United States
Environmental Protection
Agency-
Office of
Research and Development
Washington, DC 20460
EPA/600/3-91/064
December 1991
Guidelines for
Culturing the Japanese
Medaka, Oryzias latipes
-------
NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
-------
EPA/600/3-91/064
December 1991
GUIDELINES FOR CULTURING THE JAPANESE MEDAKA,
ORYZIAS LATIPES
BY
JEFFREY S. DENNY1, ROBERT L. SPEHAR1, KURT E. MEAD1,
AND SHIRIN C. YOUSUFF2
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RESEARCH AND DEVELOPMENT
DULUTH, MN 55804
2AScI CORPORATION
DULUTH, MN 55804
Printed on Recycled Paper
-------
CONTENTS
Page
Foreword .......................... ..... ........... ..... ........ iv
Abstract ..................... ........... ............. ........ v
Acknowledgements .................................. « ..... - ....... V-L
Introduction ................. . ............... ........ ....... 1
1. Physical Systems
1 . 1 Tanks ........................ ................ » , . . ...... 3
1.2 Hatching pans ........ ........................ « ........ 5
1.3 Water supply ........ ., ............. .......... ---- > ---- 5
1 . 4 ERL-D ' s flow- through system ................... ........ 6
1 . 5 Photoperiod ................................. ........ 8
1 . 6 Construction materials ..................... ........... 9
1 . 7 Temperature control .......................... - ......... 9
1.8 Aeration. --- . .......... . .............................. 10
1 . 9 Spawning substrates . .................................. 10
1 . 10 Disturbance ... * ....................................... 12
1. 11 Tank cleaning ......................................... 13
2. Biological Systems
2.1 Brood stock .......................... ...... ..... ; ..... 14
2 . 2 Selecting spawning fish ....................... ... ...... 16
2 . 3 Spawning .............................................. 17
%2 . 4 Embryo collection and incubation ...................... 19
2.5 Juvenile rearing. .... ................. ........... . ... .21
2 . 6 Feeding ............. ...................... ........... 2 1
11
-------
2.7 Nutritional content of Artemia .23
2 . 8 Disease 2s
2.9 Record keeping >2?
2 .10 Summary 28
Appendix-Nutritional tables for Artemia 29
References 3 4
111
-------
FOREWORD
This manual is a description of culture techniques for the
Japanese medaka that have been developed over the last five years
at the Environmental Research Laboratory-Duluth. These methods are
presented as a culture system that works, not as the only way to
culture medaka. Many different culture methods exist, and it is up
to the user to decide what modifications or combinations of methods
are appropriate for a specific application. The report has been
reviewed by the Environmental Research Laboratory-Duluth, and
approved for publication. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
IV
-------
ABSTRACT
This paper describes culture techniques for producing large
numbers of all life stages of Japanese medaka, Orvzias latipes. for
use in biological research. The biology of the medaka is described
as it relates to culturing practices and the physical systems used
to maintain a large culture. The physical systems include water
delivery apparatus, tanks, incubation pans, lighting, spawning
substrates and other useful tools. The biological section
addresses water temperature, spawning ratios, embryo incubation,
juvenile rearing, and larval and adult feeding.
-------
ACKNOWLEDGEMENTS
Many people have been involved in the development of
successful medaka cultures at ERL-Duluth. The authors would
specifically like to thank Larry Herman, Rod Johnson, Joe Tietge,
Nan Stokes, Duane Benoit, Gary Holcombe, Craig Wilson,, Kathleen
Jensen, Wes Smith, Jodi Collins, and Roger LePage.
