Lab animal Euthanasia methods for species specific

Acceptable conditions for noninhaled agents

1. Barbiturates and barbituric acid derivatives (Pentobarbital ≥3x anesthetic dose); IV access > IP, if possible

2.  ketamine and similar dissociative agents in combination with an a2-adrenergic receptor agonist (xylazine) or benzodiazepines (diazepam)

Acceptable with conditions for inhaled agents halo, iso, sevo, des, N20

• Halothane, isoflurane, sevoflurane, or desflurane ± N₂O, for prolonged periods to ensure death.  

Acceptable with conditions for inhaled agents N20

• Nitrous oxide should not be used alone for euthanasia, but can be used with other gasses

Acceptable with conditions for inhaled agents CO2

• Carbon dioxide—Carbon dioxide, ±  premedication with inhaled anesthetics

  • flow rate for CO2 systems should displace 30% to 70% of cage volume/min (dyspnea with lower flow rates, mucous membrane pain with higher)

  • Prefilled CO2 chambers are not recommended; no advantage to add O₂ (prolongs euthanasia)

  • Death may be confirmed by physical examination, ensured by an adjunctive physical method, or obviated by calibration and validation of the euthanasia chamber and process

Acceptable with conditions for inhaled agents CO

• CO in institutional situations where appropriately designed and maintained equipment and trained and monitored personnel can administer; not recommended for routine euthanasia

Acceptable with conditions for noninhaled agents,Tribromoethanol (Avertin)-

Tribromoethanol (Avertin)- if prepared (use within 2 weeks), stored (cool, dark room), and administered at the appropriate dosage. Controversial due to reports of peritonitis and death. Many IACUCs have approved its use in rodents. Use of secondary method..

Acceptable with conditions for noninhaled agents, ethanol

Ethanol—IP injections of 70-100% ethanol in small species (ex. mice, but NOT rats)  when physical methods are not desired or other euthanasia agents are unavailable

Acceptable with conditions S2.2.2.3 Physical Methods, cervical dislocation

Cervical dislocation- for mice and rats < 200 g, rabbits <1kg by trained personnel

Acceptable with conditions S2.2.2.3 Physical Methods, decapitation

Decapitation—mice/rats by trained/proficient personnel on clean equipment in good working condition

Acceptable with conditions S2.2.2.3 Physical Methods, Focused beam microwave irradiation

Focused beam microwave irradiation- for mice and rats if using an appropriately designed machine. Preferred method when immediate fixation of brain metabolites is required for research purposes

Unacceptable methods for inhaled anesthetics-nitrogen and argon

• Nitrogen and argon— (N₂ and Ar are acceptable with conditions in chickens, turkeys, and pigs), unable to acquire the O2 conc of <2% required for unconsciousness and death

Unacceptable methods for noninhaled anesthetics-KCL

Potassium chloride— IV or IC administration of potassium chloride as a sole agent.

Unacceptable methods for noninhaled anesthetics-NM block agents

Neuromuscular blocking agents—Paralytics are unacceptable for use as sole agent

Unacceptable methods for noninhaled anesthetics- injectable barbiturates and NM blocking agents

Injectable barbiturates and neuromuscular blocking agents—Combining injectable barbiturates and neuromuscular blocking agents in the same syringe for administration is unacceptable because the neuromuscular blocking agents may take effect before the animal is anesthetized

Unacceptable methods for noninhaled anesthetics-opioids

• Opioids—they are not rapidly acting, require high doses, and are not true anesthetic agents

Unacceptable methods for noninhaled anesthetics- urethane

• Urethane— as a sole euthanasia agent. It is a human carcinogen and has slow onset of action. Can be used as anesthetic prior to adjunctive method 

Unacceptable methods for noninhaled anesthetics- alpha-chloralose

• alpha-Chloralose—as a sole agent of euthanasia. It is a hypnotic with poor analgesic properties. Can be used as anesthetic prior to adjunctive method 

Euthanasia consideration altricial vs. precocial

Precocial young (i.e. guinea pigs) should be treated as adults. Differentiate from altricial young (i.e. mice and rats)

Acceptable euthanasia considerations for pregnant dams

  Euthanasia of the dam and fetuses in utero—Rodent fetuses along with other mammals are unconscious in utero and hypoxia does not evoke a response.

