoflurane, have been used for rendering animals rapidly unconscious prior to being killed with CO2 or by a physical method of euthanasia. However, all volatile anaesthetics so far tested appear to also cause aversion in rats and mice. Key questions remain on whether isoflurane is less aversive than CO2 and if not are there other strategies which allow CO2 to be used more humanely in rodents. To investigate these questions, Dr Huw Golledge, Newcastle University, was awarded the first NC3Rs strategic grant in 2009.
CO2 and isoflurane are similarly aversive
Dr Golledge has used conditioned placed aversion (CPA) to investigate the relative aversiveness of CO2 and isoflurane. This is the first time CPA has been used to investigate how rodents ‘feel’ during the euthanasia process. It is possible to test with CPA whether animals avoid areas where they have previously been exposed to CO2 or isoflurane, thus providing a measure of whether the animals remember their aversion in the absence of the euthanasia agent. Using this approach, Dr Golledge has demonstrated that both CO2 and isoflurane are aversive, with no statistically significant difference in the level of aversion between the two agents.
Argon is more aversive than either CO2 or isoflurane
Non-anaesthetic gases such as argon and nitrogen have also been proposed as alternatives to CO2 and are listed as permissible euthanasia agents for rodents under Directive 2010/63/EU. Dr Golledge has shown that argon is significantly more aversive to rats than either CO2 or isoflurane. As a result of this, argon is not in the list of approved methods for euthanasia in Schedule 1 of the amended Animals (Scientific Procedures) Act 1986.
Euthanasia in the home cage with isoflurane may be the best option
The stress associated with euthanasia is likely to arise from a combination of the physiological consequences of exposure to euthanasia agents with procedural factors, such as moving animals to an unfamiliar environment. A reduction in stress may be possible if agents are applied in the animals’ home cage, or if euthanasia is timed to occur during periods of minimal ambient stress, such as when the animals are resting or sleeping. This approach would be particularly relevant for animals housed in individually ventilated cages, since these sealed cage units lend themselves to being filled with gaseous agents.
To investigate this, euthanasia was carried out with CO2 or isoflurane in three scenarios, first in empty cages into which mice were placed immediately prior to euthanasia (this is analogous to the normal procedure), second in the animals’ home cage whilst they were sleeping, and third in the home cage whilst the animals were awake. The studies showed that of the different scenarios the use of isoflurane administered to sleeping mice may represent the most humane method as the time spent between waking and losing consciousness was less than any other group, thus minimising the duration of any stress or distress.
Addition of nitrous oxide shortens the time to loss of consciousness
Nitrous oxide, commonly known as ‘laughing gas’, is used in man to accelerate the induction of anaesthesia with volatile anaesthetic via a mechanism referred to as the ‘second gas’ effect. Dr Golledge has shown that the addition of nitrous oxide to isoflurane or to a rising concentration of CO2 reduces the time to loss of the righting reflex in mice by 17.6% and 10.3% respectively, without increasing stress-related behaviours. This provides a potential refinement of the euthanasia process by shortening the time to loss of consciousness.
Promoting a change in euthanasia practice
The research has provided an evidence base for improving animal welfare during euthanasia. Dr Golledge has disseminated his work to an international audience in Europe, the USA, Canada and Australia, including the plenary lecture at the 2012 annual conference of the Australian and New Zealand Laboratory Animal Association.
With NC3Rs sponsorship, Dr Golledge has also organised an international meeting to disseminate the research results to key stakeholders to ensure that they contribute as rapidly as possible to improvements in practice. The meeting also provided an opportunity for sharing data on euthanasia methods for neonatal rodents and fish. There have been three publications arising from the grant to date and three new collaborations established with research groups at Newcastle University, Fera and the University of British Columbia (Canada).
This case study was published in a review of our research portfolio in November 2013.