Anaesthesia and Euthanasia of Experimental Animals

1. NOIDAINSTITUTEOFENGINEERINGANDTECHNOLOGY
(PHARMACYINSTITUTE)
GREATERNOIDA
Presented by:- Under the Guidance of:-
ABDUL MANNAN DR. SAUMYA DAS
M.PHARM (Pharmacology) Professor (HOD Pharmacology)
PTSM-I NIET (PHARMACY INSTITUTE)

2. Anaesthesia and
Euthanasia of
Experimental Animals
• •
• •

3. BACKGROUND
A fundamental responsibility of individuals that use animals in
research, teaching or testing is to anticipate and eliminate or minimize
any potential that may cause pain, distress, or discomfort in animals.
Although animals that are in pain may not behave like humans, (e.g.,
pain in animals may be accompanied by immobility and silence, in
contrast to the groans and cries of human patients), it is assumed that
procedures that cause pain in humans cause pain in animals.

4. BACKGROUND CONTI...
The presence of pain in animals can be recognized by alterations in
animal behaviour (e.g., reduced activity, reduced grooming, hunched-
up posture, altered gait, changes in temperament, vocalizations,
reduced food and water intake, reduced urinary and fecal output), and
in physiological variables, (e.g., reduced depth of respiration,
increased heart rate, and reduced hydration status).
Reducing post-procedural/post-operative pain, distress, and
discomfort is accomplished by good nursing care, (e.g., keeping the
animal warm, clean, dry and well padded), and by the administration
of analgesic drugs.

5. BACKGROUND CONTI...
The prevention or minimization of animal pain, distress, or
discomfort by the proper use of tranquilizers, anaesthetics, and
analgesics is scientifically and ethically essential to the humane care,
use, and treatment of research animals. The use of these classes of
drugs must effectively prevent or minimize suffering and discomfort
of animals during potentially painful procedures.
The use of these three classes of drugs must be in accordance with
currently accepted veterinary medical practice and produce in the
subject animal an appropriate level of tranquilization, anaesthesia, or
analgesia consistent with the protocol or design of the experiment.

6. DEFINITIONS
Neuroleptics (antipsychotics)- drugs which produces central
nervous depression, depression of excitability of the autonomic
nervous system, a dulling of consciousness and a reduction of
spontaneous motor activity (e.g., tranquilizers/sedatives).
Analgesia- relief from pain.
Tranquilization- a state of behavioural change in which the patient is
relaxed, unconcerned by its surroundings, and often, indifferent to
minor pain.
Sedation- a mild degree of central depression in which the patient is
awake but calm; larger doses of sedative may lead to nercosis.
Local anaesthesia- loss of sensation in a limited body area.

7. DEFINITIONS CONTI...
Regional anaesthesia- loss of sensation in a larger, though limited,
body area.
Basal anaesthesia– a light level of general anaesthesia usually
produced by pre-anaesthetic agents; serves as a basis for deeper
anaesthesia following the administration of other agents.
General anaesthesia- complete unconsciousness.
Surgical anaesthesia- unconsciousness, accompanied by muscular
relaxation to such a degree that surgery can be performed painlessly.
Neuroleptanalgesia- a state of central nervous system depression and
analgesia usually produced by a combination of a neuroleptic and a
narcotic analgesic.

8. ANAESTHESIA
 The word anaesthesia has been derived from Greek word that means
“without perception of insensibility”.
 Anaesthesia is the act of providing sensation-free relief from
pain or pain-producing procedures.
 Anaesthesia must be performed by a person with knowledge of and
familiarity with the drugs to be used in the animal species under
consideration.

9. FACTORS AFFECTING THE
ACTIVITY OF ANAESTHETICS
Many factors can affect the activity of anaesthetics. The species,
strain, sex, age, nutritional and disease status, relative body size,
disposition/demeanour, presence of concurrent pain or distress,
medication are known to cause a variation in the amount of drug
needed to produce a desired effect in an individual animal.

