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IV Sources
A. SE LECTION
Guidelines for the selection of species for specific investigations are be-
yond the objectives of this document. However, certain general principles
should be borne in mind. In order of availability or accessibility to the
investigator, one may list wild-caught, wild, laboratory-reared, and labora-
tory-bred animals. Although the quality of the data may be in proportion
to the degree of definition of the animals (see Chapter III), the current
state of the art and of supply is such that the investigator should choose
the type of animal that is most abundant, yet adequate for his investiga-
tion. If simply a live frog is sufficient and if parasite or genetic control is
not necessary, choose wild or wild-caught animals, at least until the supply
situation has improved.
Perhaps the most important criterion in a selection process is to choose
a supplier whose animals are reliable. "Sources of Amphibians for Re"
search" (Nace et al., 1971) lists sources, species, and geographic origins
for approximately 150 species of anurans and urodeles. Most of these
species are only available from other investigators and on a seasonal or
"on hand" basis. The list also includes the several large dealers that sup"
ply over 80 percent of the amphibians used in research. The investigator
should learn to know the seasonal characteristics of his animal and the re-
sources and reliability of his supplier (Emmons, 1973~. He should deter-
mine whether the supplier is a collector or whether the supplier obtains
his animals from a primary supplier. The adequacy of the supplier's fa-
cilities must be determined. Those who expect to use many animals
should visit the facilities of the suppliers.
Endangered amphibian species, which are not available from commer-
cial suppliers, are listed in Appendix A. Reputable users and dealers do
not undertake the use or supply of endangered species.
39
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B. DEALER CARE
An important responsibility of the dealer is to reduce the stress on the
animals in his charge for the period he holds them in his establishment.
This presents problems when dealing with animals throughout the year
and from different geographical origins, as their physiological state is
highly dependent on season and origin (see Chapters II, V, and VI). It is
useful for the dealer to know the objectives of his users: For example, is
the user engaged in studies involving reproduction, cellular physiology,
or organ physiology? Dealers must establish those procedures in their
plants that will meet the requirements for the standards of animals they
choose to supply (see Chapter III). For example, hibernating northern
frogs can be held in dealer's facilities if water quality (see Chapter V,
Section B.2.a) and other conditions (see Chapter V, Section C.S and
Chapter VI, Section B.1) are controlled. In the past, hibernating frogs
have been kept by many dealers in small ponds that freeze over in
winter and are supplied by untreated water from underground springs.
In general, this is a poor practice; the water is often oxygen deficient
and too warm for optimal hibernation conditions.
Field studies have indicated that the hibernating frog in nature is
extremely sensitive to its surroundings, adjusting repeatedly throughout
the winter to changes in the environment. The two most significant envi-
ronmental variables appear to be temperature range and oxygen availabil-
ity, and it is not uncommon for the hibernating frog to move several hun-
dred yards from one location in a lake or stream to another to optimize
these two factors.
Interest has been shown by the research community in wild-caught
conditioned animals. Commercial dealers are attempting to meet this
demand for R. catesbeiana and in a more limited way for R. pipiens. The
latter is limited because R. pipiens is more difficult to feed artificially, and
the established price structure restricts the introduction of the necessary
technology.
C. ORDERING, SHIPPING, AND RECEIVING
1. Ordering
The user is responsible for ordering animals in such a manner that the
dealer can reasonably be expected to meet his desires.
The user must be specific in stating requirements as to size, geographic
origin, nature of the research, and level of definition required (see Chap-
ter III, Section B). Supply limitations must be recognized. For example,
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in dealers' pens most R. pipiens over 90 mm (3.5 in.) in body length are
female. Therefore, orders for " 105 mm (4 in.) grassfrogs (half male and
half female)" are frustrating to the dealer, if not impossible, to Din.
The purchasing department should include the name of the user on the
order and, if possible, reference to a previous order for similar animals.
This permits the dealer to maintain uniformity in shipments.
Order amphibians by "use date, " rather than "shipping date. " Leave
the time and method of shipment to the supplier. Though dealers limit
their guarantee to live arrival, reputable dealers attempt to ship under
optimal circumstances to ensure healthy arrival.
Do not order amphibians for delivery on Monday mornings. This yen"
orally necessitates a weekend holding period. Upon receipt of the animals,
it is important that they receive prompt attention as described below.
