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.

ANAESTHETICS USED IN EXPERIMENTAL ANIMALS

Anaesthesia used in the experimental study is to ensure analgesia, amnesia and immobilization of animals during the procedure. Unless contrary to the achievement of the results of the study, different anaesthetics should be used to control pain or distress during the experiment, making the study simpler, reliable and reproducible. The painful procedures are conducted under appropriate anaesthesia as recommended for each species of animal. It must also be ensured that the anaesthesia is given for the full duration of the experiment, and at no stage is the animal conscious to perceive pain.

Local or general anaesthesia may be used, depending on the type of surgical procedure. Local anaesthetics are used to block the nerve supply to a limited area and are used only for minor and rapid procedures. This should be carried out under expert supervision for regional infiltration of the surgical site, nerve blocks, and for epidural and spinal anaesthesia. The most common compounds for surface anaesthesia are tetracaine, procaine, lidocaine, mepivacaine, etc. But for most laboratory animals, general anaesthesia is the method of choice. General anaesthetics are drugs which produce a reversible loss of all sensation and consciousness, which are used for surgical procedures to render the animals unaware of the painful stimuli. General anaesthetics are used in the form of injectable or inhalants or a combination of both routes. The following anaesthetics are commonly used for laboratory animals.

INJECTABLE ANAESTETICS: Injectable anaesthetics commonly used for laboratory animals are:

Barbiturates: Barbiturates interfere with nerve impulse transmission both in the CNS and in the ganglia, producing depression of cardiovascular and spinal cord reflexes. Phenobarbitone sodium is used for prolonged experiments. Pentobarbitone sodium is used for inducing rapid anaesthetic effects. Thiopentone sodium induces rapid anaesthetic effects and is used for surgical operations of short duration. For laboratory animals, short-and very short-acting barbiturates are used. Marked slowing of respiration is produced by barbiturates. It can be used for all species.

Chloralose: It is prepared by heating equal parts of anhydrous glucose and chloral. α-chloralose is the active form. It induces surgical anaesthesia for 3-4 hours and has the advantage of greater constancy of the depth of anaesthesia. It is a suitable anaesthetic for dogs, cats and rats, but it is not suitable for rabbits as they are narcotised rather than anaesthetized.

Urethane: Urethane or ethyl carbamate is suitable only for acute experiments since it has delayed toxic effects on the liver and may cause agranulocytosis; it has little or no effect on nerve transmission and produces little reflex depression. Duration of anaesthesia is 3-4 hours. It is a suitable anaesthetic for rabbits and rats. Frogs can be anaesthetized by placing them in a covered beaker containing 5-10% urethane solution. 

Paraldehyde: It depresses only the cerebrum and not the medullary centres; hence, it has a wide margin of safety. Intravenous injection may produce cardiac dilatation, pulmonary congestion and oedema. It is used for dogs and cats.  

Ketamine: This is a unique anaesthetic characterized by analgesia, immobility, and amnesia with light sleep. It is a neuroleptic compound with a very fast onset of action after intramuscular injection. It can be used for all species.

Xylazine: It is frequently used for anaesthesia in combination with other substances. Alone, this compound is used to produce anaesthesia in cattle.

INHALATIONAL ANAESTHETICS: Inhalational anaesthetics play a minor role in small laboratory animals such as rodents, but are more commonly used in larger laboratory animals such as dogs, cats, monkeys, etc. The advantages of inhalational anaesthetics are controlling the depth of anaesthesia. Ether is used in a fume hood, but the use of ether as an anaesthetic agent is prohibited. Other inhalational anaesthetics widely used in animal studies are Halothane, Isoflurane, Enflurane, Desflurane and Sevoflurane.

Preanaesthetic medication: Before using anaesthetics, the animal is prepared for anaesthesia by overnight fasting and the administration of pre-anaesthetics, which block parasympathetic stimulation of the cardio-pulmonary system and reduce salivary secretion. Atropine (an anticholinergic agent) is the most commonly used as a preanaesthetic medication. Atropine (0.02-0.05 mg/kg by s.c. or i.m., or i.v. routes) is used for all species to reduce salivary and bronchial secretions and protect the heart from vagal inhibition, given before anaesthesia.

a) Inhalational anaesthetics in Guineapig	b) Inhalational anaesthetics in rat

Figure: Induction of inhalational anaesthesia in animals. 

Table: Commonly used anaesthetic drugs for laboratory animals.

Drugs (route)	Dose (mg/kg) in
	Mouse	Rat	Hamster	G. pig	Rabbit	Cat	Dog	Monkey
Ketamine HCl (i.m.)	22-24	22-24	-	22-24	22-24	30	30	15-40
Pentobarbitone sodium (i.v.)	35	25	35	30	30	25	20-30	35
Pentobarbitone sodium (i.p.)	50	50	-	40	40	-	-	-
Thiopentone sodium (i.v.)	25	20	20	20	20	25	25	25
Thiopentone sodium (i.p.)	50	40	40	55	-	-	-	60
Urethane (i.v.)	-		-	-	1.0	1.25	1.0	1.0
Urethane (i.p.)	-	0.75	-	1.5	1.0	1.50	-	-

 

PRECAUTIONS:

1. Species characteristics and variation must be kept in mind while using an anaesthetic.

2. Side effects such as excessive salivation, convulsions, excitement and disorientation should be suitably prevented and controlled.

3. The animal should remain under veterinary care till it completely recovers from anaesthesia and postoperative stress. 

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.