Much of the credit for development of culture techniques for
new species is due to the wealth of experience in fish culture
resident in the staff at the Environmental Research Laboratory-
Duluth. This collective knowledge has been invaluable.
vx
-------
INTRODUCTION
The Japanese medaka, Orvzias latipes r has been used in various
fields in biology, especially in developmental biology, genetics,
and embryology (Yamamoto, 1975). Aquatic and environmental
toxicologists have recently begun to explore use of the medaka as
a sentinel or biomarker for environmental contamination (Grady, et
al., 1991). At the U.S. EPA Environmental Research Laboratory in
Duluth, Minnesota, the medaka is being evaluated in a
multidisciplinary research program encompassing carcinogenicity
testing, metabolism, neurotoxicity, reproductive toxicity, and
comparative toxicity for both acute and chronic endpoints. The
medaka is also being tested for future use in the development of
EPA freshwater aquatic life criteria documents, and the detection
of carcinogenic components in industrial effluents and contaminated
sediments.
The culture techniques described here are based on years of
experience in the large scale culture of fathead minnows at ERL-
Duluth. The aquarium systems have been continually refined over 25
years by dozens of workers. The biological methods for rearing
medaka have been developed over the last five years by numerous
people. Some methods are employed because of their traditional
success, and others because they are cheap, simple, or minimize
labor. There is very little "hard data" on how the culture system
works. It has grown along with the size of the medaka research
effort, eventually being capable of supplying thousands of embryo,
larval, or juvenile medaka each week for research purposes. Over
-------
300 toxicology related experiments have been performed at ERL-
Duluth using medaka from this culture system.
The medaka is an extremely adaptable organism, and will thrive
under a wide variety of conditions. Indeed, this adaptability is
a crucial quality that makes the medaka so useful as a research
animal. Adult medaka measure from two to four cm long, are very
hardy, and can be easily maintained at room temperature (Kirchen
and West, 1976) . This simplicity of care makes it possible to
establish a small culture of fish in almost any laboratory. Since
medaka breed readily in captivity, small cultures can provide
enough embryos for some types of research. For large scale
experiments requiring 1000 or more fish of all life stages,
intensive culture techniques are necessary.
It is outside the scope of this paper to delineate specific
ranges for all parameters involved in the successful culture of
medaka. For basic information on culture systems, water quality-
parameters, and general fish culture guidelines, the reader is
referred to works such as Aquarium Systems by Hawkins (1981),
Aquaculture. by Bardach, et al. (1972), or Fish and Invertebrate
Culture by Spotte (1970).
Japanese biologists have been using the medaka as an
experimental animal for at least 70 years (Yamamoto, 1975).
Yamamoto (1975) gives an exhaustive description of all aspects of
medaka biology, and includes a large bibliography. Recent papers
concerning the use of medaka in carcinogen assays include Ishikawa
et al., 1984, Klaunig, et al., 1984, and Hyodo-Taguchi and Egami,
-------
1985. A basic discussion of the care and development of the medaka
can be found in Kirchen and West, 1976.
CHAPTER 1
PHYSICAL SYSTEMS
The physical systems are quite similar to those described for
fathead minnows in Denny (1987). Figure 1 provides a generalized
schematic of how the flow-through aquarium system works.
1.1 Tanks
The tanks are 57 liter (15 gallon) glass aquaria with
standpipe drains adjusted to provide 20 cm of water depth. One
hole (2.5 cm) is drilled in each tank bottom, a stopper with a hole
is inserted, and a drain is fashioned through the hole in the
plywood rack. This allows for approximately 40 liters of water per
tank. The incoming water flows through the tanks and out the
standpipe drain. The tanks, supported by racks of slotted angle
iron and 1.9 cm (3/4 inch) plywood, are arranged in two tiers, in
rows of 12 tanks. Two units are used for spawning, rearing, and
holding research fish at ERL-D. This setup supports production of
3,000 - 4,000 embryos/week, along with rearing and holding space
for thousands of fish of all life stages. Groups of future brood
stock are held in four 285 1 (75 gallon) flow-through tanks.
-------
Solenoid Valve
Incoming
Water
ling \
(30" C) V
Incoming
Water (15'C)
U
Temp. Controller
Outgoing
Water
(To Tanks)
27'C
Temperature
Probe
I
Flow
Restrictors
r
Stand
Pipe
f1
/2" PVC Plumbing
2" PVC Drains
Drain
Figure 1. Temperature controlled, flow through culture apparatus.
