AMER. ZOOL., 13:85-90 (1973).
Use of Bullfrogs in Biological Research
DUDLEY D. CULLEV, JR.
School of Forestry and Wildlife Management, Louisiana Stale University,
Baton Rouge, Louisiana 70803
SYNOPSIS. This paper points out some deficiencies of bullfrogs now used in biological
research. The need for obtaining stocks with infoimation on geographical origin, age,
nutrition, general health, and environmental conditions under which they were maintained or reared is paramount and can be obtained from commercial dealers. Growth
rates and food utilization data with regard to bullfrog age and geographical origin are
discussed in relation to the effects these characteristics may have on data interpretation in many research projects. The logic of utilizing only large bullfrogs in research
is discussed. Motile sperm of males grown under laboratory conditions can be obtained
in three months after metamorphosis, and in females, eggs approaching maturity are
produced in six months after metamorphosis.
INTRODUCTION
At the risk of exposing myself to the
scrutiny of professional physiologists, geneticists, enclocrinologists, and a variety of
other professionals, I agreed to present this
paper in the hope of pointing out the need
for developing laboratory strains of amphibians. My training is in aquatic biology
and until four years ago my research dealt
entirely with the effects of toxic substances
on aquatic and terrestrial organisms. My
research involved the use of wild populations, and I quickly became aware of the
difficulties in maintaining healthy stocks
of animals for testing purposes. During my
research with amphibians, the maintenance
problems and data interpretation difficulties I encountered led me to wonder how
researchers dealing with sophisticated biomolecular problems maintained their animals and interpreted their data. Through
correspondence with many individuals conducting research with amphibians, it became quite clear that I was not alone in
my problems. It also became apparent that
little effort was being made to develop culture methods for amphibians. Through
these communications and experiences, I
was stimulated to develop culture methods
for the bullfrog, Rana catesbeiana (Culley
and Meyers, 1972). I selected this species
because of its popularity as a research animal and its potential as a food organism
for man.
Our research program would not have been possible without the financial support of several groups
and individuals. I am especially indebted to Drs.
George Nace and Christina Richards at the University of Michigan's Amphibian Facility for their
many suggestions and help in developing our program. Mr. John Priddy, President of the Southern
Frog Company, Dumas, Arkansas, provided us with
stocks for testing and allowed us to make observations on a large commercial operation. Dr. Robert
Amborsky, Assistant Professor of Microbiology,
Louisiana State University, has been invaluable in
our disease research program. Original research
leading to this publication was supported by
funds from the Louisiana Agricultural Experiment
Station; the Animal Resources Branch of the National Institute of Health, grant number RR0063502; Mr. John Priddy, President of the Southern
Frog Company, Dumas, Arkansas; and Mr. Jim
Bankston, President of Gulf South Biological Supply Company.
Anurans obviously have been widely
used in the biological education process for
well over a century. It is one of the first
organisms used for demonstrating the tissue-organ-system concepts in biological
training (Nace, 1970). Literally hundreds
of research projects presently utilize
anurans for elucidating basic biological and
ecological processes.
Researchers and educators in the United
States alone utilize about fifteen million
anurans annually. With the present rate
of exploitation and habitat destruction we
will certainly see the termination of many
research efforts involving amphibians unless an effort is made to bring them under
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DUDLEY D. CULLEY, JR.
laboratory control
1971).
(Priddy and Culley,
USERS OF LABORATORY
project requires a large animal. I hope our
research in bullfrog culture will point out
the need for considering other factors.
ANIMALS
GROWTH CHARACTERISTICS OF BULLFROGS
When I consider the excellent research
training of many colleagues, the type of
research they are conducting, and the quality of amphibians they use in their research,
program, I am rather perplexed. Certainly these individuals have rubbed shoulders
with researchers utilizing mice, rats, rabbits, fruit flies, etc., and are aware of the
care with which these individuals select
their animals. Their animals often are
carefully bred for certain biological characteristics, may be germ-free, and reared
under rigid nutritional programs and in
well-defined environments. Records on genetic background, population characteristics, age, sex, health, disease, medication,
etc., are often available and taken into
consideration when research animals are
chosen for particular projects. Why have
users of amphibians not been as demanding? What type of amphibian are they
using? Where do they come from? What
care are they given? How reliable are the
data generated from amphibians? These
are some of the questions that crossed my
mind when we began developing culture
methods for the bullfrog nearly four years
ago.
