Combining Nitrous Oxide with Carbon Dioxide Decreases the Time to Loss of Consciousness during Euthanasia in Mice — Refinement of Animal Welfare?

Carbon dioxide (CO2) is the most commonly used euthanasia agent for rodents despite potentially causing pain and distress. Nitrous oxide is used in man to speed induction of anaesthesia with volatile anaesthetics, via a mechanism referred to as the “second gas” effect. We therefore evaluated the addition of Nitrous Oxide (N2O) to a rising CO2 concentration could be used as a welfare refinement of the euthanasia process in mice, by shortening the duration of conscious exposure to CO2. Firstly, to assess the effect of N2O on the induction of anaesthesia in mice, 12 female C57Bl/6 mice were anaesthetized in a crossover protocol with the following combinations: Isoflurane (5%)+O2 (95%); Isoflurane (5%)+N2O (75%)+O2 (25%) and N2O (75%)+O2 (25%) with a total flow rate of 3l/min (into a 7l induction chamber). The addition of N2O to isoflurane reduced the time to loss of the righting reflex by 17.6%. Secondly, 18 C57Bl/6 and 18 CD1 mice were individually euthanized by gradually filling the induction chamber with either: CO2 (20% of the chamber volume.min−1); CO2+N2O (20 and 60% of the chamber volume.min−1 respectively); or CO2+Nitrogen (N2) (20 and 60% of the chamber volume.min−1). Arterial partial pressure (Pa) of O2 and CO2 were measured as well as blood pH and lactate. When compared to the gradually rising CO2 euthanasia, addition of a high concentration of N2O to CO2 lowered the time to loss of righting reflex by 10.3% (P<0.001), lead to a lower PaO2 (12.55±3.67 mmHg, P<0.001), a higher lactataemia (4.64±1.04 mmol.l−1, P = 0.026), without any behaviour indicative of distress. Nitrous oxide reduces the time of conscious exposure to gradually rising CO2 during euthanasia and hence may reduce the duration of any stress or distress to which mice are exposed during euthanasia

The Influence of Isoflurane Anaesthesia on the Rat Grimace Scale.

Over 234,000 rats were used in regulated procedures in the UK in 2014, many of which may have resulted in some degree of pain. When using animals in research, there is an ethical and legal responsibility to alleviate or at least reduce pain to an absolute minimum. To do this, we must be able to effectively assess pain in an accurate and timely manner. The Rat Grimace Scale (RGS) is a pain assessment tool, which is suggested to be both accurate and rapid in pain assessment. Many procedures involve the use of general anaesthesia. To date, the effects of anaesthesia on the RGS have not been assessed, limiting its potential utility for assessing pain following anaesthesia. Forty-eight Lister hooded rats were used in this study (24 in part A and 24 in a separate part B). Rats were randomly assigned to one of two treatment groups in part A; short duration isoflurane exposure, short duration control exposure (air) and one of two treatment groups in part B; surgical duration isoflurane exposure or surgical duration control exposure (oxygen). Rats were placed into an anaesthetic induction chamber and isoflurane, or control gas piped into the chamber for either 4 (short duration exposure) or 12 minutes (surgical duration exposure). Following recovery, photographs of the rats' faces were taken and then scored blindly using the RGS. Short duration isoflurane anaesthesia had no effect on RGS scores. However, when rats are anaesthetised for a longer duration, akin to a simple routine surgical procedure, the RGS score increases significantly and this increase remains on repeated exposure to this duration of anaesthesia over a 4-day period. This should be accounted for when using the RGS to assess pain in rats in the immediate time period following procedures involving the use of isoflurane anaesthesia

Values of pH, P_aO₂ (mmHg) and lactate (mmol.l⁻¹) measured in arterial blood at the time of loss of righting reflex.

<p>Composition of the gas mixtures for the groups 1–3: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032290#pone-0032290-t003" target="_blank">table 3</a>.</p

Time to loss of righting reflex (LORR) in seconds within the 3 treatment groups and the 2 strains of mice (C57Bl/6 and CD1).

<p>Error bars: +1 SD. Composition of the gas mixtures for the groups 1–3: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032290#pone-0032290-t003" target="_blank">table 3</a>.</p

Composition and flow rate of the anaesthetic mixtures used for anaesthesia induction – pilot study.

<p>The Isoflurane % was read on the dial of the precision vaporizer, the O2 and N2O % referred to the Fi of the gases.</p

Diagram representing the induction chamber.

<p>The fresh gas inlet (bottom arrow) of the chosen mixture was delivered underneath a multi-perforated floor. A total of 4 mini fans were ensuring that the mixture was optimally blended. The sampling end of the sidestream gas monitoring (<b>X</b>) was positioned at mouse level, 8 cm above the floor. The gas outlet (upper arrow) was connected to an active scavenging system.</p

Composition and flow rates of the gas mixtures used for mice euthanasia- main study.

<p>CV.min−1: Chamber Volume per min. The induction chamber had a volume of 7 liters: 20% CV.min−1 = 1.4 l.min−1, 60% CV.min−1 = 4.2 l.min−1.</p