-------
1.2 Hatching Pans
Plastic dishpans, commonly available in department stores
(e.g.: 53 cm x 40 cm x 12 cm deep) are used for embryo incubation.
White pans provide the best background for viewing the newly
hatched larvae. Pans are held in a temperature controlled water
bath (27 °C), to decrease temperature shock due to fluctuation of
room temperature. Incoming water (27 °C, 150 ml/min) flows in and
through the hatching pan and out a screened drain. Temperature can
also be maintained by using walk-in incubators, or individual
aquarium heaters.
1.3 Water Supply
The two main criteria for water supplied to a culture facility
are that it meets or approaches optimum conditions for the
physiological needs of the fish, and that it be free of
contaminants. It is generally desirable to use water from a
spring, a well, or a controlled surface water with consistent water
quality. Culture water should be similar to water used in testing.
The source should be examined for contamination by pesticides,
heavy metals, sulfides, disease vectors, or any other suspected
contaminants. Filtration may be necessary if well water is used
(Mount, 1971). Dechlorinated tap water from a municipal water
supply should be used only as a last resort (Benoit, 1982) . If the
water is contaminated with fish pathogens, pass the water through
an ultraviolet or similar sterilizer immediately before it enters
the system (Allison and Hermanutz, 1977).
-------
Water quality parameters such as hardness, alkalinity, and
anions should fall within the following limits: hardnejss 40-300
mg/1 (as CaCO3), and alkalinity slightly less than the hardness.
The anions should be those found in a normal stream or lake. Avoid
well or spring waters which have high levels of iron, silica, or
sulfides not found in surface waters.
Research goals and design will dictate both the quality and
quantity of water necessary to support medaka cultures. The ERL-D
medaka culture system uses Lake Superior water with a pH range of
7.4-8.2, an alkalinity (as CaCO3) of 42 mg/1 and a total hardness
of 45 mg/1. For a more detailed characterization of Lake Superior
water see Biesinger and Christensen (1972).
1.4 ERL-D's Flow-Through System
The ERL-D water delivery system is a constant temperature,
flow-through system (see Fig. 1, Sec 1.1). It is gravity fed, with
custom welded stainless steel mixing boxes (46 cm x 28 cm x 40 cm
deep) positioned on a shelf approximately 1 m overhead. Lake
Superior water warmed to 15 °C flows through a toilet tank valve to
maintain water level in the headbox. Water heated to 30°C flows
through a solenoid valve positioned over the headbox. To achieve
the desired constant temperature, a temperature probe suspended in
the headbox feeds into a solid-state temperature controller (Syrett
and Dawson, 1975). Headbox temperature must be maintained at 28 -
29°C to provide 26 - 27°C in the tanks, to counteract heat loss to
room air. This basic water delivery system can supply most
-------
experimental water temperatures, depending only on the temperature
of the water supply to the float and solenoid valves.
Agitators or airstones must be used in the headbox to assure
complete mixing and to prevent supersaturation of gases caused by
heating water. Water flows from the headbox through 1.27 cm (1/2
inch), threaded (not glued) polyvinyl chloride (PVC) pipe. This
type of pipe (schedule 80), is available commercially. The outlet
is located on the side of the headbox, about 2 - 3 cm from the
bottom, to prevent sediment from clogging the outflow. Detailed
instructions for the design for the mixing boxes, electronic
relays, etc., are contained in Syrett and Dawson, 1972, 1975, and
McCormick and Syrett, 1970.
Water flows into .the tanks through a 1/2 inch PVC pipe
manifold. At one or two points on the manifold an open ended pipe
is extended upward above the water level in the headbox to
eliminate possible air blockage. Above each tank is placed a tee
with a 1/2 inch to 3/8 inch reducer attached. Into the reducer a
3 ml disposable syringe barrel is glued using silicone glue.
Hypodermic needles or capillary tubing of different gauges can be
attached to the syringe to provide different flow rates into the
tanks. ERL-Duluth's 40 liter tanks are supplied with 200 ml of
fresh water per minute, providing 7-8 turnovers each 24 hours.
