UWSP IACUC SOP #5
STANDARD OPERATING PROCEDURE AMPHIBIANS AND
REPTILES
USED IN TEACHING AND RESEARCH (updated October 21, 2012)
4.1 Intent
The intent of this Standard Operating Procedure (SOP) is to describe the acceptable methods to transport,
house, care for, and euthanize amphibians and reptiles. This SOP is intended for personnel that use
amphibians and reptiles in research or teaching. This procedure is approved by the UWSP Institutional Animal
Care and Use Committee (IACUC). All investigators using live amphibians and reptiles in teaching or research
must submit a protocol to the IACUC for approval prior to animal use. This SOP is intended to expedite the
preparation of a protocol for review; it does not replace the investigator-prepared protocol. Reference to this
SOP is sufficient to answer many questions on the IACUC protocol submission form. If amphibian or reptile
use deviates from this SOP, the changes, additions, or omissions must be clearly outlined in the animal use
protocol and must be submitted for approval to the IACUC prior to implementation.
This SOP follows the Guidelines for the Use of Live Amphibians and Reptiles in the Field and Laboratory
Research (2nd edition, HACC, 2004) and the Report of the AVMA Panel on Euthanasia (AVMA 2007). Please
see the last page for full references and contact information.
4.2 Requirements of personnel implementing this SOP
The principal investigator or lead instructor must ensure that all individuals responsible for euthanasia:
a. Receive appropriate training to perform the procedure.
b. Adhere to IACUC-approved protocols and institutional policies.
c. Are knowledgeable in species differences in metabolism, respiration, and tolerance to cerebral hypoxia.
4.3 Approved sources of animals
“Whether collecting for permanent laboratory use, future release, or museum preparation,
each investigator should observe, and require of students and co-workers, a strict ethic of
habitat conservation.” (HACC 2004)
The principal investigator must obtain animals from approved sources, including licensed commercial suppliers
for laboratory and teaching applications, or field capture. Animals from approved commercial suppliers can be
ordered by Tammy Naczek, purchasing agent for the CNR. Suppliers used by UWSP faculty and staff include:
Bio Corporation
www.biologyproducts.com
800.222.9094 Alexandria MN 56308
Carolina Life Science
www.carolina.com
800.334.5551 Burlington NC 27215
Nasco
www.enasco.com
800.558.9595 Ft. Atkinson WI 53538
Investigators capturing animals from the field may require that permits be obtained from the Wisconsin
Department of Natural Resources (WI DNR), and from federal wildlife agencies for threatened or endangered
species. Evidence of proper permitting may be required by the IACUC for protocol approval. Investigators
should be familiar with herpetological capture techniques. Dr. Kevin Russell, associate professor of Wildlife
Ecology and Dr. Pete Zani, assistant professor of Biology, are experienced field herpetologists and can provide
assistance to new investigators. In addition, the UWSP Herp Society has active student members, many of
whom are well-trained and skilled field biologists.
4.4 Transportation
Animals collected in the field or shipped from a commercial distributor should be confined and transported in a
manner that does not expose them to temperature extremes, dehydration or excessive moisture, or
overcrowding. Caution must be employed when requesting shipment during the dangerously cold Wisconsin
winters. The Biology Stockroom manager, Ellen Jo Holquin, and the purchasing agent, Tammy Naczek, can
arrange for expedient and safe shipment and arrival of animals.
4.5 Housing
Amphibians and reptiles may be housed on a short-term basis (< 12 hours) in teaching or research labs.
Longer-term housing is available in the animal care facility (ACF) on the 2nd floor of the TNR or in IACUCinspected classrooms and laboratories. Cages should be constructed of non-porous materials that are easily
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cleaned and should be escape-proof. The appropriate size of the cage will vary by species but should
generally be no smaller than 4 body-lengths long and 2 body-lengths wide with a height appropriate to allow
normal locomotion (walking, burrowing, jumping, climbing, etc.; Pough 1992). The caging arrangement
should provide a thermal gradient including the preferred temperature and allow for appropriate moisture
levels. Food and nutrition adequate to maintain good health will be provided. Animals should be exposed to
natural lighting conditions if possible, and should not be maintained in constant light or dark.
4.6 Permanent marking
Amphibians and reptiles may be marked via toe-clipping (Perry et al. 2011). Removing toes does not affect
the probability of recapture or survival, or appear to increase the stress response of amphibians and reptiles.
While other techniques for permanent marking exist, many of these are less desirable for a permanent mark.
