Veterinary Clinical Services Euthanasia Standard Operating Procedures

Background:
This document details the methods used by VCS for elective, fee-for-service and emergency euthanasia of all species housed at Yale. Animal use protocols that specify “YARC” or VCS-provided euthanasia refer to those methods outlined below. All methods used are in accordance with the 2000 AVMA Panel of euthanasia as either acceptable or conditionally - acceptable.

Definitions:

Elective euthanasia: Euthanasia to be performed by YARC (rats, mice, hamsters and gerbils only) or VCS (all species) at their convenience, in which no tissues or carcasses are harvested or saved for the investigator. Requests for elective euthanasia are usually accommodated within one working day of the request - no elective euthanasia is performed on weekends or holidays.

Fee-for-service euthanasia: Euthanasia which must be performed at a particular time or place so that the investigator can collect tissues for research purposes, or for which tissues or carcasses must be saved and given to the investigator.

Emergency euthanasia: Euthanasia that must be performed by VCS for humane or health reasons (e.g. a distressed moribund mouse, an emergency cesarean section)

Euthanasia card: A cage card, which is placed on an animal cage to request elective euthanasia of ALL ANIMALS in that cage.

Euthanasia authorization: A written permission or request to euthanize all species other than mice and rats.

Guidelines:
1.	Investigators who use either elective or fee-for-service euthanasia, or who will use the euthanasia methods described in this policy to euthanize their own animals, can fulfill the IACUC requirements for a description of the euthanasia method by referring to this policy.
2.	Investigators requesting elective euthanasia must place a complete, signed euthanasia card on the cage of the animal(s) to be euthanized. All animals left in the cage must have a source of food and water. Unweaned animals cannot be left without a source of nutrition. If the dam cannot be left with them to be sacrificed, the lab cannot request elective euthanasia - they must instead either euthanize the animals themselves, or bring the pups directly to the washroom and hand them directly to a YARC tech for immediate euthanasia.
3.	A written euthanasia authorization should be faxed (737 1943) or emailed (vcsstaff@email.med.yale.edu) to the veterinary clinical services office by the PI or his personnel. The authorization must include: PI’s name Date Name and phone or email of the person sending the authorization Species Animal identification information Location of the animal to be euthanized.
4.	Investigators requesting fee-for-service euthanasia must schedule this at least 1 full business day in advance, or the request may not be able to be accommodated.
5.	VCS will perform emergency euthanasia with the authorization of the PI or the PI’s lab personnel, or the authorization of a veterinarian.
Comment: “Pentobarbital” refers to medical grade pentobarbital or to commercially available euthanasia solution (Euthasol ®). Euthasol contains other ingredients that may affect tissue fixation or processing. This is a non sterile solution used exclusively for euthanasia. The active ingredients per ml are: 390 mg pentobarbital sodium, 50 mg phenytoin sodium. The inactive ingredients are: 10% ethyl alcohol, 18% propylene glycol, 0.003688 mg rhodamine B, 2% benzyl alcohol (preservative), water for injection, sodium hydroxide and/or hydrocloric acid may be added to adjust the pH.
Methods:
Companion animals (cat, dog, ferret) and Farm animals (pig, sheep, goat)
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 100mg/kg IV. Takes up to 1 minute Can also be administered intra-peritoneal in piglets, ferrets, kittens and puppies when vascular access is difficult. May be administered intrathoracic or intracardiac in anesthetized animals. May use premedication for sedation or a short-term anesthesia to facilitate the IV injection - Dog sedation – acepromazine 0.05-0.1 mg/kg IM. - Cat sedation – Ketamine 10 mg/kg + Xylazine 1 mg/kg IM. - Ferret sedation- Ketamine 20-30 mg/kg + xylazine 1-3 mg/kg IM or ketamine 30 mg/kg + Acepromazine 0.5 mg/kg. - Pig sedation – Ketamine 20mg/kg + xylazine 2mg/kg IM. Confirmation of death: verify absence of respiration, cardiac function, corneal reflex, muscle tone, and mucus membrane color.
Rabbits and guinea pigs
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 100-150 mg/kg intracardiac (anesthetized rabbit or guinea pig only) or intravenous – aural veins (sedated rabbit) or intraperitoneal (guinea pig). Anesthesia: ketamine 35-40 mg/kg + xylazine 5 mg/kg intramuscular. Sedation: acepromazine 2 mg/kg intramuscular. Confirmation of death: verify absence of respiration, cardiac function, corneal reflex, muscle tone, and mucus membrane color.
Rodents (rats, mice, hamsters, gerbils)
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 150-200 mg/kg IP.
2.	Carbon dioxide asphyxiation, with gaseous CO2 from a regulated tank, following the YARC protocol.
3.	Cervical dislocation of anesthetized mice only.
4.	Cervical dislocation without prior anesthesia (emergency euthanasia of mice for cesarean section only).
	Rationale for CD without anesthesia is that this procedure is performed to salvage the pups, and any anesthetic given to the dam would adversely effect the pups.
Confirmation of death: verify absence of respiration, cardiac function and toe/tail pinch reflexes.
Non Human Primates (NHPs)
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 100 mg/kg IV.
	All NHPs must be chemically restrained before handling IV catheter placement.
	Small species like squirrel monkeys or babies may be injected IP or intra-cardiac after being anesthetized.
	Chemical restraint: Ketamine 15mg/kg + Atropine 0.08mg/kg IM or anaesthetic gas induction when feasible.
Confirmation of death: verify absence of respiration, cardiac function, corneal reflex, muscle tone, and mucus membrane color.
Amphibians and Aquatic species (frogs, toads, fish, salamders)
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 120 mg/kg intra-coelomic or intra-cardiac (fish, frogs, toads, salamanders) or in dorsal lymph sac (frogs and toads). Death results in 15-30 minutes.
2.	Percutaneous anesthetic overdose: Immersion in 6.5%MS222 for at least 30 minutes (aquatic species only)
3.	Physical method: Immersion in ice water for immobilization and decapitation using guillotine followed by immediate double pithing. (aquatic species only)
Confirmation of death: amphibians may have reflexive movements after death. To verify death – check for respiration and reflexes like righting reflex and horizontal flotation. In case of doubt use mechanical methods described in (3).
Reptiles (turtles, lizards)
1.	Injectable anesthetic overdose: Sodium pentobarbital or Euthasol ® 150-200 mg/kg intra-coelomic or intra-cardiac.
Confirmation of death: reptiles may have reflexive movements and heartbeat after death. To verify death – check for respiration and reflexes like righting reflex, reflex withdrawal to noxious stimuli. Remove a vital organ (decapitate or open thorax and remove heart).