Grady, et al. (1991), reported success using 100 ml/min flows with
75 liter tanks. Again, space and other physical constraints may
modify the flow rate or nature of the aquarium system.
-------
Medaka can be maintained in static or recirculating systems,
but flow-through systems allow higher stocking densities and
feeding rates, while problems associated with disease organisms and
waste products are reduced.
Recirculating systems often consist of aquaria at table-top
level for the fish, and an aquarium on the floor that acts as a
trickling filter for the drain water from the fish tanks. This
filter can be made of any nontoxic, high surface area material such
as crushed coral, pea gravel, or tower packing. Water can be
pumped from a sump up to a headbox above the fish tanks, from
whence it flows back into the tanks. Nitrifying bacteria in these
filters convert ammonia to nitrate. Ammonia must be monitored
closely in static or recirculating systems. The chronic effects
threshold for fathead minnows exposed to ammonia, based on
histological damage, is estimated to be .15 mg/1 (Thurston, et al.
1986). For a discussion of filtration for recirculating systems,
see Hawkins (1981).
1.5 Photoperiod
The relationship between photoperiod and reproductive activity
in the medaka remains unclear. Papers addressing the issue
(Yoshioka, 1963, Awaji and Hanyu, 1989) report conflicting results
depending on geographical strain or maturational stage. We have
found that a 16 light/8 dark regime stimulates reproduction, while
a 8L/16D regime halts reproduction. All medaka in the ERL-D
culture unit are maintained under the 16L/8D regime. Four foot
8
-------
fluorescent tubes that simulate sunlight (e.g.: Duro-test Vita-
lite, Sylvania Gro-lux or Gro-and-sho, or GE Chroma 50) are used.
Light intensities at the water surface average 400 - 500 lux. This
stimulates the growth of periphyton which the fish are observed to
actively graze upon. If a strictly defined diet is critical to
research goals, it is necessary to keep the tanks scrupulously
clean to deny grazing opportunity. Medaka will also spawn under
cool white lighting and shorter light periods.
1.6.Construction materials
Culture water should not contact brass, copper, lead,
galvanized metal, or natural rubber. Items made of neoprene rubber
or .other materials listed above should not be used unless it has
been shown that their use will not adversely affect either survival
or growth of embryos and larvae of the test species (ASTM, 1990).
Glass, teflon, stainless steel and PVC are acceptable materials.
1.7 Temperature control
Temperature control is accomplished with paired controllers,
connected to temperature probes and solenoid valves as described in
Section 1.4. A timer set to operate from 5 a.m. to 9 p.m. keeps
the temperature at 28°C during the daylight period. During the
dark period a temperature of 22°C is maintained. This fluctuating
temperature regime helps to stimulate reproduction (Kirchen and
West, 1976). Fail-safe devices such as temperature recorders,
-------
shut-off solenoids, etc. are in order if mechanical malfunction can
result in rapid change in water temperature.
1.8 Aeration ',
Mechanical aeration provided by air-lines in individual tanks
has been avoided in the ERL-D culture system because female medaka
will deposit embryos on the air-line rather than on the embryo
collection substrate. In addition, vigorous aeration can damage
newly hatched larvae. Also, aeration is usually not as necessary
in high turnover, flow-through situations; however, the traditional
aeration system consisting of an air pump, air-line, and air-stone
is suitable for static systems. Embryos can be collected from the
air-lines if necessary, and aeration can be reduced for young
larvae.
1.9 Spawning Substrates
Spawning substrates essentially take the place of the roots of
aquatic plants, which are the preferred natural sites for egg
deposition for the medaka (Yamamoto, 1975).
Some criteria necessary for a useful spawning substrate are:
They must be 1) acceptable to fish,
2) easy to handle (cleaning, storage, etc.)
3) non-toxic
4) inexpensive, readily available
The ERL-D system uses cylindrical foam aquarium filter
cartridges for spawning substrates (see Fig. 2). These
10
-------
Tank r-^
Rim **|
Plexiglass Support
1
Stainless
Steel
Tubing
Embryos
Sponge
Figure 2 Spawning substrate, suspended in tank.