For example, paint marking is temporary; colored elastomers injected under the skin are costly and not
feasible in extremely small animals; cold branding using liquid nitrogen is often impracticable in field
situations; sewing colored beads into the tail of reptiles using wire is time consuming, may cause loss of the
tail, and has unknown effects on predators that ingest the beads and wire used to attach the marking. When
removing toes sharp scissors should be used and sterilized between uses with 70% ethanol.
4.7 Euthanasia
According to the AVMA Acceptable methods of euthanasia of amphibians include injection of barbituates (e.g.,
sodium pentobarbital), topical application of tricaine methane sulfonate (TMS, MS-222) or benzocaine
hydrocloride, inhalant administration of isoflurane or halothane, or CO or CO2 asphyxiation, and physical
methods of double pithing. Acceptable methods of euthanasia of reptiles include injection of barbituates (e.g.,
sodium pentobarbital), inhalant administration of isoflurane or halothane, and CO2 asphyxiation.
Conditionally acceptable methods of euthanasia of amphibians and reptiles include decapitation and pithing.
Unacceptable methods of euthanasia of amphibians and reptiles include cooling or freezing or administration
of clove oil.
4.7a Sodium pentobarbital
Inject sodium pentobarbital IP (into intraperitoneal cavity) or IV (intravenously) into amphibian or reptile at a
dose of 60 to 100 mg•kg-1 of body weight. Verify that an animal is dead before disposing of the carcass by
monitoring for respiratory or buccal movement. Time to effect may vary, with death occurring in up to 30
minutes. Follow injection with a physical method of euthanasia to ensure death. Examples of physical
methods include pithing and decapitation.
4.7b Tricaine methane sulfonate
Immerse amphibian in a solution of tricaine methane sulfonate (TMS, MS-222) at 500
mg•l-1 buffered with sodium bicarbonate to pH 7.0 – 7.5. Verify that an animal is dead
before disposing of the carcass by monitoring for respiratory or bucal movement. Time to
effect may vary, with death occurring in up to 30 minutes. Follow immersion with a
physical method of euthanasia to ensure death. Examples of physical methods include
Fig. 1. Benzocaine gel
pithing and decapitation.
should be applied to
Tricaine may also be used as an injection. Inject MS-222 into the lymph spaces or
pleuroperitoneal cavities at a concentration of 500 mg•l-1 in physiological saline. Follow
instructions as above to verify death.
the dorsal head region
between and behind
the eyes.
4.7c Benzocaine hydrochloride
Immerse amphibian in a solution of benzocaine hydrochloride solution at 250 mg•l-1. Verify that an animal is
dead by monitoring for respiratory or bucal movement. Time to effect may vary, with death occurring in up
to 30 minutes. Follow immersion with a physical method of euthanasia to ensure death. Examples of physical
methods include pithing and decapitation.
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Benzocaine may also be applied topically. It is available in several over-the-counter teething analgesics, such
as Orajel (maximum strength gel = 20% benzocaine in petroleum base). Apply gel to dorsal head area
between and behind the eyes (shaded region in Fig. 1) and to seat patch (ventral abdomen and thighs).
Follow procedure as above.
®
4.7d Isoflurane or halothane
Because many amphibians and reptiles are highly tolerant of anoxia (up to several hours for some species),
prolonged exposure times may be required for euthanasia by isoflurane or halothane overdose. Euthanasia by
overdose of isoflurane and halothane is more effective in active species and those with less tendency to hold
their breath.
Place amphibian or reptile in a container slightly larger than its body. Administer isoflurane or halothane into
the container by vaporizer or via a soaked cotton ball. Verify that an animal is dead by monitoring for
respiratory or buccal movement. Time to effect may vary, with death occurring in up to 30 minutes. Follow
inhalant administration with a physical method of euthanasia to ensure death. Examples of physical methods
include pithing and decapitation.
4.7e CO or CO2
Because many amphibians and reptiles are highly tolerant of anoxia (up to several hours for some species),
prolonged exposure times may be required for euthanasia by carbon dioxide asyphixiation. CO2 asphyxiation is
more effective in active species and those with less tendency to hold their breath.
Place amphibian or reptile in a closed container. Displace oxygen by adding carbon dioxide from a compressed
air cylinder. Verify that an animal is dead by monitoring for respiratory or bucal movement and watching for
movement of the heart. Time to effect may vary, with death occurring in up to 30 minutes. Follow CO2
asphyxiation with a physical method of euthanasia to ensure death. Examples of physical methods include
pithing and decapitation.
4.7f Decapitation and pithing
Anesthesia or sedation is suggested prior to the use of physical techniques unless scientifically justified by
user and approved by the IACUC. Use sharp equipment of the appropriate size for the species to be
euthanized to ensure that the head is quickly separated from the body rapidly and completely. This should be
used in conjunction with pithing (see 4.6g).
