Definitions
• Pain – An unpleasant sensory or emotional
Anesthesia & Euthanasia experience associated with actual or potential
of tissue damage
Experimental Animals • Analgesia – A complete loss of sensation to
pain
Department of Pharmacology, Manipal College of Pharmaceutical Sciences • Anesthesia ‐ A total loss of sensation in a
MAHE, Manipal part of or in the entire body
• Euthanasia – Causing death without pain
Anesthesia of experimental Anesthesia of experimental
animals animals
• Immobilization methods: Animals undergoing potentially painful procedures
should be provided with adequate anesthesia.
– Physical restraint: produces fear, distress
and anxiety in experimental animals Based on routes of administration:
• Inhalant anesthetics
– Chemical restraint: Anesthesia
• Injectable anesthetics
• Local anesthesia plays only a minor role • Immersion anesthetics
for experimental animals as compared
to general anesthesia • Local anesthetics
Injectable anesthetics (e.g. Ketamine
Inhalant anesthetics (e.g. Isoflurane) combinations, Medetomidine)
•Delivery of inhaled anesthetics by mask or • Appropriate for many procedures
endotracheal tube via precision vaporizer
•Safer than injected drugs • Variation in depth and duration of anesthesia
among rodent strains and individual animals
•Disadvantage is lack of residual analgesia
•complexity and cost of equipment, hazard
to personnel
Immersion anesthetics (e.g. Tricaine, Local anesthetics (e.g. Lidocaine,
Bupivacaine) Bupivacaine)
•For aquatic species such as amphibians and • Injected at the site of incision
fish • As supplements to either inhalant or
•Different solution strength for induction and injectable anesthetics
maintenance of anesthesia
Premedication Local anesthesia
Anticholinergic • Surface: Tetracaine and proparacaine.
(e.g. Atropine, Glycopyrrolate)
•Prevent vomiting • Injectables: Procaine, butanilicaine, lidocaine,
•Control salivary and bronchial secretions mepivacaine and etidocaine are commonly used
injectable local anesthetics
Sedatives (e.g. Droperidol, Diazepam) Local anesthesia is only recommended for gentle and
•Decrease dose of anesthetic by 20-50% calm animals (cattle, sheep)
•Minimize stress, anxiety and excitement For most laboratory animals, general anesthesia is
•Eases the transition to the first plane of GA the method of choice.
General anesthesia Course of anesthesia
• Preparation of animals • Depth of anesthesia
– General condition of the animal prior to – I. Stage of analgesia
anesthesia – II. Stage of excitation
– Animals with a vomiting reflex should be – III. Stage of tolerance
fasted prior to anesthesia – IV. Stage of asphyxia
• Fasting at least 12 h • Routes of general anesthesia
• Pigs and cattle for at least 24 h. – (i) injection and (ii) inhalation anesthesia
• Water ad libitum.
Inhalation
• A minor role for small laboratory animals • The parts of an inhalation system include:
– Bottle with oxygen (blue bottles)
• More common for the bigger laboratory – Valve to regulate pressure (reduces the pressure of the
animals such as dogs, cats oxygen-bottle)
– Flowmeter (monitors the gas flow to the animal)
• Advantages – Evaporator (evaporation of liquid anesthetic compounds)
– Oxygen-bypass (fast supply of oxygen to the animal in case
1. controlling exactly the depth of of need)
anesthesia – Tube to the system
2. fast management of complications
Techniques of inhalation Inhalant Agents
• 1. Technique of insufflation Ether
– via a mask •Volatile liquid, pungent odour, Irritant to mucus
membranes
• 2. Open system
– Inspired and expired gases are separated •Highly flammable, explosive
by a valve. •Induction and recovery rough
• 3. Half-closed and closed systems •Complete anesthesia, analgesia, muscle
• In closed systems all of the expired air passes to a relaxation, unconsciousness
CO2 absorberCO2 is removed chemicallyair is
•Toxicity – Liver
inspired again with newly evaporated anesthetic
compounds mixed with oxygen.