Acceptable euthanasia for noninhaled anesthetics neonates- injectable barbiturates

injectable barbiturates alone and in combination with local anesthetics and anticonvulsants; dissociative agents combined with a2-adrenergic receptor agonist or benzodiazepines

Acceptable with conditions for inhaled gases in neonates

Nonflammable volatile anesthetic agents like CO2 for up to 35 minutes (neonatal rats) or 50 minutes (neonatal mice) to die. Halogenated agents = must use secondary method

Acceptable with condition- hypothermia in neonates

• Hypothermia—The gradual cooling of fetuses and altricial neonates <10 days old, but animals should not come in direct contact with ice or precooled surfaces; use secondary method

• Altricial neonates < 5 days of age may be quickly killed by rapidly freezing in liquid N2. Do not have sufficient nervous system development to perceive pain

Acceptable with condition neonates- decapitation and cervical dislocation

Decapitation—using scissors or sharp blades for altricial neonates (< 7 days)

Cervical dislocation in mice and rats, pinching high spinal cord

Acceptable with conditions barbituates- rabbits

• Barbiturates and barbituric acid derivatives—IV ear access if rabbits are used to handling, otherwise IP (painful?); may need to sedate fractious rabbits to get IV access

Acceptable with conditions inhaled- rabbits

• Inhaled anesthetic overdose if already under anesthesia; will struggle and hold breath for unpleasant/unfamiliar odors. May work better with a pre-med.

• Carbon dioxide—50-60% flow vol/min with sedation. Aggressive at higher concentrations. 

Acceptable with conditions physical rabbits

• Cervical dislocation— for rabbits (<1kg) by individuals with a high degree of proficiency

  • “Hopper popper”

• Penetrating captive bolt if maintained in good working order, positioned correctly (slightly paramedian on frontal bone as close to the ears as possible) by trained personnel.

NPCB with animals restrained on non-slip flooring, if maintained in good working order, positioned correctly, and discharged twice in rapid succession at age-appropriate pressure

In general for  LABORATORY FARM ANIMALS, DOGS, CATS, FERRETS, AND NONHUMAN PRIMATES

• When animals to be euthanized are fully anesthetized, adjunctive methods such as bilateral thoracotomy, exsanguination, perfusion, and IV or intracardiac injection of potassium chloride are acceptable

• When rabbits are in a surgical plane of anesthesia, adjunctive methods such as delivery of potassium chloride, exsanguination, or bilateral thoracotomy are acceptable. Blunt force trauma only in emergency situations under extenuating circumstances (i.e. large injured rabbit)

There are no FDA-approved euthanasia drugs of aquatic animals, true or false

true

Common euthanasia methods aquatic species

MS 222 for 30 minutes after cessation of opercular movements plus secondary (physical) method is recommended; MS22 not effective for eggs, embryos, or larvae (<14 days)

Zebrafish considerations for euthanasia

Zebrafish (Danio rerio) may be euthanized by rapid chilling (2° to 4°C) until loss of orientation and operculum movements and subsequent holding times in ice-chilled water, specific to finfish size and age

• Adult zebrafish should be exposed for a minimum of 10 minutes following loss of operculum movement

• Fry 4 to 7 days after fertilization (dpf) for at least 20 minutes following loss of operculum movement

• unreliable for embryos < 3 dpf (MS 222 also unreliable); to ensure embryonic death these methods should be followed with another method

Best euthanasia method for embryos fish

Immersion in diluted sodium or calcium hypochlorite solution acceptable for embryos up to 7 days of age

Acceptable euthanasia of frogs

• African clawed frog (X laevis), leopard/bull frogs (Rana spp) best euthanized via a physical method while fully anesthetized

• sodium pentobarbital IV, intracelomically, or in the lymph spaces is an acceptable method, but high doses are often required and inconsistent time to loss of consciousness

Considerations for fiber and meat farm animals

General comments

o   Facility design and monitoring training of handler performance is monitored.

o   Most important indicator of death in food and fiber species is lack of heartbeat with secondary verification by lack of movement over 30 minutes or the presence of rigor mortis.

Acceptable barbiturate for cattle considerations

Barbiturates and barbituric acid derivatives

·       Drawbacks: administration requires animal restraint, personnel registered with DEA, use under vet supervision (use in food is extralabel), control over drug with accounting of amount used), fewer animal disposal options. Cost is also a deterrent due to the size and number of animals.

Acceptable with considerations for cattle-physical

• May be facilitated by use of neuromuscular blocking agents (succinylcholine) IM to rapidly immobilize then euthanized as soon as the bovid can be controlled.