10. Commonly used laboratory Anaesthetics
There are numerous anaesthetics available for use in rodents.
Some of the more popular agents include:
1. Chloralose
2. Urethane
3. Barbiturates (Pentobarbitone)
4. Paraldehyde
5. Magnesium Sulphate
6. Ketamine
7. Benzodiazepines (Diazepam)

11. CHLORALOSE
Causes Cardiovascular and Respiratory system Depression.
Dosage:- 80-100 mg/kg IV, IP
URETHANE
Minimal effects on Cardiovascular and Respiratory systems.
 Dosage:- 1000 mg/kg IV, IP
BARBITURATES (PENTOBARBITONE)
Barbiturates interfere with nerve impulse transmission both in the
central nervous system and in the ganglia producing depression of
cardiovascular and spinal cord reflexes.
 Dosage:- 30 mg/kg IV

12. There are many drugs
which are used in
combination with other
drugs to produce
anaesthetic effects in
Animals.
E.g., 1. Ketamine,
acepromazine.
2. Ketamine, xylazine, etc.
PARALDEHYDE
Produces CNS Depression.
 Dosage:- 0.5 ml/kg IM
MAGNESIUM SULPHATE
Blockage of N-methyl-D-aspartate (NMDA) receptor and calcium
channel.
 Dosage:- 5ml/kg IV
KETAMINE
Disrupts Pain Transmission and Suppresses Spinal Cord Activity.
 Dosage:- 87 mg/kg IM
DIAZEPAM
Inhibits NMDA‐receptors.
Dosage:- 2mg/kg IP

13. EUTHANASIA
The term euthanasia is derived from the Greek terms eu meaning
good and thanatos meaning death. A “good death” would be one that
occurs with minimal pain and distress.
Euthanasia is the act of inducing humane death in an animal.
“Sacrificing the experimental animal after use by gentle procedure
causing minimum of physical and mental suffering is called
Euthanasia (Painless killing).

14. OBJECTIVES OF EUTHANASIA
The primary criteria for euthanasia in terms of animal welfare are that
the method be painless, achieve rapid unconsciousness and death,
require minimum restraint, avoid excitement, is appropriate for the
age, species, and health of the animal, must minimize fear and
psychological stress in the animal, be reliable, reproducible,
irreversible, simple to administer (in small doses if possible) and safe
for the operator and so far as possible, be aesthetically acceptable for
the operator.

15. Methods of Euthanasia
Methods of euthanasia fall into two broad categories.
1. Chemical methods.
2. Physical methods.
Chemical methods
• Inhalant agents:-
Example- ether, halothane, methoxyflurane, isoflurane, enflurane,
chloroform, nitrogen, nitrous oxide, carbon di oxide, carbon
monoxide, argon, hydrogen cyanide.
• Injectable agents:-
Example- barbiturates, chloral hydrate, ethanol, ketamine,
magnesium, potassium sulphate, neuromuscular blocking agents.

16. PHYSICAL METHODS
• PENETRATING CAPTIVE BOLT
• EUTHANASIA BY A BLOW TO THE HEAD
• GUNSHOT
• CERVICAL DISLOCATION
• DECAPITATION
• ADJUNCTIVE METHODS
•Exsanguination
•Pithing

17. PENETRATING CAPTIVE BOLT
A penetrating captive-bolt pistol uses a blank cartridge to fire a bolt
into the brain of the animal. This causes concussion and trauma to the
cerebral hemispheres and brainstem which renders the animal
instantaneously unconscious. A correctly stunned animal will collapse
immediately often with muscular contractions and involuntary
kicking.
A penetrating captive bolt can cause varying degrees of brain injury
depending on the placement and length of the bolt and the force with
which it is projected into the brain. In some cases the animal may not
die quickly, therefore, it is recommended that immediately after the
procedure, death of the animal is ensured by a second procedure such
as exsanguination or pithing.
It Is an acceptable method of euthanasia in larger animals i.e. horses,
pigs and ruminants (sheep, cattle, goats etc.).

18. EUTHANASIA BY A BLOW TO THE HEAD
Stunning the animal by a blow to the head may be acceptable in small
or young animals with a soft skull (e.g., neonatal pigs <3 weeks old,
rats, mice, kittens, new-born pups, birds, reptiles, amphibians, fish).
A single, sharp blow should be delivered to the central skull bones.
This can be achieved with a hard and heavy, blunt instrument (e.g.
Metal pipe, wooden club etc.). Alternatively, small animals can be
held by the hind quarters and swung in an arc so that the back of the
head is struck on the edge of a hard object.
Stunning usually only renders an animal unconscious; therefore it
must be immediately followed by a second method that ensures death
(e.g. Exsanguination, cervical dislocation, pithing).
When properly performed with sufficient force, immediate depression
of the central nervous system and destruction of brain tissue occurs.
Loss of consciousness is rapid.

