Euthanasia

Statement of Purpose

These links and documents contain information about best practices for euthanasia.

Contributors

Breda Zimkus and Dirk Neumann. Some content generated during The American Society of Ichthyologists and Herpetologists (ASIH) Annual Joint Meeting - 2016, during an iDigBio sponsored workshop by the following individuals participating in the "Field to Database" Group of the aforementioned workshop: Breda Zimkus, Cesar Aguilar, Ben Frable, Meredith Mahoney, Zachary Randall, and David Wernecke.

Introduction

Studies involving animals should be conducted according to internationally-accepted standards. Prior approval or research protocols must be obtained if research involves vertebrate animals (e.g., IACUC) or human subjects (e.g., IRB). Note: journals are increasingly requiring that the name of the relevant ethics committees, as well as relevant permit numbers, must be provided at submission; therefore, this information has become important for museums to track.

Institutional Animal Care and Use Committee (IACUC)

Scientific procedures carried out on animals should minimize adverse effects and maximize the scientific benefit gained. These legal and ethical requirement are included under the laws and regulations of numerous countries worldwide, including the U.S. Animal Welfare Act (United States Code, Title 7, Chapter 54, Sections 2131-2159), the U.K. Animals (Scientific Procedures) Act 1986, the U.K. Animal Welfare Act 2006, the Animal Health and Welfare Strategy for Great Britain, Animal Welfare Strategy, Canadian Council on Animal Care in Science, among others. Researchers should be aware of laws and regulations associated with their home country and possibly the country of origin of the specimens collected.

Within the U.S., Institutional Animal Care and Use Committees (IACUCs) ensure that all projects involving the use of live vertebrates animals comply with federal regulations and guidelines. An institutional animal care and use committee (IACUC) is required by federal regulations for most institutions that use animals in research, teaching, and testing. The IACUC has a key oversight role, including the review and approval of animal use activities, and inspection of animal facilities. Federal regulations and guidelines dealing with animal welfare focus mainly on biomedical and behavioral research, teaching, and testing that takes place in the laboratory. The U.S. Department of Agriculture (USDA) Animal Welfare Act (AWA) regulations exempt field study ("any study done on free-living wild animals in their natural habitat, which does not involve an invasive procedure, and which does not harm or materially alter the behavior of the animals") from IACUC review. However, if the animals are confined, an invasive procedure is involved, or the behavior of the animal is harmed or materially altered, then the study must comply with federal regulations and standards. Since field studies often cannot satisfy the USDA definition, and the IACUC is also answerable to Public Health Service (PHS) Guidelines, researchers should have protocols reviewed by Institutional Animal Care and Use Committees (IACUCs) before specimens are collected. In addition, some funding agencies may require proposed projects involving use of any vertebrate animal for research or education be approved by the submitting organization's Institutional Animal Care and Use Committee (IACUC) before an award can be made.

IACUC review of such studies would be necessary and would focus on, but not necessarily be restricted to, such issues as:

1. Number of animals to be used in the study, and the stability of the population from which the animals are to be taken
2. Appropriateness of the methods used for capturing, immobilizing, and euthanizing animals
3. Training and supervision of the personnel involved with the study

Be aware that some euthanasia methods used may not be allowed by IACUC at your particular institution, or IACUC preferred methods (e.g. pentobarbital) may not be easy to complete in the field or may not be allowed in some countries.. Lastly, ensure that you are able to transport any controlled substances needed for approved euthanasia method.

General Recommendations for Processing Specimens

• Photograph specimens before euthanization to preserve coloration in life.
• Euthanize specimens individually, noting information in field notes as you proceed. See Field Notes for additional information about best practices.
• Select a euthanization method that will case minimal pain and leave the specimen appearing relaxed.

General Methods

check Simmons for standard v. less common methods, examples are from Auburn Tetrapod person, should we note this

• MS-222 (Tricaine methanesulfonate); use a freshly mixed solution every few days

-Example amphibian methods: Water bath of MS-222 solution. 5-10g/L for overdose (possibly use higher number to be sure and to speed up the process). Anesthetic levels would be less (Surgical anesthesia in adult frogs: 1 to 2 g/liter Tadpoles: 0.2 to 0.5 g/liter). 3 g/L seems to be the tipping point. (Penn IACUC guidelines) The solution should buffered to neutral (7.0-7.5 pH) with sodium bicarbonate before use.

-Example reptile method: It is a two-step process with an injection of a 1% solution followed by a 50% solution.

-Fish must be held 21 days before release if anaesthitized with MS-222

• Ice-bath
• Cooling then freezing (Shine et al. 2015)
• Clove oil (Note: This cannot be used if fish may be consumed by people)
• Pithing

-Flex the neck, identify the foramen magnum, insert a rigid metal rod cranially, and pivot/rotate the rod within the cranium to destroy the proximal spinal cord and brain.