Handgun considerations for euthanasia of cattle

limited to close-range shooting (within 1 to 2 feet or 30 to 60 cm) of the intended target. Calibers ranging from .32 to .45 are recommended

·       solid-point lead bullets are preferable to hollow-point bullets because they are more likely to traverse the skull

·       the .22 caliber handgun is not recommended for adult cattle

rifle considerations for euthanasia of cattle

More accurate and thus the preferred firearm for euthanasia if need to shoot from a distance; not ideal for indoor or short-range conditions

• Recommended to fire from the shoulder

·       General recommendations on rifle selection for use in euthanasia of cattle include .22, .223, .243, .270, .308

·       if a .22 LR is to be used the following conditions apply: (1) the firearm of choice is a rifle, (2) a solid-point bullet should be used, (3) it must be fired within close range of the skull (within 1 to 3 feet), and (4) the bullet must be directed so that proper anatomic placement on the skull is assured

·       Shotguns

·       all shotguns are lethal at close range, the preferred gauges for euthanasia of cattle are 20, 16, or 12

• appropriate from a distance of 1-2 yards

·       Number 6 or larger birdshot or shot-gun slugs are the best choices for euthanasia of cattle

·       for operator and bystander safety, the muzzle of a shotgun (or any other firearm) should never be held directly against the animal’s head

Penetrating captive bolt considerations for cattle

Penetrating Captive Bolt

·       used for euthanasia of mature cattle in field situations

·       bolt velocity minimums recommended for bulls are as high as 70 m/s

·       Muzzles of captive bolt guns should always be pointed toward the ground and away from bystanders in case of accidental discharge

·       Styles include in-line (cylindrical) and pistol grip (resembling a handgun); Pneumatic captive bolt guns (air powered) are limited to use in slaughter plant environments

·       captive bolt guns, whether penetrating or nonpenetrating, induce immediate loss of consciousness, but death is not always assured with the use of this device alone

·       an adjunctive method such as exsanguination, pithing, or the IV injection of a saturated solution of potassium chloride is recommended to ensure death when penetrating captive bolt is used; Adjunctive methods should be implemented as soon as the animal is rendered unconscious to avoid a possible return to sensibility

·       unlike techniques described for gunshot, the animal must be restrained for accurate placement of the captive bolt. And, unlike use of a firearm, proper use of the captive bolt requires that the muzzle of the device be held firmly against the animal’s head

·   use of the poll (bony protuberance on the top of the skull) for application of the penetrating captive bolt in slaughter plants is not allowed by regulations in the European Union because the depth of concussion in this region is less than that observed in frontal sites; use of the penetrating captive bolt at the poll is prone to operator error and misdirection of the bolt into the spinal cord instead of the brain

·   For euthanasia of bison, recommended method is gun shot with a 30-30, 270, 30-06

Adjunctive methods cattle: KCL

Potassium Chloride and Magnesium Sulfate

·       rapid IV injection of potassium chloride may assist in ensuring death after cattle have been rendered unconscious by penetrating captive bolt, gunshot, or administration of general anesthetics

• administer rapidly with 3-4 x 60 mL syringes with 14-16g needles pulled up in advance

·       magnesium sulfate death  may not occur as quickly as that with potassium chloride, both have low residue risk for predators/scavengers

Adjunctive methods cattle-exsanguination

Exsanguination

·       may be performed as an adjunctive measure to ensure death when necessary in an unconscious animal

·       not recommended as a sole method of euthanasia; rather it is reserved for use as an adjunctive method; information in the literature is inconsistent as to the length of time between the neck cut and loss of consciousness

• conduct with a rigid blade at least 15 cm and 6 inches long as soon as the animal is unconscious

Adjunctive methods of euthanasia cattle: pithing

  performed by inserting a pithing rod through the entry site produced in the skull by a bullet or penetrating captive bolt

·       operator manipulates the pithing tool to destroy brainstem and spinal cord tissue to ensure death

·       Muscular activity during the pithing process is often quite violent, but is followed by quiescence that facilitates exsanguination or other procedures

Acceptable methods barbituates sheep and goat

Barbiturates and Barbituric Acid Derivatives acceptable

Acceptable with conditions for neonatal goats: CO2

 Carbon dioxide inhalation can be used in young goat kids (<3weeks)

·   Place kids in a euthanasia chamber filled at a sufficient rate to achieve a CO2 concentration of > 70% by 5 minutes and to use a dwell time of 10 minutes to assure euthanasia