Halothane Isoflurane
•Colorless, odorless, Volatile liquid •Respiratory and CVS depression
•Fast response
•Non-flammable, non-explosive, non- •Good muscle relaxant action
irritant
•Poor analgesic action Enflurane
•Moderate skeletal muscle relaxant •Cardiopulmonary depression and minimal
•Toxicity – Liver and renal respiratory depression
•Fast response
Desflurane Carbon dioxide (CO2)
• Rapid onset and recovery
• Irritating to airway •Anesthetic for brief procedures
• Marked increase in heart rate •As a euthanasia agent
•appropriate for quick procedures like tail
Nitrous Oxide
snipping, ear marking and orbital bleeding
• Can be used as a sole anesthetic agent
• Moderate analgesia •Very fast response
• Very fast response •Toxicity – Cerebral anoxia
• Toxicity – Liver
Non-inhalant anesthetics
Ketamine Local anesthetics
•Dissociative anesthetic • Local infusion of incision site with lidocaine may
reduce amount of GA
•Produces sedation and immobility
• Lignocaine or bupivacaine provide considerable
•Increase BP, muscle tone, salivary pain relief
secretion
Urethane
•Slight respiratory depression
• Mutagenic and carcinogenic
•Administered in combination with • Use is strongly discouraged
xylazine, medetomidine or diazepam • Limited to non-survival procedures
Euthanasia
• Humane killing (sacrifice) of an animal
Xylazine
• Produce rapid unconsciousness and subsequent
•Centrally acting alpha-2 adrenergic
death
receptor agonist
• Without or minimal pain or distress to animal
•Analgesic and sedative effects
• Greek
•Muscle relaxation
–Eu = good
•Side effect in animals – transient
–Thanatos = death
hypertension, respiratory depression
Euthanasia vs. alternatives Ideal humane killing method
• Euthanasia • Emergency killing • Rapidly induces unconsciousness • Is ‘aesthetically’ acceptable to
and death without pain, distress, operator and client
“Euthanasia is the humane killing For disease control anxiety
of an animal for its own benefit” Irreversible • Can be administered
competently
• Humane Killing Reliable
by the staff available
Humane by definition: Greek:
“Killing by using a method that
‘eu’ = good, ‘thanatos’ = death • Does not put personnel at risk
causes rapid and irreversible loss • Produces a dead body which
of consciousness with minimum • Feasible for the circumstances in can be disposed of safely
question, e.g.
• Slaughter pain and distress to the animal”
Cost
“The killing of animals intended for
The animal can be restrained with
human consumption” little distress
Techniques should result in: Euthanasia – Why?
• At end of experiment or continuing adverse effect
•Rapid unconsciousness
• To provide blood and other tissues for a scientific purpose
•Cardiac arrest
• When levels of pain, distress and suffering exceed
•Respiratory arrest acceptable levels
•Loss of brain function • When the health or welfare of animals are grounds for
concern
• When they are no longer breeding
Personnel should have appropriate training and
experience in technique and animal handling • Unwanted stock or those with unsuitable characteristics,
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type or sex
Euthanasia objectives Choice of methods depends on:
• Reliability and irreversibility
• Species
• Minimum pain, distress, anxiety, apprehension • Age
• Minimum delay until unconsciousness • Availability of restraint
• Skill of personnel
• Safety and emotional effect on personnel
• Number of animals involved
• Compatibility with requirement and purpose,
• Method must be consistent with experimental
including subsequent use of tissue
goal
• Compatibility with species, age, health status
Methods of Euthanasia Inhalant agents
• Chemical methods • Either vaporized or delivered as a gas into
– Inhalants chambers or anesthetic circuits
– Non-inhalants
• Chambers designed to ensure
• Physical methods – even distribution of gas
– Animals rapidly exposed to high conc. of
agents
Inhalant agents – Dos & Dont Inhalant agents – Dos & Dont
• Used in many small animals
• Select agents that are not unpleasant to
inhale
• Not in rabbit
– react adversely to gases
– Show signs of excitation • Safety precautions should be taken
• Reptiles and amphibian hold their breath resulting
• Use appropriate gas scavenging equipment
in a long induction time
• Newborn are resistant to hypoxia • Death must be confirmed
Carbon dioxide Carbon monoxide
– At concentration above 60%, CO2 causes rapid loss of –CO binds to hemoglobin in RBCs – 250 times greater
consciousness due to narcotic effect in brain without affinity than oxygen
causing hypoxia
– Leads to hypoxia
– Death by direct depression of CNS, respiratory and
cardiac functions. – CO concentrations at 4 to 6%
– Effective and humane for most small animals – 2% sufficient to cause loss of consciousness, failure of
respiratory centre followed by death from cardiac arrest
– Heavier than air
– High risk to the operator
– Less risk to the operator
