Duke University adheres to the guidelines of the American Veterinary Medical Association. A copy of this report is available in pdf format by clicking here. You will need Adobe Acrobat Reader to open this file.  The Reader program is a FREE program.  Click here to download the Adobe Acrobat Reader.

• Inhalant Anesthetics

• Non-Anesthetic Gases

• Pharmacological Agents

• Physical Methods

Inhalant Anesthetics

In the liquid state, most inhalant anesthetics are topical irritants; therefore, animals should be exposed only to the vapors of the anesthetic. Air or oxygen must be provided during the induction period. All agents are given "to effect" until respiratory and cardiac arrest occurs.

 Halothane and isoflurane have the most rapid action, and since halothane is better tolerated, it is preferred. Methoxyflurane is less suitable, due to its slow effect and poor market availability. Care should be taken to minimize personnel exposure to vapors.

Ether is acceptable but not recommended because it poses an explosive hazard and is a respiratory irritant that is considered stressful to animals.  Administration should be performed in a fume hood, and signs indicating that ether is present or in use should be posted. To avoid explosions, the carcasses of ether-killed animals should be stored in explosion-safe refrigerators or freezers, and should not be incinerated until the ether is removed by aeration in a hood. Methoxyflurane is a similar but non-explosive and less irritating agent which is recommended as a substitute for ether.

 Non-Anesthestic Gases

Carbon dioxide (CO₂) is a common agent used for euthanizing rodents and other small laboratory animals. Use of a sealed chamber filled by a compressed gas cylinder is required. CO₂ generated by other methods, such as from dry ice, is unacceptable because gas flow can't be regulated precisely. Chambers should not be overcrowded. A CO₂ concentration of 70% or more should be utilized for euthanasia. Because CO₂ can act as a reversible anesthetic, it is imperative that the animals be kept in the chamber for several minutes after respiratory arrest. A secondary method of euthanasia is recommended, such as a thoracotomy, to ensure death or death may be verified by absence of a palpable heart beat. Due to physiologic characteristics, neonates require prolonged exposure to the gas.

Pharmacological Agents

Barbiturates:  Barbiturates such as pentobarbital are acceptable for mammalian species and birds. These drugs should be administered intravenously (IV) except in rodents where intraperitoneal (IP) administration is an acceptable alternative. Sodium pentobarbital (Nembutal) is the most common barbiturate agent for euthanasia. The dosage is usually at least twice that required for anesthesia, and ranges from 85 mg/kg for larger species to 200 mg/kg for some rodents. A dosage of 120 mg/kg is sufficient for most species, but more should be given if death does not ensue. Commercial barbiturate euthanasia formulations as are also appropriate, and should be used following label directions (e.g., 1 ml/lb for Beuthanasia-D*). Sodium pentobarbital is a Class II drug which is regulated by the Drug Enforcement Agency. Personnel using this agent are required to store it in a locked location and maintain records which include the date and amount of use.

Chloral Hydrate:   Chloral hydrate is not recommended, but may be used in ruminants and swine when administered I.V. at 900 mg/kg, but only after sedation with another drug.

 Mag Sulfate or Potassium Chloride:  Neither magnesium sulfate nor potassium chloride (KCl) can be used as a sole agent of euthanasia. Overdose with KCl is permissible in an anesthetized animal. Concentrated KCl should be given rapidly IV until rising serum potassium levels result in cardiac arrest.

 MS 222:  Tricaine methane sulfonate (MS222) can be used either as an injectable agent (200-300 mg/kg of a 1% buffered solution) or as an immersion bath (2 mg/ml in H2O) for amphibians and fish. The immersion time needed to assure death can range from 20 minutes to three hours, so it may be advantageous to use MS222 as an anesthetic followed by a physical method of euthanasia. Benzocaine immersion (100-200 mg/liter H2O) is also acceptable.  Note: Gloves should be worn at all times when handling fish and amphibians, and in particular when using MS222 as this agent may cause retinal toxicity after cutaneous exposure.

Neuromuscular blocking drugs are absolutely condemned for use as euthanasia agents.

Physical Methods

These methods require that the user have experience and skill in the techniques to be used.

Exsanguination is acceptable for all species following anesthesia of the animal.

Cervical dislocation is acceptable for mice, birds, rats (< 200 gm) and rabbits (< 1 Kg), but proper technique is essential. It is therefore recommended that animals be first sedated with another agent (carbon dioxide, pentobarbital or halothane are suggested). Its use as a sole means of euthanasia requires scientific justification and A-PLAC approval.

Decapitation with proper equipment may be performed on small mammals or birds after the animal has been sedated or lightly anesthetized (carbon dioxide, pentobarbital or halothane are suggested). Decapitation of fish, amphibians and reptiles should be followed by pithing. Use as a sole means of euthanasia in any species requires scientific justification and IACUC approval. Decapitation should generally be used only when study design requires it due to the potential hazard to personnel. Many species react adversely to the smell of blood, so animals should not be decapitated in the presence of other animals and the person performing decapitation should change gloves and/or wash hands between animals.

 Pithing of both the brain and the spinal cord (double pithing) may be used as the sole means of euthanasia in frogs of the genus Rana or other amphibians with anatomic features that facilitate easy access to the central nervous system. In all other amphibian and reptile species pithing should be followed by decapitation.

Stunning, rapid freezing or air embolism (under anesthesia) may be allowed in small species if scientifically justified and there are no available alternatives.