Although I will confine my comments to
the bullfrog most of them apply to all
laboratory animals. In our culture program
we have uncovered considerable information about biological and ecological characteristics of bullfrogs which leads me to
suspect that researchers have not scrutinized their amphibians sufficiently to insure that their data will be as useful as
they desire.
About the strangest thing I have run
across is that almost every user of bullfrogs
wants a big bullfrog, i.e., with at least a
snout-vent length of 12 cm (about 4i/^
inches). Very few suppliers can move bullfrogs less than this size. I fail to see the
logic in this requirement except when the
How does age compare with growth in
bullfrogs? If a bullfrog is collected from
the Northern United States and has a snoutvent length of 12 cm, it is almost certainly
four years of age from the egg and two or
three years from metamorphosis, even if it
lived under optimal conditions and had
rapid growth characteristics. If collected in
the South, it will probably be about two
years of age from metamorphosis. If, however, the southern-caught bullfrog has rapid growth characteristics and optimal environmental conditions, its age may not
exceed sixteen months from the egg. If the
southern bullfrog is a slow grower, it may
be four to five years of age. Rapid-growing
southern bullfrogs will obtain a 12-cm
snout-vent length in ten months from the
egg when grown under laboratory conditions (Culley and Gravois, 1971)! Evidence
shows that basic physiological mechanisms
are common to all vertebrates (Hoar,
1966). If age, or other factors such as temperature, crowding, nutrition, handling,
etc., affects results of studies dealing with
aging, ion transport, sensory function, toxicology, wound healing, and organ function
in other laboratory animals, surely they do
in amphibians (Dechambre, 1971; Fox,
1971; Simionescu, 1971; Weihe, 1971).
If size is to be the only criterion, why
will researchers not purchase small bullfrogs, but will readily accept the smaller
leopard frog (Rana pipiens)? If a large
volume of blood or some other tissue is
not necessary, it certainly would be more
economical to purchase a small, rather
than a large, bullfrog. However, the small
size does not solve the age difference problem because some bullfrogs do not grow
very much. We have maintained bullfrogs
in our laboratory for over three years and
they never exceed a snout-vent length of
7.5 cm (about 2^4 inches), while rapid
growers will reach this size in three months
USE OF BULLFROGS IN BIOLOGICAL RESEARCH
from metamorphosis. Surely it would be instructive to know the age and growth characteristics of research animals and utilize
this knowledge when selecting stocks.
The utilization of food by bullfrogs
should be taken into consideration in any
study dealing with such factors as aging,
growth, tissue regeneration, and enzymology. Certainly these characters are associated with food utilization in bullfrogs.
During the first three months after metamorphosis, it is not uncommon for bullfrogs to have a food conversion (c) of two
or less (two pounds of food per pound of
gain). By five months of age the c value
on the average approaches 3.5 and by nine
months it may well exceed 7. However, it
it not uncommon for a three-month frog
to have a c value of 4 and a seven-month
frog a c value as low as 3. Changing c values indicate that physiological processes associated with food use and growth are
changing and may well affect research results for some studies, particularly since individuals of a population may respond
quite differently from the average of some
measured response (Marshall et al., 1971).
The size and age of bullfrogs as related
to food utilization increase the complexity
of interpreting data. A small bullfrog of
the same age as a large bullfrog may have
a similar c value, indicating the physiological processes associated with food use and
growth are equally efficient. The only difference we have been able to detect between two such frogs is that the smaller
bullfrog simply consumes less food, but utilizes it as efficiently as the rapid-growing
frog. Not only would the larger individual
be more expensive to purchase and maintain, but it would not necessarily offer any
advantages over the smaller individual.
If there is anything consistent about
bullfrog growth, it is the inconsistency in
growth rates during the first few months
after metamorphosis. Young bullfrogs gain
weight in spurts, and we have been able to
detect this in as few as thirty days. As a
population parameter, newly metamorphosed bullfrogs on the average increase
their body weight two to three times dur-
87
ing the first month. By the fifth month
their weight increase is about 25%. However, individual responses are quite different. During the first three months it is not
uncommon for individual frogs to have a
much higher gain during the second or
third month than during the first month.
