Huw Golledge, Ph.D

Senior Scientific Programme Manager

Universities Federation for Animal Welfare

& The Humane Slaughter Association

golledge@ufaw.org.uk

Aversion to Carbon Dioxide and

Inhalational Anaesthesia in Rodents

Aims

Consider the welfare impact of using

inhaled substances for killing (and

anaesthetising) lab rodents.

Examine the decisions facing the

AWERB in considering the use of

such methods.

The Issue

Millions of animals are used for scientific purposes per

annum .

A few are re-homed, and some die without intervention,

but the vast majority are actively killed.

Why?

Because they’ve reached the end of the study

(generally “re-use” is not permitted)

To end their suffering (they’ve reached an “endpoint”)

Because they are surplus to requirements

- GMO breeding, “sexism”, overstocking

There is on-going debate about whether any of the

methods used to kill them are humane.

Annual Statistics of Scientific Procedures on Living Animals Great Britain 2012, (Home Office, 2013)https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/212610/spanimals12.pdf

Regulation - Directive 2010/63/EU

“Member States shall ensure that

animals are killed with minimum

pain, suffering and distress.”

But also,

Competent authorities can allow other methods;

If they’re shown to be at least as humane

or, if they’re scientifically justified

or, in an emergency.

Regulation: ASPA – Schedule 1

How should we choose a method?

Lots of licences just say “animals will be killed

by a Schedule 1 method”

Is this acceptable? Are all Schedule 1

methods equally humane?

If not, how should we decide which is most

humane?

Choice of Method – Welfare Constraints

The method should cause

minimal pain and/or distress

If distress or pain is

unavoidable the method

should be as quick as possible

Choice of Method - Scientific Constraints

Samples for post-mortem use – i.e.

intact brain etc.

Drug contamination of

tissues/samples

Arresting of physiological processes

Choice of Method - Practical Constraints

Cost

Number of animals – could be hundreds

or more per day

Equipment availability/safety

Feelings of the person carrying out the

procedure

Inhalation Techniques

Popular due to practicality, reliability, low skill requirement etc.

Only practical method for large numbers of animals

Permissible Inhalation Techniques

Carbon Dioxide (CO2)

Acceptable under schedule 1 (in a rising concentration)

Golledge, H.D.R.. Laboratory Animals 46, no. 4 (October 1, 2012): 358–59

Volatile/inhaled anaesthetics (Isoflurane, Sevoflurane etc.).

Routinely used for surgical anaesthesia in animals and

humans.

Don’t kill (Golledge, 2012) but render unconscious to allow

another technique to be used humanely.

Inert Gases (Argon, Nitrogen) – not

transposed by the UK

Carbon Dioxide

Most widely used method to kill lab rodents. Also widely used for poultry and pigs.

Animal breathes CO2 in a chamber until it loses consciousness and eventually dies.

Simple, safe, effective, cheap, doesn’t contaminate tissues, relatively un-distressing for operator.

Conlee, K M, M L Stephens, A N Rowan, and L A King. “Carbon Dioxide for Euthanasia: Concerns Regarding Pain and

Distress, with Special Reference to Mice and Rats.” Laboratory Animals , 39, 137 –161.

But, is it humane?

Carbon Dioxide

Causes pain at high concentration - CO2 + H2O H2CO3 H+ + HCO3-

Can avoid this by using in a gradually rising concentration.

Even at low concentrations CO2 is aversive to

rodents. Likely because it causes anxiety/fear

as it does in humans.

Anxiety occurs at such low concentrations that it can’t be avoided.

See e.g. Newcastle Consensus Report 2006 for a summary of the evidence - https://goo.gl/EBGMOh

Bailey,J.E., et al. “Behavioral and Cardiovascular Effects of

7.5% CO2 in Human Volunteers.” Depression and Anxiety

21, no. 1 (2005): 18–25.

Makowska, I. J. et al. (2009) Applied Animal Behaviour Science 119: 229–35.

Makowska, I. J. et al. (2009). Applied Animal Behaviour Science 121: 230–35.

Niel L, Weary D (2006), Applied Animal Behaviour Science 100, 295-308.

Niel L, Weary DM (2007), Applied Animal Behaviour Science, 107, 100-109.Wong, D et al. Biology Letters 9, no. 1 ,. doi:10.1098/rsbl.2012.1000.

Approach avoidance studies clearly show that CO2 is aversive at low concentrations

Conditioned Place Aversion

CO2 causes long-lasting conditioned place aversion => rats consciously experience negative emotions during exposure and remember them

Agen

t (CS+)

Air (C

S-)

Makowska, I. J. et al. (2009) Applied Animal Behaviour Science 119: 229–35.

Makowska, I. J. et al. (2009). Applied Animal Behaviour Science 121: 230–35.

Niel L, Weary D (2006), Applied Animal Behaviour Science 100, 295-308.

Niel L, Weary DM (2007), Applied Animal Behaviour Science, 107, 100-109.Wong, D et al. Biology Letters 9, no. 1 ,. doi:10.1098/rsbl.2012.1000.

The case for CO2

Some studies find no behavioural signs of distress when

exposing rats or mice to CO2 for humane killing 1

Others find no physiological signs of stress either2

1 Hackbarth, H-J et al. Euthanasia of rats with carbon dioxide--animal welfare aspects. .Lab Anim. 2000, 34:91-6.

2 Valentine, H et al. “Sedation or Inhalant Anesthesia Before Euthanasia with CO2 Does Not Reduce Behavioral or Physiologic Signs

of Pain and Stress in Mice.” Journal of the American Association for Laboratory Animal Science 51, no. 1 (2012): 50–57.