Acceptable with condition sheep and goat: gun

Gunshot

·       Firearms recommended for euthanasia of adult small ruminants include the .22 LR rifle; .38 Special, .357 Magnum, and 9 mm or equivalent hand-guns; and shotguns; solid point bullets and larger caliber preferred for large-horned adult rams

·       Don’t hold gun flush with the skull and no closer than 6-12 inches from the target

Acceptable with condition sheep and goat: Captive bolt

 Poll position (anatomic placement) preferred in sheep with horns as compared to the frontal position

·       using the poll position for application of captive bolts to sheep may be associated with rapid recovery of brain function. Therefore, adjunctive methods to ensure death should be applied immediately following loss of consciousness in small ruminants

·       Effective application of the captive bolt in sheep and goats is indicated by immediate loss of consciousness lasting until death by exsanguination or another adjunctive method

·       use of concussive methods (nonpenetrating captive bolt) has been determined to be an effective means of inducing insensibility that will persist until death caused by exsanguination

• Optimal Site: optimal site is on the intersec­tion of 2 lines, each of which is drawn from the later­al canthus of one eye to the middle of the base of the opposite ea

Adjunctive methods sheep and goat: KCL / MgSulfate

   Potassium Chloride and Magnesium Sulfate

o   rapid IV injection of potassium chloride is an effective method to ensure death in sheep and goats previously rendered unconscious

o   magnesium sulfate death may not occur as quickly as that with potassium chloride, both have low residue risk for predators/scavenger

Physical methods adjunctive: pithing and exsanguination

Exsanguination

·       may be performed as an adjunctive measure to ensure death when necessary in an unconscious animal

·       not recommended as a sole method of euthanasia; rather it is reserved for use as an adjunctive method; information in the literature is inconsistent as to the length of time between the neck cut and loss of consciousness

·       Pithing

·       performed by inserting a pithing rod through the entry site produced in the skull by a bullet or penetrating captive bolt

·       operator manipulates the pithing tool to destroy brainstem and spinal cord tissue to ensure death

·       Muscular activity during the pithing process is often quite violent, but is followed by quiescence that facilitates exsanguination or other procedures

Unacceptable methods for sheep and goats

Manually applied blunt trauma to the head, injection of chemical agents into conscious animals, xyalzine or any other α2 agonist followed by IV KCl or magnesium sulfate, drowning, air embolism, and electrocution with a 120V electrical cord, and exsanguination in a conscious animal

Acceptable for neonatal farm animals

Barbiturate overdose, gunshot, and captive bolt – calves, lambs, kids. Captive bolt require adjunctive methods: exsanguination, pithing, IV admin of barbiturate

Unacceptable for neonates farm animals

Manual blunt force trauma to the head is not acceptable for calves – skulls are too hard to cause immediate unconsciousness and death

Dam and fetus consideration for farm animals

Behavioral and EEG indicates that mammalian fetuses are insentient and unconscious throughout the first 75-80% gestation.

the fetus does not suffer when the dam is euthanized; nor is it likely to experience pain associated with other types of invasive procedures in utero

·       when animals are euthanized by physical methods that include exsanguination, delaying removal of the fetus from the uterus for a minimum of 5 minutes after hemorrhaging has ceased generally assures a substantial amount of anoxia-induced damage to the cerebral cortex that will normally prevent progression toward a return to sensibility

·       fetuses should be retained within the uterus for at least 15 to 20 minutes after maternal death has occurred to prevent the delivery of viable fetuses.

Common acceptable euthanasia techniques in pigs

• CO2, Ar, N2, gas mixtures, gunshot, nonpenetrating and penetrating captive bolts, overdose of an anesthetic administered by a veterinarian, electrocution, and blunt force trauma (in suckling piglets only)

• Method selection is dependent upon size and weight of the animal, avail-ability of equipment and facilities, operator skill and experience with the procedure, aesthetic concerns, human safety, and options for disposal of remains

Acceptable non-inhaled agents: Mature Sows, Boars, and Grower-Finisher Pigs

Barbiturates and Barbituric Acid Derivatives 

·       Mature sows, boars, and grower-finisher pigs can be euthanized by IV administration of barbiturates; dose 1ml/5kg up to 30 kg, then 1ml/10kg thereafter

·       Extralabel use

·       Disadvantage – tissues may not be suitable for diagnostics evaluation; concern for residues may complicate disposal options (scavenger risk)