Ichthyology Specific Methods

The following information is taken from Neumann (2010).
Fishes are extremely sensitive animals that require fundamentally different handling requirements compared to other vertebrates due to their physiochemical make-up. Unnecessary by-catch together with careless handling and injury to specimens can result in increased mortality rates and must be avoided. Unnecessary (physiological) stress, inadequate handling or manipulation of specimens in the field will result in discoloured, damaged specimens with limited or no scientific value. Unless the fishes are not already dead (e.g. gill net fishing), fishes have to be euthanased prior to tissue sampling. An overdose of approved anaesthetic immobilizes the fish and allows more efficient processing and sampling of the catch and reduces potential pain at contact with the fixative.

Appropriate ichthyological anaesthetics include:

• Chlorobutanol (trichloro-2-methyl-2-propanol, CAS-No. 57-15-8); a saturated solution of approximately 1-2 tablespoons per litre will narcotise within seconds and has no known negative degrading effects on the DNA extracted from tissues. Possible issues:

a) For euthanasia, dip specimens 3-4 times for few seconds into the anaesthetic, but do not leave them for longer periods in the fluid; the adhering narcotic on the gill surface is sufficient for sedation.

b) Species with thick mucilaginous layers (e.g. eels, sturgeons) show increased mucus secretions after Chlorobutanol treatment caused by the high salt concentrations; especially those species should be narcotised by repeatedly short dipping for only few seconds.

c) Low ambient temperatures and metabolism rates of fishes during autumn/winter demand higher Chlorobutanol concentrations which may lead to vascular gill swellings and subsequent gill haemorrhage due to the high salt concentrations of the anaesthetic.

d) Air breathing fishes are hardly affected and cannot be efficiently narcotised with this method.

e) Chlorobutanol is extremely dangerous if ingested and may cause irritation of skin and eyes.

• MS-222 (9-Tricaine Methane Sulfonate, CAS-No. 886-86-2) is the only approved substance (Europe, North America) for anesthesia of fishes; the fine powder can be dissolved in much lower concentrations (10 mg/l, thus avoiding possible negative effects of high salt concentrations of the narcotic; irritant but less irritating than Chlorobutanol.
• Clove oil (CAS-No. 8015-98-2) is a natural analgesic, the main ingredient Eugenol is used as narcotic mainly for marine organisms; Eugenol is water insoluble, for usage emulsify 1-5 ml clove oil in alcohol; irritant and hazardous in case of skin contact.
• Carbon dioxide can be an effective narcotic and is easily available (e.g., carbonated water or soda); narcosis can take longer with this method and may cause body contortions and muscle spasms that may affect the quality of the preserved specimens.
  

After short sedation in above detailed anaesthetics, or after electrofishing, fishes recover in well-oxygenated and ambient temperate water usually within minutes. For recovery, they should be placed in a separate tank or bucket. Only fully recovered specimens should be carefully released back into their environment to prevent injuries, damage or predation while still tranquilised.

Methods not discussed by Neumann (2010) but reported by attendees of ASIH fluid workshop, include:

• Directly into formalin (5-10%) (e.g., larval fishes)
• Directly into ethanol (70-95%)
• Freezing (ice and water bath)

Herpetology Specific Methods

Methods used for amphibians and reptiles generally differ since the latter can readily absorb euthanization agents through the skin. MS 222^TM (also called trichina, trichina methane sulfate) may be injected for animals with thick skins, or amphibians can be immersed into a solution if aquatic. Simmons (2002) suggests that the preferred method of euthanizing reptiles and large amphibians that have thick skins is by injection of aqueous sodium pentobarbital (e.g. Beuthanasia®, Euthasol®, Fatal-Plus®, Somlethal®).

From https://www.aaalac.org/accreditation/RefResources/SS_Amphib.pdf GUIDELINES FOR USE OF LIVE AMPHIBIANS AND REPTILES IN FIELD AND LABORATORY RESEARCH Second Edition, Revised by the Herpetological Animal Care and Use Committee (HACC) of the American Society of Ichthyologists and Herpetologists, 2004. (Committee Chair: Steven J. Beaupre, Members: Elliott R. Jacobson, Harvey B. Lillywhite, and Kelly Zamudio).