4.7g Pithing
Anesthesia or sedation is suggested prior to the use of physical techniques unless
scientifically justified by user and approved by the IACUC. Insert a rigid metal rod into
the foramen magnum, which is identified by the slight midline skin depression posterior
to the eyes when the neck is flexed (Fig. 2). Ensure that both the brain and the
proximal end of the spinal cord are destroyed.
4.8 Disposal of carcasses
Animals euthanized by injection, immersion, or application of drugs must be disposed of
in a manner such that they may not be consumed by humans or other animals, or
contaminate water sources.
Fig. 2. To pith a frog,
the probe is inserted
into the foramen
magnum, located
posterior to the eyes.
Researchers are strongly suggested to consider depositing the carcasses in the vertebrate museum collection,
or providing the carcass for use in other classrooms. Herpetology, Parasitology, and Animal Physiology
courses can often make use of whole or partial carcasses.
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4.9 Resources and references
Contacts
Tammy Naczek
Title
Manager of animal care
facility
CNR purchasing agent
Ellen Jo Holguin
Kevin Russell
Pete Zani
Herpetologist
Herp Society
student organization
Emily Starke
Phone
x 4823
Room
Responsibility
222 TNR
Live animals in TNR
x 4685
194 TNR
ordering frogs
Biology stockroom manager
x 3794
193 TNR
ordering chemicals, chemical safety & disposal
Herpetologist
x 2251
325 TNR
advising re: amphibian & reptile care
x 2237
445 TNR
advising re: amphibian & reptile care
check website for current members: www.uwsp.edu/stuorg/herp/
American Veterinary Medical Association (AVMA). 2007. AVMA Guidelines on Euthanasia.
https://www.avma.org/KB/Policies/Documents/euthanasia.pdf
Herpetological Animal Care and Use Committee (HACC) of the American Society of Ichthyologists and
Herpetologists. 2004. Guidelines for the Use of Live Amphibians and Reptiles in the Field and Laboratory
Research (2nd ed). www.asih.org/files/hacc-final.pdf
Perry, G., M. C. Wallace, D. Perry, H. Curzer, and P. Muhlberger. 2011. Toe clipping of amphibians and reptiles:
science, ethics, and the law. Journal of Herpetology 45:547-555.
Pough FH. 1992. Recommendations for the care and use of amphibians and reptiles in academic institutions.
National Academy Press, Washington, DC.
4.10 Procedure for use of Rana species in teaching laboratories
Frogs in the genus Lithobates (formerly Rana), which includes Northern leopard frogs, are obtained through a
commercial supplier via CNR purchasing, Tammy Nazcek, and delivered by overnight express (Fedex or USPS).
Upon receipt, animals are delivered to the UWSP Animal Care Facility and inspected for overall condition and
presence of red-leg and other parasitic or infectious diseases. Day to day care of frogs until laboratory use is
the responsibility of the ACF staff. Obviously diseased or injured animals are euthanized by isoflurane
induction (4.6d) followed by pithing (4.6e). Animals are housed in a flow-through stream bed in the ACF (TNR
220A) maintained at 25°C on a 12:12 light cycle. The stream bed allows for access to both wet and dry
habitats. Animals are fed crickets three times per week, or as required to allow maintenance of body mass.
Animals not used in a given laboratory exercise will be maintained in the stream bed under conditions suitable
for long-term health.
Prior to the use of frog cadavers in teaching laboratories, the specified number of animals is removed from the
stream bed and transferred to small aquaria containing water to a depth of ~ 3cm . Animals are transported
to the prep room of the teaching laboratory where they are euthanized by isoflurane induction (4.6d) followed
by pithing (4.6e). Euthanasia is performed by experienced personnel as indicated on the appropriate IACUC
protocol. Animals are not expected to experience pain or discomfort from the procedure, as isoflurane
effectively induces anesthesia and unconsciousness. Upon verification of death, animal carcasses or tissues
are prepared for use.
Following the laboratory, animal waste is collected in durable plastic bags and labeled as “Not for use as
food”. Animal waste is held in the carcass freezer in the ACF until disposal in semi-annual incinerations [Ellen
Jo Holguin, Biology stockroom manager]. Researchers are strongly suggested to consider depositing the
carcasses in the vertebrate museum collection, or providing the carcass for use in other classrooms.
Herpetology, Parasitology, and Animal Physiology courses can often make use of whole or partial carcasses.
end SOP#5 October 21, 2021
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