[NHCOLL-L:1319] RE: amphibian euthanasia agents compromising tiss ue conservation...

[Simmons, John E]

This is a subject of increasing concern for field workers, as some of the
preferred chemicals for euthanasia are difficult to obtain or illegal to
take out of the US.  Below is an excerpt from the draft revision of my 1987
publication on Herpetological Collecting and Collection Management (the
revision should be published in 2002, if all goes well).  As the draft is
currently undergoing review, any comments that readers might have on this
section will be appreciated.  If there is interest, I will post the list of
references cited in this section, too.

--John

John E. Simmons
Collection Manager, Natural History Museum
and
Coordinator, Historical Administration and Museum Studies Program
University of Kansas
Dyche Hall
1345 Jayhawk Boulevard
Lawrence, Kansas 66045-7561
Phone 785-864-4508
FAX 785-864-5335
jsimmons at ku.edu

Killing
Specimens should be dispatched quickly by a method that will cause minimal
pain and leave the specimen relaxed (Anon. 1987).  

Reptiles
The preferred method of killing reptiles is by injection of aqueous sodium
pentobarbital (e.g., Nembutal(tm)) into the heart (Karlstrom and Cook 1955,
Lowe 1956).  Use 1.0 to 5.0 cc for large specimens, less than 1.0 cc for
smaller specimens (Lowe 1956).  The solution may be diluted 1:5 to 1:10,
depending on the size of the specimen.  The heart may be located in most
reptiles by gentle pressure from one or two fingers (not your thumb) on the
chest.   As a general rule, in lizards the heart is just below the junction
of the arms in the middle of the chest.  In most snakes, the heart is about
1/4 of the way down the body from the head. 
Other Methods for Killing Reptiles
· Injection of the animal tranquilizer, MS 222(tm) (also called tricaine,
tricaine methanesulfonate, or Finequel(tm)) (Karlstrom and Cook 1955, O'Shea
1992).  Use about ½ teaspoon in 20 ml of water to make an injectable
solution (L. Grismer, pers. comm.).  
· Lidocaine(tm), also known as lidocaine hydrochloride,
2-diethylamino-N(2,6-dimethylphenyl) acetamide, Anestacon(tm),
Dilocaine(tm), L-Caine(tm), Lida-Mantle(tm), Nervocaine(tm), Octocaine(tm),
and Xylocaine(tm). 
· Livezey (1958) recommended the use of procaine hydrochloride for reptiles
and amphibians.  Other names for this drug include procainamide
hydrochloride, Procan(tm), Procanbid(tm), and Pronestyl(tm).  A 10% mixture
is prepared by dissolving tablets (which contain 0.07 g of procaine
hydrochloride) or crystals in water, or by obtaining the drug as a prepared
solution.  About 0.05 cc per gram of body weight should be injected into the
pleuroperitoneal cavity just posterior to the heart.
· Lambert (1967) suggested using Anectine(tm) (succinylcholine chloride).
Other names for this drug include diacetylcholine chloride, Quelicin(tm),
and Sucostrin(tm).  It is available as a white powder.  The recommended
solution is 25 mg/cc of water.  Inject into the abdominal and pericardial
cavities.  Small reptiles require 0.05 to 0.20 cc, large reptiles require up
to 2.5 cc or more.
· Reptiles may be euthanized by placing them in a killing jar containing
cotton soaked in nail polish remover (ethyl acetate; see O'Shea 1992), or
acetone (L. Grismer, pers. comm.).   
· Application of a small amount of benzocaine (ethyl p-aminobenzoate) based
oral analgesic (e.g., Oragel®) to the head (Altig 1980) or venter (Chen and
Combs 1999) may be used to kill small, thin-skinned reptiles.
· Succinylcholine chloride (Lambert 1967)
· Chloroform.
· Ether.
· Drowning in warm water.  
· Less desirable methods of killing include the injection of isopropyl
alcohol into the brain through the optic foramen, or by freezing (Meylan
1989).  The drawbacks to these two methods are that you may damage the brain
case of the specimen by the former, and the latter may result in a frozen
specimen.  Specimens that have been frozen do not preserve well, due to
ruptured cells and the inability of fixatives to penetrate frozen tissues
(see Frozen Specimens).  

Amphibians
The preferred method for killing amphibians is to relax them in a solution
of hydrous chlorobutanol (1,1,1-trichloro-2-methyl-2-propanol; also known as
Chloretone(tm); see Snyder 1915).  Prepare the killing solution by making a
saturated solution of hydrous chlorobutanol crystals dissolved in 95% ethyl
alcohol; use a few milliliters of this saturated solution to about 500 ml of
water.  Alternately, a killing solution may be prepared by dissolving one
teaspoon of hydrous chlorbutanol crystals in one liter of water (McDiarmid
1994).  
Other Methods for Killing Amphibians
· Application of a small amount of benzocaine (ethyl p-aminobenzoate) based
oral analgesic (e.g., Oragel®) to the head (Altig 1980) or venter (Chen and
Combs 1999). 
· Submersion in a 10% ethyl alcohol and water solution.
· Submersion in solution of procaine hydrochloride.  Refer to the
recommendations of Livezey (1958) described above.
· Drowning in warm water.
· Use of a killing jar containing cotton soaked in nail polish remover
(ethyl acetate; see O'Shea 1992).  
· Very large amphibians, particularly those with thick skins, may be killed
with an injection of sodium pentobarbital or any other of the methods
described above for reptiles.  


-----Original Message-----
From: josh butler [mailto:boiga13 at hotmail.com]
Sent: Wednesday, November 14, 2001 4:02 PM
To: NHCOLL-L at lists.yale.edu
Subject: [NHCOLL-L:1318] amphibian euthanasia agents compromising tissue
conservation...


Greetings,
Currently I'm involved with putting together an internal manuscript for 
procedures in the Museum of Southwestern Biology.  When I got to the 
euthanasia of amphibians, my experience had told me to look for 
alternatives.  Just for the purpose of euthanasia, it is suggested by the 
AVMA to use a Benzoic acid (ethyl p-aminobenzoate).  My question to the 
group is, has anyone considered/researched whether or not this may 
compromise tissue preservation.  Are there references in the natural history

collections literature that discuss euthanasia methods vs. preservation 
integrity.
I have considered other alternatives, and in my experience (animal 
technician @ UC Berkeley) Benzoic acid has been the cheapest and most humane

method of euthanasia for amphibians.  However, my experience is limited and 
admit that my research into this topic is still ongoing.  I appreciate any 
information you can forward my way.

Joshua A. Butler
Undergraduate Biology        Curatorial Assistant
University of New Mexico     Museum of Southwestern Biology
Albuquerque, New Mexico      (505)277-5130
cell:(505)301-3816           boiga13 at hotmail.com

"To knock a limpet from the rock does not even take cunning, that lowest 
power of the mind." -- Charles Darwin


_________________________________________________________________
Get your FREE download of MSN Explorer at http://explorer.msn.com/intl.asp