EUTHANASIA PROCEDURES USED IN EXPERIMENTAL ANIMALS

In experimental pharmacology, animals need to be sacrificed to obtain different organs for evaluating drug activity. Euthanasia is used where an animal is required to be sacrificed on termination of an experiment or otherwise for ethical reasons. There are various procedures carried out by which an animal is sacrificed quickly and painlessly in an atmosphere free from fear or anxiety. For accepting an euthanasia method as humane, it should have an initial CNS depressive action for immediate insensitivity to pain. The choice of a method will depend on the nature of the study and the species of animal to be killed. Euthanasia methods are classified into chemical and physical methods.

Chemical method: different chemicals, due to overdose or their toxic effect, may cause death, but very few are recommended for euthanasia. Chemical methods may be inhalants or non-inhalants.

Inhalant methods: Inhalant anaesthetics (nitrous oxide, ether, halothane, enflurane, sevoflurane, methoxyflurane, isoflurane and desflurane ), carbon monoxide, carbon dioxide or carbon dioxide with chloroform are widely used and preferred for euthanasia in animals.

Non-inhalant methods: Intravenous anaesthetics (overdose of Barbiturates, Chloral hydrate, Ketamine), potassium chloride, magnesium sulphate, neuromuscular blockers (Curare, Succinyl choline, etc.) are also used for euthanasia in animals.

Physical method: Physical methods are performed by a skilled and experienced person with suitable equipment. Some of these methods are Cervical dislocation (used for mice, rat, guinea pig), Decapitation (used for rodents and small rabbits), Exsanguination (used for rat, guinea pig, hamster and rabbits), Microwave irradiation (used for fixation of mouse or rat brain metabolites without losing anatomic integrity of the brain), Penetrating captive bolt (conditionally used for horses and swine) etc.

Methods not acceptable for any species of animals: Certain methods like Decompression, Electrocution, Stunning, Nitrogen flushing, Argon flushing, administration of Curariform drugs, Nicotine sulphate, Magnesium sulphate, Potassium chloride, Strychnine, Paraquat, Dichlorvos and Air embolism are not recommended by CPCSEA.

                Figure: The euthanasia procedures used for experimental laboratory animals

Table: Different euthanasia procedures used for experimental laboratory animals

ANIMAL	EUTHANASIA METHOD
Frog	a)     Chilling: by keeping the frog at 4 degrees Celsius until its movements are completely abolished. This can also be achieved by immersing the frog in ice-cold water. b)    Pithing.
Mice	a)     Decapitation (Allowed only in stress analysis). b)    Cervical dislocation. c)     Euthanasia within an atmosphere of 80% Carbon dioxide and Chloroform. d)    Euthanasia within an atmosphere of a suitable inhalation anaesthetic. e)     Sodium pentobarbitone at a dose of 150 mg/kg i.p. f)     Overdosed ketamine (i.v./i.m.). g)    Microwave irradiation
Rat	a)     Cervical dislocation followed by bleeding and decapitation. b)    Euthanasia within an atmosphere of 80% Carbon dioxide and Chloroform. c)     Euthanasia within an atmosphere of a suitable inhalation anaesthetic. d)    Sodium pentobarbitone at a dose of 150 mg/kg i.p. e)     Overdosed Ketamine (i.v./i.m.). f)     Microwave irradiation
Guinea pig	a)     Cervical dislocation followed by bleeding. b)    Euthanasia within a carbon monoxide, 80% carbon dioxide and chloroform atmosphere. c)     Euthanasia within an atmosphere of a suitable inhalation anaesthetic. d)    Sodium pentobarbitone at a dose of 150 mg/kg i.p. or overdosed Ketamine (i.v./i.m.).
Rabbit	a)     Cervical dislocation with subsequent bleeding. b)    Euthanasia within an atmosphere of 80% Carbon dioxide with Chloroform. c)     Euthanasia within an atmosphere of a suitable inhalation anaesthetic. d)    Sodium pentobarbitone at a dose of 120 mg/kg i.v. or overdosed Ketamine (i.v./i.m.).

 PRECAUTIONS:

1. While rendering an animal unconscious, care should be taken to carry out the process in the absence of other experimental animals.
2. The vocalization and release of pheromones during the procedures can cause undue stress in the other experimental animals. 

REFERENCES

1.   Panigrahi G., Patra A., 2019. Experimental Pharmacology- I: bridges the gap between animal models and computer simulation models. 1^st edition, Nirali Prakashan, Pune, India.

2.     Medhi, B., Prakash, A., 2010. Practical Manual of Experimental and Clinical Pharmacology. 1^st edition, Jaypee Brothers Medical Publishers (P) Ltd, New Delhi, India.