19. GUNSHOT
Shooting is a quick and effective means of humanely destroying animals
and in most situations is the only practical method available for use in the
field.
Head shots are preferred when euthanizing animals that are immobilized
by injury or physical restraint. Correctly placed head shots cause brain
function to cease and result in an instantaneous loss of consciousness.
Shooting should only be performed by skill operators who have the
necessary experience with firearms and who hold the appropriate licences
and accreditation.
1. Rifles
Smaller caliber rifles (minimum .22 magnum) are adequate for euthanasia
of most species of animals at short range (<5 m), as long as the shot is
correctly positioned. Soft-nose or hollow point ammunition which
expands on impact should be used.
2. Shotguns
12 gauge shotguns with shot sizes of BB or AAA may also be used.
3. Handguns
Handguns are class H firearms and their use is tightly controlled in all
jurisdictions.

20. Cervical Dislocation
CERVICAL DISLOCATION
Acceptable for small animals which are easily handled e.g., small to
medium sized birds, rodents and rabbits (weighing <1 kg).
It requires mastering of technical skills to ensure that loss of
consciousness is rapidly induced.
This method involves separation of the skull and the brain from the
spinal cord by pressure applied posterior to the base of the skull. The
brain stem – which controls respiration and heart activity – is
consequently damaged, stopping breathing and reducing blood flow
to the brain, leading to death. Studies in rats have shown that
electrical activity in the brain persists for around 13 seconds
following cervical dislocation. This may represent a period of
remaining consciousness.
Violent muscular contraction can occur after cervical dislocation.

21. DECAPITATION
•Decapitation involves the rapid severing of the head from the body
using a guillotine or sharp blade.
•Loss of consciousness is thought to develop rapidly in warm-
blooded animals.
•Violent muscular contraction can occur after decapitation.
•This method is acceptable method for very small mammals
providing that appropriate and well-maintained equipment is used.
•In cold-blooded vertebrates, the animals must be stunned or rendered
insensible prior to decapitation as they are very tolerant of anoxia.
•This method is not acceptable for birds as they may be conscious for
up to 30 seconds after decapitation.

22. EXSANGUINATION
Exsanguination or ‘bleeding out’ of the carcass is achieved by cutting
the major blood vessels in the neck (i.e., the carotid arteries and
jugular veins).
Exsanguination must not be used as a sole method of euthanasia as
extreme hypovolaemia is associated with anxiety. Pain is also
associated with cutting the deeper blood vessels.
This method is not acceptable for birds because of the tendency for
the blood to clot relatively quickly and thus result in incomplete
bleeding-out. It is also not acceptable for reptiles and other cold-
blooded vertebrates because of their slow metabolic rate and tolerance
to hypoxia.
Should only be used on unconscious animals i.e., those that have been
stunned or anaesthetised.
Often used in the field after prior stunning to ensure death.

23. PITHING
Pithing is sometimes used as an adjunctive procedure to ensure death
in an animal that has been rendered unconscious by other means (e.g.,
stunning).
It is often more acceptable than exsanguination as little blood is
spilled.
It is carried out by inserting a sharp needle or probe through the
foramen magnum into the base of the brain to ensure brain
destruction. Pithing can also be performed by inserting a probe
through the hole made by a captive bolt.
This method is used for some fish, amphibians and reptiles.

24. CHEMICAL METHODS
• INHALANT AGENTS
• Carbon Dioxide (CO2)
• Carbon Monoxide (CO)
• ANAESTHETIC GASES
• Using Soaked Gauze
• Using a Vaporiser
• INJECTABLE AGENTS
• Barbiturates
• Sedative Drugs

25. Carbon Dioxide (CO2)
Carbon dioxide used in a sealed environment is suitable for animals
up to 3 kg and is mostly used for small birds and rodents.
There is less risk to the operator when using CO2 compared to
anaesthetic gases or carbon monoxide.
When animals are placed into a chamber containing up to 70% CO2
they lose consciousness very quickly due to the narcotic effect of the
high intake of CO2 on the brain without causing hypoxia. Death is
caused by direct depression of CNS, respiratory and cardiac
functions.
A continuous inflow of CO2 should then be allowed to flow into the
sack. A constant level of CO2 should be maintained for at least 3
minutes and anaesthesia will occur within 60 seconds.
With animals inside the chamber, an optimal flow rate should displace
at least 20% of the chamber volume per minute.