But see also: Makowska J, Golledge H, Marquardt N, Weary D. Comment on: Sedation or inhalant anesthesia before euthanasia with

CO2 does not reduce behavioral or physiologic signs of pain and stress in mice. JAALAS 2013, 51, 396-397

CO2 is not an ideal, humane ‘euthanasia’ agent

Balance of evidence strongly suggests CO2 is not entirely

humane…

BUT, is there a practical humane alternative?

Physical methods or injected overdoses are potentially more

humane but impractical in many cases.

Alternative Inhalation Methods:

Directive permits: Inert gases or volatile (inhaled)

Anaesthesia?

Inert gases

Raj, A.B.M. and Gregory, N.G. 1995. Welfare implications of gas stunning pigs 1.

Determination of aversion to the initial inhalation of carbon dioxide or

argon. Animal Welfare, 4: 273-280.

Niel, L and Weary, D.M.. “Rats Avoid Exposure to Carbon Dioxide and

Argon.” Applied Animal Behaviour Science 107, (2007) 100–109.

Makowska, I. J., et al.. “Rats Show Aversion to Argon-induced

Hypoxia.” Applied Animal Behaviour Science 114, (2008): 572–581.

Strongly aversive to rodents, but not to pigs!

Isoflurane

Several studies (from the same lab)

show aversion to Isoflurane is much

lower than to CO2 - animals stay

much longer in the chamber to eat

their cheerios.

They often ‘allow’ themselves

become anaesthetised.

The Case against Isoflurane

Subjective behaviour looks worse than CO2

? Elevated stress markers

Isoflurane causes conditioned place aversion

Valentine, H et al. “Sedation or Inhalant Anesthesia Before Euthanasia with CO2 Does Not Reduce Behavioral or Physiologic

Signs of Pain and Stress in Mice.” Journal of the American Association for Laboratory Animal Science 51, no. 1 (2012): 50–57.

But see also: Makowska J, Golledge H, Marquardt N, Weary D. Comment on: Sedation or inhalant anesthesia before

euthanasia with CO2 does not reduce behavioral or physiologic signs of pain and stress in mice. JAALAS 2013, 51, 396-397

Aversion-Avoidance – Light/Dark box

Animal must choose between two aversive stimuli

Boulanger Bertolus ,J et al. / Applied Animal Behaviour Science 164 (2015) 73–80

Wong, D et al. (2012) Biology Letters 9, no. 1 . doi:10.1098/rsbl.2012.1000.

Aversion to isoflurane appears to be learned

Wong, D et al. Biology Letters 9, no. 1 . doi:10.1098/rsbl.2012.1000.

Isoflurane aversion induced in place aversion experiments involves multiple exposures – explains why it is as strong as CO2 in these experiments

Aversion in naive animals will likely be lower.

=> Isoflurane/Sevoflurane may be the best current alternative for naieveanimals.

But many lab rodents have been previously exposed to isoflurane.

Is use of Isoflurane a refinement in non-naïve animals?

Implications beyond humane killing?

Animals frequently anaesthetised

many times for longitudinal

imaging studies etc?

Is repeated anaesthesia as a harm

rather than a refinement?

How often, how frequent is acceptable?

Summary - choosing the least harmful killing

method

Lists of “acceptable” methods may not help the selection of the most humane method.

All current methods appear to entail some harm.

Killing method should be considered and justified for each study.

Killing technique should be part of the harm component of harm/benefit analysis.

For AWERBS

Not all Schedule 1 methods are equally humane.

You could set a local policy on the ‘default’ killing method

for a given species.

Establishment Licence can be amended to allow non

schedule 1 techniques .

Many techniques can be refined

– consider local practice.

Are you comfortable with repeated anaesthesia for

longitudinal studies etc??

Evidence is complex and evolving – keep under review.

Ask for data if it’s not available.

Acknowledgements

Research supported by a UK NC3Rs strategic

award

Animals donated by Charles River, UK

Many colleagues for helpful discussion especially Melissa Bateson, Georgia Mason, Dan

Weary, Lee Niel, Aurelie Thomas and Joanna Makowska,

golledge@ufaw.org.uk

2012 Vacation Scholarship award to Alice Fodder

More Information

• A more detailed version of this talk - https://www.youtube.com/watch?v=o6LWzttaR48

• The American Veterinary Medical Association’s Guidelines for the Euthanasia of Animals: 2013 Edition provide an exhaustive and considered overview of methods for killing most species. Whilst the guidance is not applicable in the EU the document remains a valuable and well referenced source of detailed information on the practical and ethical issues concerning most methods.

https://www.avma.org/kb/policies/documents/euthanasia.pdf

• The Royal Society for the Protection of Animals (RSPCA) - Good Practice for Humane Killing provides supplementary resources for members of ethical review bodies, but also more broadly useful guidance on the selection of humane killing methods for lab animals.

http://tinyurl.com/GPHumKill

• NC3Rs - http://www.nc3rs.org.uk/euthanasia • Includes 2006 Newcastle Consensus Report.

• Humane Slaughter Association (livestock) – www.hsa.org.uk

golledge@ufaw.org.uk