Standard on Rat and Mouse Euthanasia

Title

University of North Carolina at Chapel Hill Standard on Rat and Mouse Euthanasia

Introduction

Purpose

The standards and procedures described below provide guidance to all researchers and animal handlers for the euthanasia of rodents. Performing euthanasia correctly is an ethical imperative. Proper euthanasia minimizes pain/distress and reliably causes death. Adequate training and proficiency are required for all personnel performing any of the euthanasia methods described in this Standard. This document does not contain details for conducting certain procedures. Specific requirements for each technique are described in the Standard Operating Procedure (SOP) for Euthanasia Processes and Procedures. Euthanasia must follow the method(s) described in the approved Animal Care Application (ACAP). This Standard is based primarily on the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2020 Edition, implemented in October 2020.

Scope

This Standard applies to all personnel engaged in the euthanasia of rodents. This includes Division of Comparative Medicine (DCM) personnel and trained research personnel listed in the Institutional Animal Care and Use Committee (IACUC) approved ACAP. Anyone involved in rodent euthanasia at the University must comply with this Standard. Requests for exceptions to this Standard must be reviewed and approved by the IACUC prior to implementation.

Anyone involved in rodent euthanasia should also review the Standard Operating Procedure (SOP) for Euthanasia of Research Rodents, which provides a more detailed description of expectations related to each procedure.

Standard

Housing and Food and Water Requirements

All animals scheduled for euthanasia must be housed according to University of North Carolina at Chapel Hill ("UNC-Chapel Hill" or "University") cage density standards (Standard on Mouse Cage Density and Standard on Rat Cage Density). Cages marked for euthanasia should not be overcrowded or stacked on top of each other, as this blocks air flow into the cage. In addition, animals should have access to food and water if they will be maintained for more than 3 hours prior to euthanasia. Unweaned animals to be euthanized should stay with the lactating female until final preparations for euthanasia are complete.

Euthanasia of Sick or Injured Animals

Sick or injured animals that cannot be treated or relieved of pain and distress should be euthanized promptly. Research personnel are responsible for euthanizing sick, injured, or moribund animals as soon as these conditions are noted. These animals should not be held for later euthanasia by DCM personnel. DCM veterinarians have the authority to euthanize moribund animals, as well as animals experiencing more than momentary or slight pain and/or distress. If the DCM veterinarian is unable to contact research personnel regarding the care or treatment of a moribund animal, DCM veterinarians or designated DCM representatives are authorized to euthanize the animal. Ensure appropriate emergency contact numbers for research personnel are posted in the animal facility. For non-urgent unexpected illnesses, research personnel may contact DCM Veterinary Services to arrange for euthanasia and necropsy of the animals.

Euthanasia of Animals in Animal Housing Areas

The AVMA Guidelines for the Euthanasia of Animals: 2020 Edition indicates: “Methods of euthanasia likely to elicit distress vocalizations or pheromones that other animals in the room could hear or smell may be best performed in another location, if transportation distress can be minimized.”  Therefore, euthanasia of rodents should take place in designated procedure rooms.  It is acceptable to conduct rodent euthanasia in rodent housing rooms containing individually ventilated caging (IVC) and/or a fume hood that exhausts to the outside. The euthanasia method used must be described in the approved ACAP.

Section 1: Definitions - Details for conduct of procedures can be found in Supplemental SOP information.

• AVMA Euthanasia Guidelines classifications: Euthanasia methods are classified in the Guide­lines as acceptable, acceptable with conditions, and unacceptable.
  • Acceptable methods are those that consistently produce a humane death when used as the sole means of euthanasia.
  • Methods accept­able with conditions are those techniques that may require certain conditions to be met to consistently produce humane death, may have greater potential for operator error or safety hazard, are not well docu­mented in the scientific literature, or may require a secondary method to ensure death. Methods accept­able with conditions are equivalent to acceptable methods when all criteria for application of a method can be met.
• Adult - At UNC-Chapel Hill and for the purposes of this Standard, weaned mice and rats, older than 23 days of age.
• Preweanling - Animals no longer considered neonates that remain with the dam prior to weaning. Typically, this lasts until the animal is 23 days old, although some strains need to remain with the dam for longer and require an IACUC-approved Exception.
• Neonate - A newly-born mammal.  In the literature, rodents within 5-7 days of birth are considered neonates. For the purposes of this Standard, rat and mouse pups 7 days or less are categorized as neonates.  
• Fetus - Animals not yet born. Research has shown that rodents less than embryologic day 14 lack the neural development to respond to noxious stimuli; therefore, removal of fetuses from the dam up until this point results in painless death of the fetus. Euthanasia of fetuses at embryologic day 15 and beyond requires additional euthanasia methods (NIH, 1997).
• Altricial Animal - Altricial animals are born in an undeveloped state and require care and feeding by the parents (e.g., rat and mouse neonates).
• Precocial Animal - Precocial species are those in which the young are relatively mature and mobile from the moment of birth (e.g., guinea pigs).