Acceptable with conditions: Mature Sows, Boars, and Grower-Finisher Pigs-inhaled

CO2, N2, Ar Mixtures include N2 with CO2, Ar alone, Ar with CO2, and CO

Acceptable with conditions: Mature Sows, Boars, and Grower-Finisher Pigs-guns

Gunshot

·       Commonly used in growing and adult swine

·       3 sites: frontal, temporal, and from behind ear towards opposite eye

• The projectile should be directed toward the spinal canal

·       Twelve-, 16- or 20-gauge shotguns are recommended for mature pigs. The muzzle should never be held flush to the skull

·       Performed by trained personnel

Acceptable with conditions: Mature Sows, Boars, and Grower-Finisher Pigs-penetrating captive bolt

 Use of well-maintained penetrating captive bolt guns with ammunition appropriately selected for the size of the animal is acceptable with conditions as a method of euthanasia for growing and adult swine

·       requires restraint of the animal because the device must be held firmly against the fore-head over the site described for gunshot

·       Confirmation that the animal has been rendered insensible includes observation of the following: rhythmic breathing stops, no righting reflex is observed, vocalization is absent, and no palpebral reflexes or responses to noxious stimuli

·       Important that penetrating captive bolt gun and cartridge charges are maintained regularly/appropriately

·    

Acceptable with conditions: Mature Sows, Boars, and Grower-Finisher Pigs-penetrating captive bolt

  Electrocution

·       Electrocution as a sole method of euthanasia can achieve death via 2-step (electrodes head to head followed by head to flank) or single-step processes (head to opposite flank)

·       Electrical current must pass through the brain to achieve loss of consciousness, but then must cross the heart to cause fibrillation and cardiac arrest

·       Head-only electrocution induces a grand mal seizure and immediate unconsciousness, but death does not occur unless followed by head-to-heart electrocution or the application of an adjunctive method

·       The secondary step, whether head-to-heart electrocution or another method, must be performed within 15 seconds of onset of unconsciousness; otherwise, the animal may regain consciousness

·       With specific electrode placement, current of 110 V at a minimum frequency of 60 Hz applied for a minimum of 3 seconds is sufficient for euthanasia of pigs up to 125 kg

Adjunctive methods: exsang / pithing: Mature Sows, Boars, and Grower-

     Exsanguination

·       Not for sole method of euthanasia

·       Can be performed as second step to ensure death

·       Pithing

·       Not for sole method of euthanasia

·       Can be performed as second step to ensure death

Acceptable Nursery Pigs (70lb or Lighter)

o   Barbiturates and Barbituric Acid Derivatives

o   Nursery pigs may be euthanized by IV barbiturates

Acceptable with conditions: Nursery Pigs (70lb or Lighter): CO2

  CO2

·       Carbon dioxide alone or in combination with N2 or Ar

·       Pigs may be exposed to CO2 by gradually displacing ambient gases (introducing CO2 into the container) or by introducing the animals into a prefilled environment; exposure of pigs with normal respiration to a constant supply of 80% to 90% CO2 for a minimum of 5 minutes is necessary for effective euthanasia

·       An appropriate pressure-reducing regulator and flow meter capable of generating the recommended displacement rates for the size container being utilized is absolutely necessary

·       Small or incapacitated piglets have low tidal volumes and will not die as rapidly as larger, more viable pigs

·       For pigs euthanized in a chamber, recommended that the chamber volume be exchanged at least 2.5x to accommodate the wash-in-washout principle regardless of the pig’s size

·       Carbon dioxide containers should never be placed in an unventilated area – risk of human OD

Acceptable with conditions: Nursery Pigs (70lb or Lighter) :Captive bolt

Nonpenetrating Captive Bolt

·       Works better in younger pigs

·       Requires immediate application of an adjunctive method to ensure euthanasia

Acceptable:     suckling pigs

    Barbiturates and Barbituric Acid Derivatives

·       Suckling pigs can be euthanized by IV barbiturates

·       Extralabel use – need vet supervision

Acceptable with conditions: suckling pigs, Gas

CO2

·       May be effective for small groups of neonatal piglets

Acceptable with conditions: suckling pigs: Captive bolt

  Nonpenetrating Captive Bolt

·   Don’t need a secondary step to ensure death if used in appropriately sized/aged pigs

·       Effective for euthanasia assuming sufficient power and correct ammunition used

·

Acceptable with conditions: suckling pigs: blunt force trauma

Manually Applied Blunt Force Trauma

·       meets the definition of euthanasia- causing minimal distress with rapid loss of consciousness leading to death

·       frontal bones are not fully developed in suckling/young pigs, leaving the brain susceptible to blunt, high-velocity impact