2. Shipping
Shipping is the responsibility of the dealer who must meet his guarantee
of live arrival. Any procedures that interfere with an amphibian's ability
to regulate its body temperature will result in the loss of animals. Am-
phibians, as ectotherms, regulate their temperature by selecting an appro-
priate habitat or posture-processes impossible in transit. The supplier
must provide the correct environment for shipping. The-most difficult
times for shipping ranids are periods of temperature and humidity em
tremes. Inadequate cooling in the summer results in what is known in the
jargon of amphibian dealers as "hot frogs"-the state of being hyperactive
to the point of convulsion.
Although frogs and salamanders may be shipped in boxes containing
sphagnum moss or other protective material, damage caused by improper
handling en route may occur (Gibbs e! al., 1971~. Aquatic forms such as
tadpoles, axolotls, and Xenopus may be shipped in thermos jugs or plastic
bags in insulated containers. The numbers of animals that can be shipped
per unit volume is critical and varies greatly with species and size. In the
summer, the containers should include ice to minimize activity and sealed
containers should also include oxygen to facilitate respiration.
A given shipping container should contain animals of only one species
In some cases sexes should also be separated, e.g., amplexing animals.
Hibernating animals may be shipped submerged in cold water 2-4 °C
(36-39 °F), nonhibernating animals in well~ventilated cartons. Before
shipment, food should be withheld from amphibians for a sufficient time
to prevent contamination of the shipping container. This is particularly
true of aquatic forms such as Xenopus.
Most amphibian deliveries to laboratory facilities should be either by
Air Parcel Post Special Delivery or Air Express.
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Accompanying the shipment, suppliers should include information as
follows if the amphibians are to be used for research:
· Simple directions for handling and holding the animals after their
arrival with references to more extensive sources of information such as
this document and
. The standard met by the animals and the information specified by
the criteria for the standard (see Chapter III).
3. Receiving
The user is responsible for receiving the animals in such a manner that the
specifications in his order and the efforts of the dealer are not nullified.
The details of procedures to be followed upon receipt of amphibians de-
pends on the species. However, certain general principles can be stated.
Unless the user is prepared to develop suitable facilities and meet demand-
ing personnel requirements for the long-term care of amphibians, they
should not be held for more than a few days. If long-term storage or ob-
servation is necessary, prepare the required facilities prior to placing the
order for animals (see Chapters V and VI). It is important to realize that
seasonal variations determine the hardiness and the nature of the care
required by most amphibians. Thus, northern R. pipiens that have spawned
are not as hardy as animals collected in the fall of the year after feeding
actively throughout the summer prior to hibernation.
a. Aquatic Amphibians
The aquatic animals such as axolotls, Xenopus, and tadpoles should be
transferred immediately from the shipping containers to previously pre-
pared containers. Last minute cleaning of spare containers often results
in shoddy cleaning and inadequate rinsing to remove soap or detergent!
Do not discard the water in which the animals were receded. The rapid
transfer of animals to water that differs radically in quality may result in
severe shock or death. As a regular practice, animals should be conditioned
to the water in the laboratory by slow dilution of the transport water.
Strain it through gauze and use it in the clean containers. Save half of this
water in case the following steps cause the animals to show discomfort.
If this water in which you will place the animals is warmer than the tem-
peratures specified in Chapter VI for the species in question, add ice made
from dechlorinated laboratory water (see Chapter V, Section B.2.a), but
do not expose the animals to temperature changes greater than about 1 °C
(2 °F) per hour. After the animals become quiescent and give signs of
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adapting to the new environment, add no more than one~fourth the vol-
ume of laboratory water. If no distress is obvious after several hours, more
fresh water may be added. Repeat these steps until the animals have ads
lusted to your water supply. If they show distress at some step, return
them to the previous water mixture in which they did not show distress
and prepare new water from a different source.
Water quality is only one aspect of their new environment. In addition,
container size, shape, and transparency must be considered. individuals
with obvious stress or damage should be isolated and treated as discussed
in Chapter IX, Section B.2. After acclimatization, normal care procedures
may be followed.
b. Terrestrial Amphibians
Because of their shape and posture, terrestrial urodeles ship quite well un-
less seriously overheated or dehydrated. They may be handled in much the
same way as the aquatic animals, i.e., they should be moved to permanent
housing through a series of gradual temperature steps.