Altricial animals, such as mice and rats, are hairless at birth. Per the AVMA Guidelines for the Euthanasia of Animals: 2020 Edition, afferent pain pathways of mouse and rat pups are not well developed until after postnatal day 5 to 7, with cortical development occurring later. Altricial rodents need to be distinguished from precocial rodents. Precocial rodents, such as guinea pigs, are haired at birth. Neonates of precocial rodents should be euthanized using the same procedures as would be used with adults.

• Unanesthetized Physical Euthanasia - Physical euthanasia (e.g., cervical dislocation, decapitation) of unanesthetized animals, irrespective of age, may only be performed by trained and certified personnel when the procedure is described in the approved ACAP. Training requirements are dependent on the animal species, age and euthanasia technique performed.

Section 2: Methods of Euthanasia by Age/Conditions (in order by prevalence of use)

Section 2a. Euthanasia of Adult Rodents

A. Gaseous Carbon Dioxide (CO2) (Non-automated)

Euthanasia by CO2 is acceptable with conditions in those species where aversion or distress can be minimized and must be conducted using appropriately designed and maintained equipment and trained and moni­tored personnel.  A compressed gas tank with a gradual-fill method and displacement rate from 30% to 70% of the chamber volume/minute to minimize pain and distress should be used. Methods include manual CO2 chambers and automated CO2 units (e.g., Euthanex Smartbox).The use of dry ice as a source of CO2 for euthanasia is not permitted. See Euthanasia of Research Rodents SOP for step-by-step procedures.

DCM procedure rooms have dedicated CO2 euthanasia chambers equipped with acceptable flow meters. Investigators wishing to perform CO2 euthanasia outside of DCM facilities must adhere to the Euthanasia SOP and must purchase the same equipment utilized by DCM. Appropriate flow meters may be purchased from VWR and can be found through the UNC-Chapel Hill purchasing system, E-Pro, or at the following website: https://us.vwr.com (part number: 89012-426). To purchase appropriately sized euthanasia chambers, contact DCM at 919-962-5335.

Unintended recovery of animals receiving CO2 must be prevented by using a secondary physical method. Unless otherwise described in the approved ACAP, confirmation of death is achieved by maintaining CO2 flow for 1 minute following respiratory arrest and utilizing one of the following secondary physical methods to ensure death: 1) cervical dislocation; 2) decapitation; 3) thoracotomy [open the chest cavity using sharp scissors or scalpel]; or 4) collection of vital organs.

B. Automatic Carbon Dioxide (CO2) Systems (e.g., Euthanex Smartbox)

The automated euthanasia system includes three stages, starting with the push of a button: charge, dwell, and exhaust. Automation also eliminates the need for physical verification of death and its emotional impact on personnel. All individuals who operate this equipment must first review the Euthanasia SOP and receive hands-on training and certification. Completion of training must be documented by the LAC or DCM. Specific machine operating instructions are available in the room with each unit. Refer to all postings and instructions within the room, whenever present. When the approved ACAP describes euthanasia utilizing automated CO2 systems, personnel are not required to perform a secondary physical method. (For detailed instructions, see Euthanasia SOP).

C. Injectable Anesthetics 

Injectable anesthetics can be effectively used to anesthetize animals prior to physical euthanasia. Common routes of injection include, but are not limited to, intraperitoneal (IP) or intravenous (IV). Injectable anesthetics are easy to administer, require minimal equipment, and avoid safety concerns associated with inhalants. The agent should be an anesthetic recommended for the species, and the dosage used should be equal to or greater than the standard published reference dose for anesthesia (e.g., a common dose of pentobarbital for euthanasia is 100 mg/kg, which is approximately twice the anesthetic dose for rats and mice). Once the injectable anesthetic is administered, allow sufficient time for the animal to lose consciousness.  It is imperative that death be verified after euthanasia and before disposal of the animal. An animal in deep narcosis following administration of an injectable agent may appear to be dead but might eventually recover. Death must be confirmed by examining the animal for cessation of vital signs or use of a secondary physical method. Consideration should be given to the animal species and method of euthanasia when determining appropriate criteria for confirming death.