Volatile inhalational agents
Nitrous oxide (N2O)
• Sufficient air or oxygen during induction period
•not preferred due to lack of fast onset
to prevent hypoxia
•May produce hypoxemia and cardiac or respiratory arrest
•used in combination with other anesthetics
• Require gas scavenging apparatus
• Neither flammable nor explosive
Ether – Halothane
•Formerly used extensively & now prohibited in many
countries – Enflurane
•Irritant to mucus membranes – Isoflurane
•Risk of fire and explosion 18
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Halothane Isoflurane
• Quick acting and stress free in overdose for – Pungent odour
euthanasia
– Not used on animals which may be able to
• Depressant effect on CVS and Respiratory system hold their breath
– No hepatic metabolism
Enflurane – Useful for toxicological or microsomal studies
• Very little drug is metabolized in liver
• Preferred for metabolism or toxicological study
Non-inhalant agents
• Many are triple strength anesthetic agents Intravenous injection
• Some are neuromuscular blocking agents – most rapid and reliable
• Animals should be fully anesthetized before Intraperitoneal injection
neuromuscular blocking agents take effect – easier in species where the veins are small and difficult to
• Generally, two times the anesthetic dose produces penetrate
respiratory arrest – Takes longer to act
• Three time the anesthetic dose usually causes death – Irritation, transient pain, distress
quickly and uniformly in non-ventilated animals Intrapulmonic injection
– Avoid because of discomfort to animals
Barbiturates
Intracardiac route • Most widely used and accepted agents for
– Very painful and not always successful on first attempt most animals
– Only on unconscious animal
• Sodium pentobarbitone is most suitable
Oral and rectal routes agent
– Prolonged onset of action
• Depression of CNS
– Potential irritation of tissue
– Inadvisable • Cardiac and respiratory arrest
Intramuscular & Subcutaneous routes • Rapid euthanasia with minimal discomfort
– Not used as prolonged onset of action
Physical methods
Sodium pentobarbitone • Cause immediate loss of consciousness through
– IV (200 mg/kg) or IP (higher volume) physical trauma to the brain
– IV results in quicker death (anesthesia followed by • Most useful when pharmacological methods
anoxia) interfere with the purpose of experiment
– IP simpler to perform reducing the stress caused by
• Aesthetically less pleasant
handling
– Intracardiac injection only on fully anesthetized animal • Quick and certain and possibly least distressing
for animal in skilled hands
• Not in the sight or smell of other animals
Cervical dislocation
Decapitation
• Humane techniques for euthanasia
•Used for killing rodents, small rabbits,
amphibians, fish and birds • Frequently used for mouse, rats, guinea pig,
rabbit (weighing less than 1 kg), other rodents
•Severing of the neck of animal, close to the head
using a sharp instrument • Death confirmed by destruction of brain
•Guillotines is generally used • Aesthetically unpleasant
• If possible, sedated or anesthetized prior to
dislocation
Penetrating captive bolt Exsanguination
• Effective tool for larger animals • ‘Bleeding out’ by cutting carotid artery or jugular
vein
• Not effective in large pigs & mature bulls due to
thickness and density of the skull • Not to be used alone or in unconscious animals
• Site of penetration differs with each species
• Site of penetration differs with each species
• Not acceptable
• Only be carried out by suitable trained personnel
Birds: Due to quick clotting tendency
Reptiles/cold-blooded vertebrates: Due to slow metabolic
rate and tolerance to hypoxia
Microwave irradiation • Only on small animals such as mice, rats,
• Used as a mean to fix brain metabolites without small rabbits, amphibians and birds
loss of anatomical integrity of brain
• Requires specialist expertise
• Specialized apparatus designed for this
purpose is used • Humane as death occurs in milliseconds
• Focusing microwave beam precisely to the • Not routine procedure for euthanasia
specific part of brain
• Care as it is dangerous to operator
Methods acceptable for
unconscious animals
Pithing Potassium Chloride (10% solution,
– Effective way of killing some fish, i.v.)
amphibians and reptiles – Potassium is cardiotoxic
– Insert a sharp needle through foramen – Cause gasping, vocalizations, muscle
magnum into the base of brain spasms, convulsive seizures
– Rendered unconscious – Unpleasant for observer
– Only by competent personnel – Only when animal is fully anesthetized
Ethanol Air embolism
–500µl 70% ethanol (i.p.) into mouse – 5-50 ml/kg air (i.v.)
– Depress CNS – Occasionally in rabbits
– Gross loss of muscle control – Accompanied by convulsions,
– Become comatose followed by respiratory opisthotonos and vocalization
arrest – Very painful and unreliable method
– Only under anesthesia
– Only under full anesthesia
