The Influence of Euthanasia Methods on Rat Liver Metabolism

An important consideration in any terminal experiment is the method for euthanizing animals. Although the prime consideration is that the method is humane, some methods can have a dramatic impact on experimental outcomes. To determine the optimal method of euthanasia in metabolic experiments, a physical method (decapitation), two asphyxiation methods (CO2 and O2/CO2), and anesthetic (isoflurane) exposure followed by exsanguination were compared for their effects on liver metabolism. Changes in metabolism were monitored by following the activities of several key metabolic enzymes that are known to be susceptible to alterations in extracellular hormones as well as to changes in intracellular energy availability. The substrates and products of these enzymes also were monitored to better estimate their in vivo activity. Decapitated animals were used as the baseline for all comparisons. The results showed that euthanasia after exposure to 3 min isoflurane, euthanasia by exposure to a pure CO2 atmosphere for 2.5 min (CO2), and euthanasia by exposure to 1 min pure O2 followed by 2.5 min CO2 (O2/CO2) stimulated the enzymes involved in glycogen breakdown and glucose utilization. After CO2 or O2/CO2 asphyxiation, liver glycogen stores fell to approximately one-half those in the decapitated animals. No significant losses in liver glycogen were apparent after exsanguination under isoflurane anesthesia. In addition, differences between euthanasia methods were noted when the pattern of enzyme activity was compared: enzymes at the start of the glycolytic pathway were stimulated after CO2 or O2/CO2 euthanasia, but the terminal glycolytic enzyme was stimulated only after O2/CO2 euthanasia. Euthanasia by CO2 or O2/CO2 methods significantly decreased the regulatory enzyme of branched-chained amino acid degradation. This study clearly indicates that the method of euthanasia can have a dramatic impact on experimental data and, in particular, on liver metabolism.