Injectable anesthetics intended for use in adult rodents may not have the desired effect in neonates. In a pilot study conducted at UNC-Chapel Hill, few anesthetics were found to be reliably effective in neonates. Contact a DCM veterinarian at 919-962-5335 for more information about appropriate doses of injectable anesthetics.

D. Inhalant/Volatile Anesthetics

The use of inhaled Anesthetics delivered as a volatile gas to the respiratory tract to induce anesthesia is acceptable with conditions for euthanasia of laboratory rodents. Inhaled anesthetics (isoflurane, sevoflurane, enflurane) can be administered as the sole euthanasia agent or as part of a 2-step process, where animals are first rendered unconscious through inhaled anesthetic agent exposure and then subsequently euthanized by a secondary method. They may be administered by vaporizers via nose cone, through induction chambers, or by drop method in various types of containers. Chamber lids should fit tightly. Induction chambers must allow animals appropriate floor space without being too large. Chambers that are too large require an increased anesthetic volume and may result in slow induction time. Where applicable, induction chambers must prevent animals from direct contact with anesthetic soaked material. A secondary method must be used with isoflurane and other halogenated anesthetics to avoid possibility of recovery.

Personnel should minimize their exposure to these agents as some are considered chemical hazards. These agents should only be used in a chemical fume hood, ducted biosafety cabinet or in a system with an active gas scavenging device. If agents are used at the bench top and/or with a passive scavenging system, contact Environment Health and Safety (EHS) to have the system assessed. (For detailed instructions, see Supplemental SOP information link.)

E. Cervical Dislocation

The AVMA Guidelines indicate that “Manual cervical dislocation is acceptable with conditions for euthana­sia of mice and rats weighing < 200 g when performed by individuals with a demonstrated high degree of technical proficiency. In lieu of demonstrated technical competency, animals must be unconscious or anesthetized prior to cervical dislocation.” Cervical dislocation must be described in the approved ACAP. Personnel must be trained by the DCM training team, Veterinary Services (or delegates of these groups) and must demonstrate proficiency on anesthetized and/or dead animals first. Personnel using cervical dislocation must be certified in the technique on the approved ACAP and must consistently apply this method humanely and effectively.

F. Decapitation

This method is ac­ceptable with conditions if performed correctly, and it may be used in research settings when its use is required by the experimental design and approved by the IACUC. Decapitation is justified for studies where undamaged and uncontaminated brain tissue is re­quired. When performed properly this technique is nearly instantaneous and is considered humane. It must be described and justified in the approved ACAP and personnel should be trained on anesthetized or dead animals to demonstrate proficiency before conducting unanesthetized decapitation. Personnel using decapitation must be indicated as certified in the technique on the approved ACAP and must consistently apply this method humanely and effectively.

The equipment used to perform decapitation (e.g., scissors, guillotines) must be maintained in good working order and ser­viced on a regular basis to ensure sharpness of blades. The use of plastic cones to restrain animals appears to reduce distress from handling, minimizes the chance of injury to personnel, and improves position­ing of the animal. Guillotines that are designed to accomplish decapitation in adult rodents in a uniformly instantaneous manner are commercially available. The equipment used to perform decapitation must be kept clean, in good condition with sharp blades and should be maintained in good working order and serviced on a regular basis to ensure sharpness of blades. Dedicated scissors for euthanasia should be clearly labeled and tested regularly with the “Testing Scissor Sharpness” instructions posted.

The use of plastic cones to restrain animals appears to minimize stress from handling, minimize the chance of injury to personnel, and improves positioning of the animal in the guillotine. Individuals performing this technique must be properly trained and should recognize the inherent danger of the guillotine and take adequate precautions to prevent personal injury. Rodents acclimated to being handled are calmer, less stressed, and facilitate the process. (See Supplemental Euthanasia of Research Rodents SOP information link for more details on use of guillotines and scissors.)