To avoid confusion and damage to terrestrial anurans, the shipping box
should be emptied into an enclosure such as a large plastic garbage can con-
taining 0.15-0.25 m (6-10 in.) of water in order to deny the frogs a surface
from which they can jump. Wash them free of packing material and sepa-
rate the apparently healthy from obviously sick or damaged animals. In the
winter, the wash water should be cooled to the temperature of the animals;
in the summer, water at tap water temperature may be used. Do not wash
them vigorously under a full flow of water from a tap or hose. Plastic
vegetable crispers with slide-on lids are good isolation containers. The con-
tainers should be filled with water to about shoulder height for leopard
frogs; toads need only a damp bottom.
From October through March and depending on the research objectives,
wild-caught nonconditioned animals from north of the ice line may be re-
turned to hibernation. This may be done by taking them from shipping
temperature to hibernation temperature through a series of 9-12 h at each
of several intermediate temperatures with each step being 3-5 °C (6.4-
10.7 °F). Alternatively, if 20 or more gallons in the hibernaculum are used
(see Chapter V, Sections C.5 and 6.a), the animals may be placed in the
water at room temperature. They will acclimate as the water cools when
the container is placed in the coldroom. This procedure should be followed
within hours after receipt of the animals if they are to be used for their
eggs (see Chapter It, Section B.2.e). Never use this chilling procedure with
summer or Mexican frogs.
Adult R. pipiens to be used at room temperature, or whose eggs are not
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5.
1. Connecticut
Maine
Massachusetts
New Hampshire
Vermont
2. Delaware
Maryland
New Jersey
New York
Rhode Island
~. Michigan
Minnesota
North Dakota
South Dakota
Wisconsin
4. Idaho
Montana
Oregon
Washington
Wyoming
Kentucky
North Carolina
Tennessee
Virginia
6. Illinois
I ndiana
Ohio
Pennsylvania
West Virginia
7. Colorado
lows
Kansas
Missouri
Nebraska
8. Alabama
Florida
Georgia
Mississippi
South Carolina
9. Arkansas
Louisiana
New Mexico
Oklahoma
Texas
1 0. Arizona
California
Nevada
Utah
1 1. Alaska
Hawaii
JAN FED MAR APR MAY JUN Jo
FIGURE 13 Amphibian closed season regulations. Hatched time spaces represent
the closed seasons. Numbers in the squares refer to dates. The states are listed by
tiers: northern, 1 4; central, 5-7; southern, 8-10; and other. Data on the Canadian
provinces may be found in Appendix B. Data assembled with the assistance of
Stafford Cox, student of environmental law, University of Michigan.
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45
needed, may be maintained at room temperature at any season (see Chap-
ter V, Section C.4~. They must be fed, however, if they are to be kept
more than a few days.
D. Legal Aspects
It is incumbent upon both dealers and users of amphibians to be aware of
and abide by the laws established to regulate their use. At the federal level
the Endangered Species Conservation Act provides for the conservation,
protection, and propagation of any wild mammal, fish, wild bird, amphib-
ian, reptile, mollusk, or crustacean threatened with extinction or likely
within the future to become threatened with extinction. It further directs
that, insofar as is practicable and consistent with the primary purposes of
bureaus, agencies, and services, all federal departments and agencies shall
exert their authorities in furtherance of this Act. The secretaries involved
determine, at their discretion, the endangered species based on the best
scientific and commercial data available; and after consultation with the
affected states, interested persons, and organizations, the secretaries pub-
lish the endangered species list in the Federal Register. Upon the petition
of an interested person, the secretaries conduct a review of any listed or
unlisted species proposed to be removed or added to the list. The evidence
required for such a review pertains to any one of the following factors:
the destruction, drastic modification, or severe curtailment of habitat;
overutilization for commercial, sporting, scientific, or educational pur-
poses; the effects of disease or predation; the inadequacy of existing
regulatory mechanisms; or other natural or man-made factors affecting
continual existence of the species in question. With respect to amphibians,
the documentation and quantification of these factors is not defined. A
listing of amphibians judged to be endangered is to be found in Appendix A.
State regulations concerning amphibians are highly variable as seen in
Appendix B. These regulations are summarized in Figure 13, which shows
the closed seasons for "frogs" in each state. in this figure, the states are
assembled by geographic regions. Figure 13 and Appendix B do not show
that collection for scientific purposes is exempt or given special treatment
by the law in some states. Caution should be exercised in interpreting
Figure 13 as state regulations affecting frogs are currently under review
in many states.
Representative terms from entire chapter:
chapter iii
