Inhaled carbon dioxide is likely one of the most studied and disputed euthanasia methods available. Because it is rapid, safe, inexpensive, and readily available it is the most common method of euthanasia for laboratory rodents (Artwohl et al., 2006). Respiratory exposure to CO₂ causes respiratory acidosis, which in turn creates a physiological domino effect. Acutely, this small molecule rapidly diffuses from the lungs into the plasma and intracellular spaces, and when combined with water it produces carbonic acid which rapidly splits into bicarbonate and hydrogen ions. If the buffering capacity is exceeded in this dose-dependent process you get respiratory acidosis. The resulting acidification of the intracellular space and cerebrospinal fluid leads to suppressed nerve cell function and inhibition of central N-methyl-D-aspartate (NMDA) receptors causing anesthesia (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Martoft et al., 2003; Pritchett et al., 2005; Thomas et al., 2012). Prolonged exposure to CO₂ and/or high concentrations of CO₂ then leads to severe respiratory acidosis and depression of the respiratory centers of the brain, eventually resulting in death. Other actions include acidosis-induced depression myocardial contractility and direct myocardium effects (American Veterinary Medical Association, 2013; Pritchett et al., 2005; United Kingdom House of Commons, 1986). Although eventual hypoxia results, it is not the direct mechanism of action resulting in death.

The ideal rate of CO₂ delivery appears to be species specific. For rodents, there has been conflicting reports with some recommending use of a prefilled chamber (100% CO₂) for euthanasia (Artwohl et al., 2006; Canadian Council On Animal Care in Science, 2010), while others recommend a gradual fill method, with a fill rate of approximately 10–20% of chamber volume per minute (American Veterinary Medical Association, 2013; Burkholder et al., 2010; Close et al., 1996, 1997; Conlee et al., 2005). Conflicting recommendations have also been published for swine and poultry (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Grandin, 1994). Furthermore, 100% exposure is recommended in rabbits and ferrets, but these animals may also be distressed at any exposure (Close et al., 1996, 1997; Fitzhugh et al., 2008). Regardless of the rate of delivery, use of compressed CO₂ gas in cylinders with flowmeters to precisely deliver the gas is the only acceptable source (American Veterinary Medical Association, 2013; Artwohl et al., 2006; Canadian Council On Animal Care in Science, 2010; United Kingdom House of Commons, 1986). Euthanasia can be accomplished over a wide range of concentrations (American Veterinary Medical Association, 2013; Canadian Council On Animal Care in Science, 2010; Close et al., 1996, 1997; Leach et al., 2002; United Kingdom House of Commons, 1986).

Many studies have looked at effect of gas combinations to mitigate the negative aspects of CO₂ euthanasia, with variable results. It has been suggested that the addition of nitrous oxide to CO₂ decreases the time to unconsciousness in mice and pigs, thus likely decreasing the distress the animal experiences (Llonch et al., 2012; Rault et al., 2013; Thomas et al., 2012).

Respiratory acidosis and other physiological events that occur during exposure to CO₂ leads to successful euthanasia, but may also play a role in potential pain and distress an animal experiences when exposed to CO₂. Resulting lactic acidosis leads to activation of the acid sensing ion channels (ASIC), which may cause pain or fear (American Veterinary Medical Association, 2013; Thomas et al., 2012). In addition, as indicated earlier, carbonic acid is created when CO₂ combines with water. This reaction occurs in the ocular, nasal, and oral mucous membranes and carbonic acid activates polymodal nociceptors and has an irritating effect on these areas (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Hawkins et al., 2006; Leach et al., 2002; United Kingdom House of Commons, 1986). CO₂ also has a pungent odor and hypoxia may lead to a feeling of breathlessness, thus possible distress if unconsciousness is not rapid (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Hawkins et al., 2006; Leach et al., 2002). Because of carbonic acid production, CO₂ should not be dissolved in water to euthanize aquatic species (Canadian Council On Animal Care in Science, 2010). Some recommend that it not be used as a sole agent in species that have shown significant aversion to CO₂ exposure such as pigs, mink, ferrets, and rodents (American Veterinary Medical Association, 2013; Canadian Council On Animal Care in Science, 2010; Conlee et al., 2005; Fitzhugh et al., 2008; Leach et al., 2002; Makowska et al., 2009; Valentine et al., 2012). However, other studies have shown that it can be used successfully in pigs and rodents (Burkholder et al., 2010; Grandin, 1994; Hackbarth et al., 2000; Sharp et al., 2006).

Since CO₂ exposure is the most common method of euthanasia for laboratory rodents, it is important to consider the biochemical effects and possible artifacts that may be created if this method of euthanasia is elected. CO₂ exposure can stimulate enzymes in the glycolytic pathway, and affect red blood cell parameters (Artwohl et al., 2006). In addition, lung pathological artifacts such as edema and hemorrhage have been reported (Danneman et al., 1997; Fawell et al., 1972).

Advantages of CO₂ as a euthanasia agent is that it is heavier than air so less likely to displace out of container (however, consider the height of the animal), generally has a rapid onset, is relatively inexpensive, and nonflammable and nonexplosive (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Llonch et al., 2012). However, the advantages must be heavily weighed with the substantial and conflicting reports on the potential for pain and distress with this method, which make it difficult to have generalized recommendations.

Carbon Monoxide (CO) is a colorless, odorless gas that is produced by the chemical interaction of sodium formate and sulfuric acid. It preferentially combines with the iron in hemoglobin producing carboxyhemoglobin, blocking uptake of oxygen and inhibiting other binding sites to off-load oxygen, resulting in hypoxia (American Veterinary Medical Association, 2013; Close et al., 1996, 1997; Makowska and Weary, 2009). This leads to loss of consciousness, eventually resulting in death. During loss of consciousness the motor centers of the brain are stimulated, thus this may be accompanied by convulsions and muscle spasms. In general CO produces rapid loss of consciousness and death with minimal pain and distress at low concentrations (4–6%), although this may be species dependent (American Veterinary Medical Association, 2013; Makowska and Weary, 2009). Use of a presedative may decrease the signs of distress in dogs (Dallaire and Chalifoux, 1985). The main concern with use of CO for euthanasia is the occupational risk involved to personnel. It is a human hazard and can be explosive in high concentrations. The environment should be equipped with an effective exhaust or ventilation system, CO monitors, and spark free and explosion-proof electrical equipment (American Veterinary Medical Association, 2013; Close et al., 1996, 1997). Only commercial cylinders should be used for euthanasia (Close et al., 1996, 1997).