-------
cylindrical sponges which are approximately 100 mm long and 50 mm
in diameter, are available from Carolina Biological Supply. Though
not similar to any natural substrate, the fish acclimate to these
sponges, and will readily brush eggs off onto them. This method of
obtaining embryos was modified from that used at The Gulf Coast
Research Laboratory in Ocean Springs, Mississippi (William Walker,
personal communication).
The sponges are suspended below the water surface in each
spawning tank by means of an elbow constructed of 9mm diameter
stainless steel tubing. The tubing is held in place by pressing it
into a slot in a plexiglass support that lies across the top of the
tank. Many other types of substrates and hanging methods are
possible as long as they fit the above mentioned criteria. The
substrates are moved directly from the spawning tanks to the
hatching pans. This process avoids handling embryos, reducing
labor and damage to the embryos.
1.10 Disturbance
Fish should be shielded from continual or drastic disturbance.
Avoid construction noises, continual human presence, and extraneous
lights that might alter the photoperiod. This is particularly true
of spawning adults; excessive disturbance or activity can result in
reduced embryo deposition. On the other hand, fish in strict
isolation become hypersensitive to people and may take time to
acclimate to increased levels of human activity.
12
-------
1.11 Tank Cleaning
The four inch wide razor scrapers used to remove wallpaper are
an excellent tank cleaning tool, and are available in hardware
stores. After scraping the tanks, the debris should be siphoned
away to remove potential disease substrate and to improve
observation. If there is a tendency to vacuum up fish during the
siphoning stage, siphon the residue into a screened pan that allows
overflow, yet catches any errant fish.
Many culturists allow light growths of algae, rotifers, etc.,
to remain as a dietary supplement for the fish. This may not be
acceptable for some specific research needs: e.g. maintenance of
a strictly defined diet. Excessive growths of blue-green algae or
fungus must be removed under any regime. An appropriate cleaning
schedule can be determined depending on water source and feeding
regime.
CHAPTER 2
BIOLOGICAL SYSTEMS
Four life stages are used for testing at ERL-Duluth - embryos,
larvae, juveniles, and adults. The culture system is designed to
produce large numbers (1,000+) of each life stage, depending on
testing demands. Research projects that require only one life
stage could be supplied by smaller and less labor intensive culture
systems. For continuous,,full life cycle culture, plans must be
made to set aside a randomly selected group of fish from each
generation to use for future breeding stock. Selection for
13
-------
specific brood stock characteristics can also be employed,
depending on intended research use.
t
2.1 Brood Stock
Medaka cultures were initiated at ERL-Duluth in April, 1986,
using two breeding sets (4 males/6 females per set), from Carolina
Biological Supply. They were derived from a stock of 2000
cultivated orange strain adults obtained by Carolina Biological
Supply in 1970 from a wholesaler in Tokyo, Japan (R. V. Kirchen
1990, pers. comm.) . Four more breeding sets were added to the ERL-
Duluth gene pool in Sept, 1986. These original 60 fish have been
used to produce a standing pool of 48 breeding sets.
Current
production consists of 3,000 - 4,000 embryos,per week. Individual
lots of larvae, juveniles, and adults (1,000 or more), are provided
to researchers on request, or set aside for future brood stock.
Yamamoto (1975) describes 13 distinct genetic strains of the
medaka: brown, blue, orange-red, variegated orange-red, white,
variegated white, gray, pale, cream, milky, albino, ankylosed, and
lordotic. He states, "The origin of orange-red and other color
varieties of cultivated stocks is wrapped in a shroud of mist. The
orange-red fishes have been painted by Ukiyoe artists of the Yedo
era, so the race must have arisen by mutation from the wild type
more than a few hundred years ago. The orange-red stock and a few
other varieties have since been kept by goldfish breeders. These
"himedaka" are available at goldfish shops and night stalls in any
large city in Japan." (Yamamoto, 1975) . This stable strain history
14
-------
and easy availability may explain why the orange-red or himedaka is
the strain most often used in research.