Section 2b. Euthanasia of Rodent Fetuses and Neonates

Mammalian fetuses are unconscious in utero due to a combination of factors, such as low oxygen tension and hormonal influences in the uterus sup­pressing consciousness. Rat and mouse pups are born neurologically immature when compared with hu­mans, and their afferent pain pathways are not well developed until after postnatal day 5 to 7, with corti­cal development occurring later. Any laboratory rodents with altricial young, such as mice and rats, must be differentiated from rodents with precocial young, such as guinea pigs. For the purposes of euthanasia, precocial young should be treated as adults.

Euthanasia of Rodent Fetuses and Neonates - Acceptable Methods with No Conditions

Fetuses up to 14 days in gestation - Neural development at this stage is minimal and pain perception is considered unlikely. Euthanasia of the mother by an approved method or removal of the fetus(es) for research should ensure rapid death of the fetus(es) at this stage of development. If death of the dam is ensured, it is unnecessary to remove fetuses for euthanasia after the dam is euthanized and has undergone a secondary physical euthanasia method.

Fetuses 15 days in gestation to birth - The literature on the development of pain pathways suggests the possibility of pain perception at this point in gestation. If kept in utero, no secondary method is needed. If the fetuses are harvested for research purposes, analgesia is needed and must be described in ACAP. The specific technique(s) employed must be described in the approved ACAP. Consult with one of the DCM veterinarians to learn more about fetal sensitivity to specific anesthetic agents.

Injectable Agents - When mouse or rat fetuses or neonates are required for study, injectable barbiturates alone and in combination with local anesthetics and anticonvulsants or dissociative agents combined with α2-adrenergic receptor agonist or benzodiazepines are acceptable agents. Review information on the use of these agents in adult rodents in AVMA Guidelines.

Euthanasia of Rodent Fetuses and Neonates - Acceptable Methods with Conditions

Inhaled anesthetics - Nonflammable volatile anesthetic agents are effective for fetuses in utero. Neonatal mice may take up to 50 minutes to die from CO2 exposure and neonatal rats may take as long as 35 minutes. Adequate exposure time should be provided, or an adjunctive method (e.g., cervical dislocation, or decapitation) should be performed after a neonate is nonresponsive to painful stimuli. An adjunctive method (e.g., cervical dislocation, decapitation) must be performed when halogenated anesthetics are used on neonatal rodents to avoid the possibility of recovery.

Rapid freezing - Due to poorly developed afferent pain pathways, mouse and rat fetuses and neonates <5 days of age may be quickly euthanized by rapid freezing in liquid N2.

Decapitation - Because rat and mouse pups are born neurologically immature and their afferent pain pathways are not well developed until after postnatal day 5 to 7, unanesthetized decapitation using scissors or sharp blades is acceptable with conditions for altricial neonates. Some rodent neonates may have a tissue mass that is too large for scissors, so appropriate decapitation tools should be selected.

Cervical dislocation - Because rat and mouse pups are born neurologically immature and their afferent pain pathways are not well developed until after postnatal day 5 to 7, unanesthetized cervical dislocation by pinching and disrupting the spinal cord in the high cervical region is acceptable with conditions for fetal and neonatal mice and rats.

Hypothermia - the gradual cooling of fetuses and altricial neonates (mice and rats)to induce deep anesthesia and subsequent euthanasia is not a preferred method of euthanasia but is acceptable with conditions per the AVMA Guidelines. This method requires a secondary method to ensure death. Scientific justification is required in ACAP. There are no data to support the use of hypothermia as a single method, and it should be followed with a secondary physical method following loss of movement. As cold surfaces can cause tissue damage and presumably pain, the animals should not come in direct contact with ice or precooled surfaces.

Exceptions

Requests for exceptions to this Standard must be reviewed and approved by the IACUC.

Related Requirements

External Regulations and Consequences

• American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals, 2020 Edition

University Policies, Standards, and Procedures

• For more detailed guidance, please refer to the University Policy on the Care and Use of Vertebrate Animals for Research, Training and Teaching Purposes
• Mouse Cage Density Standard
• Rat Cage Density Standard
• Disposal of Rodent Carcasses Standard
• Standard Operating Procedures (SOP) for Euthanasia of Research Rodents

Contact Information

Important Dates

• Effective Date and title of Approver: June 28, 2002; UNC IACUC
• Revision and Review Dates, Change notes, title of Reviewer or Approver: July 2013, March 2015, October 2015, March 2016, July 2016, August 2017, March 2018; February 2021, UNC IACUC

Approved by: UNC IACUC