title: Assessment of Carbon Dioxide, Carbon Dioxide/Oxygen, Isoflurane and Pentobarbital Killing Methods in Adult Female Sprague-Dawley Rats.
abstract: BACKGROUND
  Exposure to carbon dioxide (CO2) gas as a killing method is aversive and exposure to high concentrations is likely to be painful. Bradycardia during exposure to CO2 is associated with nociception and pain. However, it is unclear if bradycardia occurs before loss of consciousness as definitions of loss of consciousness vary in the literature. The objectives of this study were to explore the relationship between recumbency, loss of righting reflex (LORR) and a quiescent electromyograph as measures of loss of consciousness, and identify the onset of bradycardia in relation to these measures. Our primary hypothesis was that CO2 exposure would result in bradycardia, which would precede LORR.
  
  
  METHODS
  Thirty-two adult, female Sprague-Dawley rats were instrumented with a telemetry device and randomly assigned to one of four killing methods (concentrations of 100% CO2, CO2 (70%)/O2 (30%), isoflurane (5%) and intraperitoneal pentobarbital (200 mg/kg). Time to achieve recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph, heart rate and apnea were recorded.
  
  
  RESULTS
  The general order of progression was recumbency, LORR, quiescent electromyograph, isoelectric electrocorticograph and apnea. Recumbency preceded LORR in the majority of animals (CO2; 7/8, CO2/O2; 8/8, isoflurane; 5/8, pentobarbital; 4/8). Bradycardia occurred before recumbency in the CO2 (p = 0.0002) and CO2/O2 (p = 0.005) groups, with a 50% reduction in heart rate compared to baseline. The slowest (time to apnea) and least consistent killing methods were CO2/O2 (1180 ± 658.1s) and pentobarbital (875 [239 to 4680]s).
  
  
  CONCLUSIONS
  Bradycardia, and consequently nociception and pain, occurs before loss of consciousness during CO2 exposure. Pentobarbital displayed an unexpected lack of consistency, questioning its classification as an acceptable euthanasia method in rats.
authors.affiliation: Department of Veterinary Clinical and Diagnostic Sciences, University of Calgary, Alberta, Canada, s.
authors.forename: Jessica M
authors.initials: JM
authors.lastname: Chisholm
authors.affiliation: Department of Veterinary Clinical and Diagnostic Sciences, University of Calgary, Alberta, Canada, s.|Hotchkiss Brain Institute, University of Calgary, Alberta, Canada.
authors.forename: Daniel S J
authors.identifier: http://orcid.org/0000-0002-6420-477X
authors.initials: DS
authors.lastname: Pang
mesh_terms: D000759:Adjuvants, Anesthesia / Q000008:administration & dosage; D000818:Animals; D001049:Apnea / Q000503:physiopathology; D001919:Bradycardia / Q000503:physiopathology; D002245:Carbon Dioxide / Q000008:administration & dosage* / Q000378:metabolism; D004562:Electrocardiography; D000069280:Electrocorticography; D004576:Electromyography; D005260:Female; D006339:Heart Rate / Q000187:drug effects / Q000502:physiology; D007530:Isoflurane / Q000008:administration & dosage*; D010100:Oxygen / Q000008:administration & dosage* / Q000378:metabolism; D010424:Pentobarbital / Q000008:administration & dosage*; D011897:Random Allocation; D017207:Rats, Sprague-Dawley; D057897:Reflex, Righting / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014474:Unconsciousness / Q000503:physiopathology
chemical_list: D000759:Adjuvants, Anesthesia; D002245:Carbon Dioxide; D007530:Isoflurane; D010424:Pentobarbital; D010100:Oxygen