26. Carbon Monoxide (CO)
When inhaled, carbon monoxide binds to haemoglobin in the red
blood cells, with an affinity 250 times that of oxygen. This results in
reduced oxygen-carrying capacity and altered delivery of oxygen to
cells. Hypoxia-the reduction of oxygen supply to the tissues –
eventually leads to unconsciousness and death.
Death occurs rapidly at CO concentrations of 4 to 6%. Carbon
monoxide concentrations greater than 2% are sufficient to cause loss
of consciousness within minutes. Failure of the respiratory centre then
occurs followed by death from cardiac arrest.
Carbon monoxide is extremely hazardous to humans as it is highly
toxic and difficult to detect. Exposure from inhalation of combustion
products can cause fatal poisoning. Non-fatal poisoning may result in
permanent nervous system damage.

27. Anaesthetic gases
Halothane and the halogenated ethers, for example, isoflurane,
methoxyflurane can be used with small animals (< 7kg). The animal
can be placed in a sealed chamber containing cotton or gauze soaked
with an appropriate amount of an anaesthetic or the anaesthetic
vapour can be introduced from a vaporiser into the container or a
plastic bag that has been placed around a cage containing the animal.
The animal will become unconscious quickly and quietly (some
voluntary, then involuntary resistance behaviour usually occurs).

28. Using soaked gauze
Anaesthetic vapour from the soaked cotton or gauze can reach
toxic concentrations within the chamber leading to death.
However, lethal concentrations cannot be guaranteed so the
animal should be removed from the cage once unconscious and
euthanised with a secondary method of euthanasia for example,
an intravenous or intraperitoneal injection of pentobarbitone
sodium. Note that if intraperitoneal pentobarbitone is used, the
animal must be returned to the gas-filled chamber until it has
died.

29. Using a vaporiser
When vapour is introduced using a vaporiser, lethal concentrations
are unlikely to be achieved and the animal must be removed from the
cage once unconscious and euthanised using a secondary method of
euthanasia for example, an intravenous or intraperitoneal injection of
pentobarbitone sodium. Note that if intraperitoneal pentobarbitone is
used, the animal must be returned to the gas-filled chamber until it
has died.
When using a vaporiser, the addition of an appropriate concentration
of nitrous oxide will produce a more rapid loss of consciousness.
This method is less hazardous to the operator as the gases can be
vented or scavenged thus lowering exposure.

30. Injectable agents
Barbiturates
One of the most humane methods of euthanasia is the administration
of a barbiturate overdose either by the intravenous, intraperitoneal or
intracardiac routes.
Injections by the intraperitoneal route work much more slowly than
the intravenous route, but are easier for an operator working alone.
The intraperitoneal route is used when an intravenous injection would
result in stressful handling or be dangerous to the animal or the
operator and when there are no easily accessible veins.
When using the intraperitoneal route, a non-irritant barbiturate
solution should be used i.e., a normal anaesthetic solution of
barbiturate, e.g., sodium pentobarbitone (Nembutal®).

31. Sedative drugs
A review of sedative, tranquillising or anaesthetic drugs (e.g.,
ketamine) for wildlife is beyond the scope of this document.
Recommendations on appropriate drugs (including combinations
thereof), dose rates and methods of application can vary dramatically
between species. Users of sedative drugs should refer to the most
recent, species specific literature and manufacturer recommendations
before proceeding to field application.

32. SIGNS OF DEATH
1. Absence of respiratory movement.
2. Absence of heart beat.
3. Absence of pulse.
4. Loss of colour in mucous membranes.
5. Corneal and palpebral reflexes are lost.
6. Glazing of eyes: this will occur rapidly after death.

33. References
https://www.sciencedirect.com
https://www.webmed.com
1. K.L. Stewart, in Principles of Animal Research for Graduate and Undergraduate
Students, 2017
2. Mark Oliver, Samantha Rossenrode, in Advances in Sheep Welfare, 2017
3. Amanda R. Hau, Jann Hau, in Laboratory Animals, 2014
4. Molly Varga BVetMed DZooMed MRCVS Textbook of Rabbit Medicine (Second
Edition) 2014, Pages 3-108
5. Sivula CP, Suckow MA, P. Christine Management of Animal Care and Use
Programs in Research, Education, and Testing. 2nd edition, Chapter 35, 2018
Williams DJ, Walker JD. A nomogram for calculating the maximum dose of local
anaesthetic. Anaesthesia. 12 May 2014; 69:847-853.