Adult amphibians (A) and reptiles (R) may be painlessly killed by use of a chemical anesthetic such as sodium pentobarbitol (R), hydrous chlorobutanol (A), MS-222 (A) (Tricaine methane sulfonate, marketed as Finquel(tm) by Ayerst, Inc.), urethane-ethyl-carbamate (A) (referred to hereafter as urethane), 10% ethanol (A) or similar anesthetics. In addition, amphibians can be euthanized by ventral application of Oragel, a 20% benzocaine gel, available over the counter world-wide (Chn and Combs, 1999). The euthanasia agent T-61 (National Laboratories) is very effective on reptiles (J. Johnson, pers. comm.). Use of such chemicals requires little additional time and effort, adds little to the bulk or weight of collecting equipment, and allows for preparation of better quality specimens. Urethane is carcinogenic, and caution should be observed with its use and field disposal. Other anesthetics may also be acceptable, especially since new agents are frequently developed. Gunshot is an acceptable and often necessary collecting technique, and is also recognized for euthanasia (Smith, 1986). The euthanasia procedure selected will depend upon the disposition of the carcass. For instance, while intracoelomic barbiturates are commonly used in the euthanasia of amphibians and reptiles, these chemicals are very destructive to tissues. If histologic studies of internal organs are to be conducted, then intracoelomic injection of barbiturates should be avoided. When special circumstances require that specimens (very small or larval animals, for example) be formalin-fixed without prior anesthetic killing, prior light anesthetization with an anesthetic such as MS-222 is recommended (Fowler, 1986).

Killing unanesthetized specimens by immersion in a formalin solution is unacceptable by IACUC standards, unless justified for scientific reasons. Formalin immersion of unanesthetized animals may, however, be the only way to adequately fix certain details of morphology critical to the successful completion of research.

Freezing – Rapid freezing following prolonged immersion in MS-222 and verification of cessation of respiration is acceptable. Freezing is NOT allowed as a sole method of euthanasia. Lowering the ambient temperature may facilitate handling, but the formation of ice crystals on the skin and in the tissues of an animal may cause pain and distress.

Additional information on euthanasia of amphibians and reptiles can be found elsewhere (Cooper et al., 1989).

Amphibians

Chemical

Physical

Amphibians and Fish: Larvae and Embryos

A. Larvae –Acceptable methods of euthanasia for larvae of these species are the same as for adult amphibians and fish as described above (i.e., chemical agent followed by secondary physical method). B. Embryos – Amphibians and fish are embryos prior to the larval stage (i.e., up to hatched embryos being able to independently feed), and therefore have yet to develop pain systems (AHAW Panel Report). Embryos of these species are disposed of depending on their experimental treatment and transgenic status, but care should be taken to recognize the variation among these species of the stage at which independent feeding begins. Use of rapid chilling and buffered MS-222 alone have been shown to be unreliable euthanasia methods for zebrafish embryos <3 dpf. To ensure embryonic lethality, immersion in sodium hypochlorite is recommended after immersion in MS-222 has been completed.

Animal Health and Welfare (AHAW) Panel. (2005). Aspects of the biology and welfare of animals used for experimental and other scientific purposes. European Food Safety Authority Journal Annex, 292, 1-136. (http://ec.europa.eu/environment/chemicals/lab_animals/pdf/efsa_scientificreport _en.pdf)

Reptiles

• Example reptile method: twice the dosage for euthanasia in mammals (for dogs the dosage is 1 mL/10 pounds). This comes out to be about 4.4 mL/Kg. Since herps are usually small the amounts are not that great, so over dose to be sure.

-Commercial Euthanasia Solution (Sodium pentobarbital 390 mg + sodium phenytoin 50 mg/ml) 0.22 ml/kg IV, ICL (~86 mg/kg pentobarbital)

-Beuthanasia can affect animals feeding on carcasses that have been injected, so any animals not ending up in the collection must be incinerated.

-Sodium pentobarbital is alkaline (11.0 pH) and can be cut with water to reduce pain at injection. Possibly also the case with Beauthanasia-D.

• Benzocaine (Orajel®) for amphibians or thin-skinned reptiles; apply to head or venter
• Chloretone/Chlorobutanol

-Make saturated solution, dilute or use. Use as a bath (25%) for amphibians, can inject reptiles (not sure about dilution).

-Use fresh solution periodically, solution loses efficacy with use.

• Ether
• Cotton soaked in ethyl acetate (i.e., nail polish remover) placed in closed jar with animal
• MS-222

-Immersion of amphibians (50mg/L??)

-Injection of reptiles

• Directly into formalin (5-10%) (e.g., larval)
• Directly into ethanol (10%)
• Freezing

Decapitation – The central nervous system of reptiles is extremely tolerant to anoxia. Therefore, methods of euthanasia that induce unconsciousness by interruption of blood supply to the head (e.g., decapitation, cervical dislocation, and exsanguinations) are inappropriate for reptiles when used alone. These methods can only be used on small reptiles that are already unconscious by a chemical agent or concussion, or when followed immediately with double-pithing, or freezing in liquid nitrogen or dry ice.