We have also observed large gains during
a single month in older bullfrogs, though
it is not as pronounced. When large numbers of frogs are being utilized in a study,
apparently some will be experiencing a
rapid growth phase. Thus, a whole series
of physiological processes are varying greatly between individuals and may increase
variability in data collected.
If bullfrogs are being conditioned for
studies, it is incorrect to assume that they
are utilizing food efficiently just because
they are eating well. We have had frogs
feed heavily during a month, yet grow little and have, therefore, a very poor c value.
We can find little correlation between food
intake and gain, or for that matter, food
intake and food conversion. There is a high
correlation between gain and food conversion, however. In other words, if a frog has
a high gain during one month, it will show
efficient food utilization (reflected in a
low c value). Thus, it appears that bullfrogs must be physiologically "ready" for
growth, and a whole host of processes apparently must be synchronized before the
food is efficiently used.
Preliminary studies we have conducted
suggest that bullfrogs collected from different latitudes (really temperature zones)
have different growth responses at a given
temperature. During a four-week study,
young bullfrogs collected from the Louisiana State University campus gained about
one-half as much weight at 17 C as the bullfrogs of the same age collected from a cool
mountain lake in Arkansas, 350 miles north
of our campus. The difference was great
enough that we plan to conduct more complete studies with bullfrogs from several
locations around the country. Until more
information is available, a researcher
should be sure that he knows the origin of
his frogs.
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DUDLEY D. CULLEY, JR.
work to date. First, red legs appear to be
symptomatic for a variety of bacterial inResearch involving reproductive proc- fections and disorders, and at present we
esses has traditionally required the procure- cannot relate the condition to any one
ment of large bullfrogs. I suppose the rea- pathogenic species. Second, when bullfrogs
son for this is that the individual will not are reared under sanitary conditions and
be sexually mature until it obtains a are provided with adequate nutrition, lit"large" size. I am not sure how you should tle mortality occurs after they are two
define sexual maturity of males but motile months past metamorphosis. Mortality in
sperm seems to be an acceptable criteria. our frogs over two months of age is norIn our laboratory studies, we have found mally less than 10%. The critical period is
motile sperm in bullfrogs three months in the late stages of metamorphosis, and
from metamorphosis. Such frogs are about we often lose better than 90% of a popu5 cm in snout-vent length. We have not lation. We do not know the cause as yet.
as yet tested the sperm to see that they are Even if we have had few losses during this
capable of fertilization. In any event the period, only on rare occasions have we had
possibility exists for obtaining mature mortality above 10% during the first two
sperm from a very young bullfrog. Very months past metamorphosis. Third, if we
little space would be required to care for inject bacterial material and incubate frogs
at low temperatures (4 C) and then eleanimals this small.
As for females, we have observed eggs vate the temperature to room temperature
with distinct animal and vegetal poles at (21 C), we get a more rapid death response
six months from metamorphosis when than if we inject and incubate the frogs
grown under laboratory conditions. These at room temperature. I believe this points
females were the largest in the population out nicely what Gibbs et al. (1971) were
but still measured less than 10 cm in snout- saying about the dangers of amphibians
vent length. If we can develop methods for being exposed to temperature extremes
natural breeding under laboratory condi- during shipment. Frogs caught in the wild
tions, purchase and maintenance of large harbour many potentially pathogenic organisms, and rapid exposure to temperabullfrogs will be unnecessary.
Although some females we have reared ture extremes may well serve as a stimulus
have been ovulated three times, egg devel- for lowering resistance in the amphibians.
opment is asynchronous. I believe the On the other hand, laboratory-reared amasynchronous condition is an environmen- phibians are normally in a healthy condital problem that may be related to photo- tion, have obtained adequate nutrition,
period and temperature. If I am correct, and may harbour fewer pathogenic orgathen captured bullfrogs, being exposed to nisms. If laboratory-reared amphibians
rapidly changing temperatures and photo- cannot be obtained, I believe it is advisperiods during storage and shipment, may able to hold wild stocks for four to six
well develop hormonal imbalance in a weeks and to give proper care before they
are utilized for research purposes.
short period of time.