Inbred strains of medaka have been established through sibling
matings, and some have been maintained in the laboratory for over
30 generations (Hyodo-Taguchi and Egami, 1985). The authors
reported that during the inbreeding, reproductive potential was
reduced or the mortality of the fish was high in many pedigrees.
Two of the orange-red and three of the brown (wild) strains were
successfully inbred by full sister-brother matings for 22
successive generations from 1974 to 1979.
The ability to maintain viable inbred strains in the
laboratory may allow development of specific strains of medaka for
specific toxicological research issues, much like inbred strains of
mice (Shimkin, 1974). Development of an Fl hybrid strain may be a
logical next step in the development of a medaka model. Montesano
et al. (1986) states: "Although we know that the choice of strain
may be important in a particular bioassay, we have in general
insufficient knowledge to permit reliable prediction in advance of
which strain to prefer. Indeed, there is not even general
agreement as to whether inbred, Fl hybrid or outbred animals are
usually to be preferred." Given that medaka genetics have been
well studied (Yamamoto, 1975), it seems that the medaka model holds
promise for the development of strains paralleling those of the
mouse, rat, or Syrian hamster models.
15
-------
2.2 Selecting spawning fish
Spawning sets of 4 males and 6 females each (Kirchen and West,
1976) are selected randomly at ERL-Duluth from a population of
approximately 1,500 adult fish maintained in four 300 liter (75
gallon) aquaria. These breeding sets are established when the fish
are 3 to 4 months old, and are replaced periodically as egg
production drops. Commonly, the age range of the oldest set of
spawning fish is 14-15 months. The ERL-D culture system has two
banks of spawning tanks, with twelve tanks each. While one bank is
being replaced, the other bank continues to supply embryos. One
bank is replaced every 4-6 months.
When spawners are being selected, a random sample of 50-75
mature fish is placed into a one gallon aquarium. A contrasting
background (i.e. black paper or wrinkled aluminum foil) is placed
behind the tank. Side lighting allows for the easiest viewing of
the translucent fins, which are used to differentiate the sexes.
After waiting a few minutes for the fish to settle down, a small
net is used to select and separate males and females.
Sexually mature medaka exhibit dimorphism which is most
readily apparent in the dorsal and anal fins. Males have a small
notch in the posterior margin of the dorsal fin and have a sail-
like, transparent anal fin which is pointed at the distal posterior
edge. Females have no notch in the dorsal fin and have a more
compact anal fin which is rounded on the margins. Gravid females
are easily spotted after spawning by the clutch of eggs attached to
the oviduct pore.
16
-------
When females are sexually mature there is an enlargement or
swelling of the abdominal region caused by the presence of
unfertilized eggs and mature reproductive tissues. Males retain
their original fusiform shape throughout their life cycle. In
healthy fish, dimorphism is usually discernable at approximately 2
months of age. Oka (1931), and Egami (1954) discuss sexual
dimorphism of the medaka in detail.
2.3 Spawning
Because medaka are polygamous, Kirchen and West (1976) suggest
a breeding ratio of three females to two males. Original breeding
sets consisting of six females/four males were maintained in
individual 40 liter tanks. This low stocking density works well,
allowing collection of 3 - 4 thousand embryos on demand from 36
breeding sets. Low density decreases the likelihood of disease,
minimizes problems with ammonia and other waste build-up, and
allows for easier inspection, counting, etc. of the breeding stock.
Medaka can be spawned at much higher densities, however, and recent
experiments have shown that increasing the density from 10 to 20 or
40 breeding adults per tank results in increased embryo output (See
Table 1).
17
-------
Table 1. Mean number of embryos collected using one, two, or four
breeding sets per tank. Numbers represent embryos voluntarily
deposited on spawning sponges, not total reproductive output.
| OF SPAWNING SETS
1 (control)
2
4
MEAN # OF EMBRYOS
87.4
192.7*
365.0*
STD. ERR. OF MEAN
5.2
12 . 6
16.9
* significant difference from control, p=0.05, TOXSTAT (program),
ANOVA, Tukey's method of multiple comparisons.
