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Title: Nitrous oxide as a humane method for piglet euthanasia: behavior and electroencephalography

Author
item RAULT, JEAN-LOUP - University Of Melbourne
item KELLS, NIKKI - Massey University
item JOHNSON, CRAIG - Massey University
item Dennis, Rachel
item SUTHERLAND, MHAIRI - Agresearch
item Lay Jr, Donald

Submitted to: Physiology & Behavior
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2015
Publication Date: 6/27/2015
Citation: Rault, J., Kells, N., Johnson, C., Dennis, R.L., Sutherland, M., Lay Jr, D.C. 2015. Nitrous oxide as a humane method for piglet euthanasia: behavior and electroencephalography. Physiology and Behavior. 151:29-37. doi:10.1016/j.physbeh.2015.06.026.

Interpretive Summary: Identification and validation of humane methods of piglet euthanasia are critical to address public concern that current methods are not optimal. Humane is usually defined as a process imposed on the animal that induces minimal pain and distress. Blunt force trauma is considered humane for piglets. However, most people find it visually difficult to accept. Carbon dioxide (CO2) chambers have been widely adopted as an alternative, but CO2 has received criticism as being aversive to swine, hence the search for a method of on-farm euthanasia that is humane, practical, economical and socially acceptable. Nitrous oxide (N2O) has been identified as a potentially less aversive gas based on the behavioral response of piglets upon gas exposure in a free-choice, approach-avoidance test. Although behavior is a useful approach to assess perception based on decision-making processes, it becomes inherently limited for procedures such as gas exposure which aims at reducing cognitive function. Electroencephalography (EEG) data can provide information on the state of consciousness as well as providing evidence of pain perception. Combining neurobiological and behavioral data offers a robust approach to assess the perception by animals of their experience as they go through the process of potentially aversive or distressful procedures. This research aimed to evaluate the aversiveness of inhaling nitrous oxide, using a free-choice, approach-avoidance test; and to validate the effectiveness and humaneness of nitrous oxide to induce loss of consciousness by electroencephalographic (EEG) recording. We tested nitrous oxide at a 90% concentration and in combination with 30% oxygen and compared the piglets’ responses to exposure to carbon dioxide. Experiments 1 and 2 measured piglet behavior and heart rate to determine whether the gas mixtures tested were aversive to piglets when they were exposed to them at either full concentration (Experiment 1), or gradients thereof (Experiment 2). Experiment 3 determined whether the gas mixtures were aversive to piglets based on electroencephalographic (EEG) activity. These series of experiments confirmed that exposure to nitrous oxide is not aversive in comparison to carbon dioxide for piglets. The electroencephalographic (EEG) results backed up the behavioral data by demonstrating that the behavioral changes seen reflect differences in the animal’s perceptive experience of the treatments rather than, for example, alterations in motor function. Nitrous oxide at a concentration of 90% with 10% air (hence with residual oxygen around 2%) is effective in euthanizing piglets. Latency to loss of awareness, based on isoelectric electroencephalographic (EEG), under 90% nitrous oxide exposure is slightly longer than when using 90% carbon dioxide but because piglets do not show an aversive response to nitrous oxide gas, it is likely more humane. Nitrous oxide use at 90% concentration may be a useful tool for humane euthanasia as evidenced by the piglets’ mild response to the gas and its relatively quick mode of action.

Technical Abstract: The search for humane methods to euthanize piglets is critical to address public concern that current methods are not optimal. Blunt force trauma is considered humane but aesthetically objectionable. Carbon dioxide (CO2) is used but criticized as aversive. This research sought to: 1) evaluate the aversiveness of inhaling nitrous oxide (N2O; 'laughing gas') using an approach-avoidance test relying on the piglet's perspective, and 2) validate its humaneness to induce loss of consciousness by electroencephalography (EEG). The gas mixtures tested were N2O and air (90%:10%; '90N'); N2O, oxygen and air (60%:30%:10%; '60N'); and CO2 and air (90%:10%; '90C'). Experiment 1 allowed piglets to walk freely between one chamber filled with air and another prefilled with 60N or 90N. All piglets exposed to 60N finished the 10 min test whereas all piglets exposed to 90N had to be removed within 5 min because they fell recumbent unresponsive and then started to flail. Experiment 2 performed the same test except the gas chamber held N2O prefilled at 25%, 50%, or 75% or CO2 prefilled at 7%, 14%, or 21%. The test was terminated more quickly at higher concentrations due to the piglets' responses. Time spent ataxic was greater in the middle concentration gradients. Flailing behavior tended to correlate with increasing concentrations of CO2 but not N2O. Experiment 3, using the minimal anesthesia model, showed that both 90N and 90C induced isoelectric EEG, in 71 and 59 sec respectively, but not 60N within 15 min. Both 90N and 90C exposure resulted in a decrease in median frequency (F50), but only 90C resulted in a decrease in total EEG power (Ptot). The 95% spectral edge frequency (F95) never changed. These EEG results together with the observed behavioral changes reflect differences in the animal's perceptive experience. The implications for animal welfare are that N2O is less aversive than CO2, and 90% N2O can euthanize piglets.