·   AVMA encourages you to use an alternative

Poultry common euthanasia techniques

Includes: gas inhalation, manually applied blunt force trauma, cervical dislocation, decapitation, electrocution, gunshot, captive bolt, and injectable agents

Poultry acceptable

 Overdoses of Injectable Anesthetics, Including Barbiturates and Barbituric Acid Derivatives

·       Poultry can be euthanized with IV anesthetic overdose to include barbiturates

Poultry acceptabel with condition: CO2

With inhaled agents, birds should be checked to verify death because they may appear dead but can regain consciousness

·       CO2

·       The most commonly used gas for poultry euthanasia- no flow rate requirement for poultry

·       has successfully been used for euthanasia of nonhatched eggs (pips), newly hatched poultry in hatcheries, and adult birds

·       neonatal birds may be more accustomed to high concentrations of CO2 (incubation environments typically include more CO2), concentrations necessary to achieve rapid euthanasia of pipped eggs or newly hatched chicks may be substantially greater (as high as 80% to 90%) than for adults

Poultry acceptabel with condition: CO

May see more convulsions compared to CO2

·       flow rate should be sufficient to rapidly achieve a uniform concentration of at least 6% after birds are placed in the chamber

·       The direct application of products of combustion or sublimation is not acceptable due to unreliable or undesirable composition and/or displacement rate

·       Not recommended in most instances due to occupational health risk

Poultry acceptabel with condition: nitrogen and Arg

Nitrogen or Ar, mixed or used alone, with approximately 30% CO2 is acceptable with conditions for euthanasia of poultry provided the residual atmospheric O2 level can be reduced to and held at sufficiently low levels (2% to 3%)

·       Causes more convulsions (wing flapping) compared to CO2 in air

Low Atmospheric Pressure Stunning (LAPS): poultry

Reduction of atmospheric pressure: drawing a vacuum in a chamber euthanizes chicks and mature poultry in a manner comparable to anoxia base methods similar to inert gases such as N2 or Ar

Poultry acceptabel with condition:   Cervical Dislocation

       Cervical Dislocation

·       must result in luxation of the cervical vertebrae without primary crushing of the vertebrae and spinal cord (true for all animals, not just poultry)

·       The legs of the bird should be grasped (or wings if grasped at the base) and the neck stretched by pulling on the head while applying a ventrodorsal rotational force to the skull

Poultry acceptabel with condition:   decap

Decapitation

·       should be executed with a sharp instrument, ensuring rapid and un-obstructed severing of the head from the neck

Poultry acceptabel with condition: blunt force trauma

Manually Applied Blunt Force Trauma

·       acceptable with conditions for turkeys or broiler breeder birds that are too large for cervical dislocation

·       Operator fatigue can lead to inconsistency in application, creating concern that the technique may be difficult to apply humanely to large numbers of birds; AVMA encourages you to use alternatives

Poultry acceptabel with condition: electrocution

 Electrocution

o   Observed to ensure death or use an adjunctive method

Poultry acceptabel with condition: gunshot

     Gunshot

·       Free-ranging poultry and ratites (ostrich/emus) - when capture or restraint would potentially be highly stressful for the animal or dangerous for humans

Poultry acceptabel with condition: captive bolt

·       Penetrating and Nonpenetrating Captive Bolt

·       acceptable with conditions for euthanasia of large poultry (turkeys, broiler breeders, ratites, waterfowl, etc) when performed by competent personnel 

·       captive bolt must be used according to manufacturer’s recommendations and bird restrained

Adjunctive methods poultry

1.   Potassium Chloride or Magnesium Sulfate

·       IV or IC to a bird that is fully anesthetized or unconscious as a way to ensure death

2.               Exsanguination

·       it is acceptable to exsanguinate birds that are fully anesthetized or otherwise unconscious as a means to ensure death

Embryo and chick euthanasia considerations

  Embryonated eggs - prolonged exposure (20 minutes) to CO2, cooling (4 hours at 40°F), or freezing; in some cases inhaled anesthetics can be administered through the air cell at the large end of the egg, egg addling can also be used

·       Maceration with a specially designed apparatus causes immediate fragmentation and death of newly hatched poultry and embryonated eggs; death by maceration in poultry up to 72 hours old occurs immediately with minimal pain and distress

·       Maceration is an alternative to the use of CO2 for euthanasia of poultry up to 72 hours old

·       Maceration is believed to be equivalent to cervical dislocation and cranial compression as to time to death, and is considered to be an acceptable means of euthanasia for newly hatched poultry according to Federation of Animal Science Societies, Agriculture Canada, World Organisation for Animal Health, and EU