SEXUAL MATURITY IN BULLFROCS
BULLFROG DISEASES
SOURCES OF LABORATORY-REARED BULLFROGS
There has been much discussion of bullfrog diseases, and the dreaded "redleg" disease is always prominent in discussions. We
are actively working to identify pathogenic
organisms of bullfrogs, and at present we
have not completed our identifications.
However, three points have come from our
There are sources of laboratory-reared
bullfrogs. We have made sufficient progress to warrant such operations. For some
reason the scientific community has been
slow in utilizing these sources despite the
high-quality animals produced. Part of the
problem is that the dealers are unknown,
USE OF BULLFROGS IN BIOLOGICAL RESEARCH
and the high prices may be a deterrent,
particularly if the scientist demands large
bullfrogs. However, other operations are
being planned and as rearing techniques
are improved upon, I am sure that the
prices will decline and more dealers will
be available across the country.
Presently available from commercial
dealers are two types of bullfrogs that are
superior to those currently available from
most biological supply houses. The first
type is a frog (usually large) that has been
wild-caught and maintained under laboratory conditions for several weeks. Although
the cost for this animal is more than that
of wild-caught frogs not maintained before shipment, they are well worth the
price. Information on geographic origin,
nutrition, laboratory rearing conditions,
and disease treatments is available with
these frogs. The second type of bullfrog is
one in which eggs or tadpoles are collected
in the wild, brought into the laboratory,
and reared under known conditions. In
addition to the data available on the first
type of frog, information is provided on
the length of time in the tadpole stage,
and date of metamorphosis. Mortality of
both of these groups has been extremely
low during and after shipment.
RESEARCH DIRECTION AT LOUISIANA STATE
UNIVERSITY
Present efforts in our culture program
are being concentrated in three areas: disease identification and control, laboratory
breeding requirements, and genetic breeding for defined characteristics. Until we understand the problems associated with disease and breeding and progress in defining
desirable characteristics, mass production
of defined strains will be slow in coming.
Although I anticipate an increase in the
availability of laboratory-reared bullfrogs
in the next few years most of these will be
from eggs or tadpoles collected in particular geographic locations and reared under
laboratory conditions. Although such bullfrogs are less desirable than defined strains
they are far superior to frogs from the wild
89
that have not been treated for disease or
acclimated to laboratory conditions before
being sold.
RETHINKING OUR STANDARDS
I hope I have been able to point out
that, as users of bullfrogs, we have possibly
not scrutinized our test animals sufficiently and have been too inflexible in our demand for only large bullfrogs. In fact, it
may be undesirable in many cases to use
large frogs. I can understand the need for
large bullfrogs for some studies, and these
can be obtained from commercial suppliers
who maintain records on age, geographical
origin, nutrition, disease treatment, etc. In
spite of the notion that large frogs are several years old we have raised bullfrogs approaching 300 grams (snout-vent length of
13 cm) in eight months from metamorphosis. Admittedly only about 5% of a population grows this rapidly, but through selective breeding, rapid-growing strains
could probably be produced. As breeding
techniques are worked out and desirable
characteristics are defined, I believe bullfrog culture will have an impact on the
laboratory animal industry.
As a final point, I was surprised to find
that the scientific community has not
pushed for the development of standardized colonies of any frogs used in research.
Our research in developing culture methods for bullfrogs indicates to me that the
least we can expect is vital statistics on age,
size, geographical origin, nutrition, general health, sex, and rearing conditions. I
hope this paper will serve as a stimulus for
researchers to be more selective in choosing test animals in the future.
REFERENCES
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Culley, D. D., and S. Meyers. 1972. Frog culture
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Dechambre, R. 1971. Effect of social environment
on the development of mouse ascitic tumors, p.
313-331. In Defining the laboratory animal. National Academy of Sciences, Washington, D. C.
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DUDLEY D. CULLEY, JR.
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behavior of laboratory animals, p. 294-312. In
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Gibbs, E. L., G. W. Nace, and M. B. Emmons.
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