·       Maceration requires special equipment that must be kept in excellent working order

·       Newly hatched poultry must be delivered to the macerator in a way and at a rate that prevents a backlog at the point of entry into the macerator and without causing injury, suffocation, or avoidable distress before maceration

Equids consideration acceptable

Barbiturates or Barbituric Acid Derivatives: IV catheter placed in jugular vein (since large amount of fluid)

Acceptable with conditions equids: captive bolt and gunshot

• penetrating Captive Bolt and Gunshot

  • Conditions: well-trained personnel, firearm maintained, appropriate restraint

  • Anatomic site: intersection of 2 diagonal lines each running from outer corner of eye to 

base of opposite ear; personnel trained and safety precautions so no ricochets

Adjunctive for equids: usually under significant anesthesia

1. Potassium Chloride

• Saturated solution of potassium chloride injected IV or IC 

2. Magnesium Sulfate

• Use of a saturated solution given IV

3. Lidocaine

• 60mL of 2% Lidocaine injected intrathecally

Unacceptable for equids

1. Chloral Hydrate

• Almost immediate sedation, but onset of anesthesia is delayed unless it is combined with other anesthetics

• Adverse effects can be severe and aesthetically objectionabl

Special cases regarding NM blockers for euids

Emergency Situations (e.g. race track injury)

• Neuromuscular blocking agent (eg, succinylcholine) may be administered to a fractious/dangerous equid IM or IV, but it must be euthanized via an appropriate method as soon as it can be controlled

Avian biology considerations with euthanasia

• Birds lack a diaphragm and have a single coelomic cavity instead of separate thoracic and abdominal cavities.

• Must be careful with intracoelomic injections to not inject into the air sacs – could drown bird

• Hollow pneumatic bones like femur and humerus communicate directly with respiratory tract

• Pre-euthanasia and euthanasia drugs should not be administered via the IO route into the humerus/femur - drowning or irritation to the respiratory system may occur

• Birds have a greater capacity to process air due to unidirectional flow of air through lungs and greater surface area which makes them more sensitive than mammals to inspired toxicants.

Noninhaled agents acceptable avians

• IV euthanasia agent is the quickest and most reliable way to euthanize avians if it can be performed without fear or distress; if IV is impossible, injectable euthanasia agents can be administered via intracardiac, Intracoelomic (avoid air sacs), or Intraosseous (avoid humerus/femur) routes only if a bird is unconscious or anesthetized

  • Injectable agents can precipitate in tissues and induce artifacts at necropsy and on histopath

• Barbiturates and barbituric acid derivatives can be administered IV for euthanasia of anesthetized or properly restrained unanesthetized birds

Acceptable with conditions inhaled agents avian

Exposure to high concentrations of inhaled anesthetics (eg, halothane, isoflurane, sevoflurane, with or without N₂O) may be used as a sole method of euthanasia, or as a first step prior to other methods.

Acceptable with conditions CO2 avian

• CO₂

• High (> 40%) concentrations induce anesthesia initially followed by loss of consciousness

• Needs to be balanced with situational needs as rapid increases of CO₂ decreases amount of time to loss of posture and consciousness. Slower increases may cause less aversion or reaction, but increase exposure time

• Most turkeys voluntarily entered a feeding chamber filled with Ar (90%), or a mixture of Ar (60%) and CO₂ (30%), compared with 50% of turkeys that voluntarily entered the chamber filled with a high concentration of CO₂ (72%) alone, suggesting an aversion to 72% CO₂

• Neonatal birds may be more acclimated to high CO₂ concentrations. Unhatched bird’s environment has a high CO₂ concentration (as high as 14% in the embryonic chicken). Concentrations required to achieve euthanasia of newly hatched chicks are much higher (as much as 80% to 90%) than those for adults of the same species.  Diving birds have physiologic adaptations to hypercapnia and may require higher concentrations.

Acceptable with conditions CO avian

Same concept as the mammal section.  More convulsions noted compared to CO₂- research recommend CO₂ due to risk to personnel and convulsion

Acceptable with conditions Ar / N2 avian

• N₂ and Ar, and gas mixtures involving these gases (including mixtures with CO₂), have been used for euthanasia of poultry, but are NOT recommended for euthanasia of companion birds

• Birds appear to not have intra-pulmonary chemoreceptors for N₂ and Ar, and this may account for a lack of aversion during their initial exposure to hypoxia from these gases.

• Ar gas mixed with < 2% O2 was shown to induce rapid loss of posture (average, 11 seconds), convulsions (average, 22 seconds), unconsciousness, and death

Convulsions can occur during euthanasia with these inert gases, but because these signs occur after collapse and loss of consciousness, these gases are considered to be humane for the birds involved

Acceptable with conditions cervical dislocation avian

• Cervical Dislocation

• Generally used for small birds (< 200g) when no other method is available

• has been performed on birds as large as 2.3kg (5.1 lb)

Acceptable with conditions decaptitation avian

• Decapitation

• Acceptable with conditions for euthanasia of small birds (< 200g) 

• May be preferred over cervical dislocation under certain field conditions due to clear evidence of a successful procedure

Acceptable with conditions gunshot avian

• Gunshot

• Not recommended as a method for captive birds

Adjunctive methods avian

• All adjunctive methods are unacceptable in unanesthetized birds

1. Potassium Chloride

• May be administered via the IV or IC routes if bird unconscious or anesthetized

2. Exsanguination

• if unconscious or anesthetized

• Can be used if blood samples are necessary for diagnostic or research purposes

3. Thoracic Compression

• May be used as an adjunctive method for animals that are insentient (incapable of feeling)

Unacceptable methods avian

• Thoracic compression can result in significant levels of pain and distress before animals become unconscious

Thoracic compression is generally an unacceptable means of euthanizing animals that are not deeply anesthetized or insentient due to other reasons, but is appropriate as a secondary method for animals that are insentient

Euthanasia method considerations bird embryos

Bird embryos that have attained >80% incubation demonstrate EEG activity that is sustained with increases in amplitude suggesting potential pain perception in conscious embryos and should be euthanized by similar methods used in avian neonates such as anesthetic overdose, decapitation, or prolonged (>20 minutes) exposure to CO₂

Eggs at < 80% incubation may be destroyed by prolonged exposure (>20 minutes) to CO₂, cooling (<4°C for 4 hours), or freezing

Fish and aquatic invertebrate considerations

3 main terms specific to fish to describe ending life- euthanasia, slaughter, and humane killing.

• Immersion in an appropriate euthanasia solution is often an easier method than injectable forms of euthanasia

• IM injections with ketamine, a2 adrenergic receptor agonists, or Telazol can be given via pole syringe/dart gun to larger fish to facilitate handling and reduce handling stress for fish, but rarely achieve surgical planes of anesthesia in teleosts

• It is more difficult to be sure a fish or an aquatic invertebrate is dead as compared with birds and mammals

fish and aquatic prep environment considerations for euthanasia

• Withhold food for 12 to 24 hours prior to euthanasia to reduce regurgitation, defecation, and nitrogenous waste production

• Reduce light intensity if possible with adequate lighting for personnel in a quiet and nonstimulatory environment

• Water quality similar to that of the environment from which the fish originated or optimized for that species and situation for duration of euthanasia; water in which they have been house or captured should be used, and supplemental aeration and temperature control may be necessary

• Either prepare the immersion euthanasia solution with water from the fish housing system and transfer the fish into it OR a concentrated form of the anesthetic agent as a solution (with buffering agent, if appropriate) is introduced directly into the container of fish to minimize stressors

 Indicators of Death in Fish and Aquatic Invertebrates

• Loss of movement, loss of reactivity to any stimulus, and initial flaccidity (prior to rigor mortis)

• More useful indicators for many fish include respiratory arrest (cessation of rhythmic opercular activity) for a minimum of 30  minutes and loss of eyeroll (vestibulo-ocular reflex, the movement of the eye when the fish is rocked from side to side). The latter is no longer present in fish that have been deeply anesthetized or euthanized

• Presence of a heartbeat is not a reliable indicator of life, but sustained absence of heartbeat is a strong indicator of death

• Secondary methods of euthanasia recommended when appropriate after fish or aquatic invertebrate is anesthetized to ensure euthanasia

 Fish and Aquatic Invertebrates Intended for Human Consumption euthanasia considerations

• Slaughter is the  term used to refer to killing of animals intended for human consumption. 

• There are no FDA drugs approved for euthanasia of fish or aquatic invertebrates- use of any unapproved chemicals for euthanasia prohibits entry of the fish into the food chain

• CO2 avoids unacceptable residues but it’s not FDA approved for killing aquatic animals intended for food

• Physical methods that are acceptable with conditions:  manually applied blunt force trauma to the head, decapitation, electrocution, and pithing