Grady, et al. (1991), report breeding success with densities
of 8 - 15 adult medaka per gallon of water. Stocking density and
maintenance of water quality at higher densities are system
dependent. Each culturist must experiment and work within the
limitations of the available system.
Female medaka lay their eggs immediately after ovulation,
which takes place almost invariably in the early morning. The
entire courtship and fertilization process takes less than 60
seconds (Robinson and Rugh, 1943).
Yamamoto (1975) describes egg deposition as follows? "After
the ova are expelled en masse at the time of mating, a cluster of
eggs remains attached to the belly .of the female for some hours,
suspended from the oviduct pore by fine threads attached to the
chorion. Finally, the egg mass is detached by the action of the
female in swimming and contacting the roots of water plants, or if
there is no vegetation by contacting the bottom of the Container."
In the ERL-D laboratory cultures, the spawning sponges described in
18
-------
a previous section act as surrogates for deposition sites on
aquatic plants.
2.4 Embryo Collection and Incubation ,
Embryos are collected from spawning adults once a week. More
frequent collections are possible, and are undertaken for research
projects with special demands. Usually the spawning substrates are
placed in the tanks on Thursday mornings and allowed to remain for
24-28 hours. On Friday afternoon, the sponges are removed and the
number of embryos deposited per sponge is recorded by tank number.
A tack can be used to mark a spot on the cylindrical sponge
substrate: counting from the tack, the sponge can be rotated 360
degrees, counting or estimating the number of embryos. Inviable
embryos are removed with tweezers. At this stage, embryos can be
provided for research projects designed to begin with embryos.
Embryos can be segregated by developmental stage at this point if
required (Kirchen and West, 1976).
Embryos are usually incubated as a group to provide lots of
larvae of known age for initiating exposures (e.g.: 800+ for an
early life stage test). Embryos are incubated on the substrates,
in flow-through hatching pans, as described in section 1.2. At
27°C to 28°C hatching of the embryos will begin in about 7 days.
By day 10, 95% of the embryos will have hatched. This hatching
period can be modified by sorting embryos by developmental stage,
by increasing incubation temperature to 28°C, or by physical
manipulation of the embryos (Benoit et al., 1991). The least labor
19
-------
intensive method is to simply allow the eggs to hatch undisturbed
during the 7-10 day period and to pick off fungused embryos as they
become visible. This is the method employed in the ERL-D culture
system. On day 10, sufficient numbers will have hatched, so that
larvae can be provided for testing, designated as <72 hours post
hatch. One of the best tools for handling larvae is a large bore
50 ml volumetric pipette. Larvae to be counted can be pipetted
from the hatching pan into a beaker in small lots of 10-15.
At times, female medaka will carry embryos for 1-2 days rather
than deposit them, especially if a suitable substrate is not
available. When a substrate is made available, the fish will then
deposit these older embryos. Most lots of embryos are made up,of
24 and 48 hour old embryos. Even if new substrates are provided
daily, some fish will retain their embryos. If embryos of exact
known age are required, observation and stripping of individual
females may be in order.
Stripping embryos is not difficult, and is a standard
technique in many Japanese laboratories (Tadakoro, pers. comm.,
1989). Individual females carrying embryos are netted from the
tank and placed on a wet towel. The embryos can be quickly brushed
or picked off with a tweezer, taking care to point the sharp tips
away from the abdomen (if the fish flips, the sharp tips could
injure the abdomen) . The female can be returned to the tank by-
lifting the towel and gently flopping the fish into the net. With
practice, and especially with two people, this can be a quick,
efficient operation, which does not damage the fish. Often,
20
-------
females stripped in this manner will spawn again the next day.
Stripped embryos can be segregated by developmental stage under a
dissecting microscope (Benoit, et al., 1991).
-*
2.5 Juvenile Rearing
Larvae not required for testing at this stage are stocked at
a density of 200 larvae per 40 liter tank and reared to 30 days of
age. Higher stocking densities can be used, but decreased survival
and increased variability in size of juveniles can result from
overstocking. These juveniles can satisfy testing protocols
requiring the juvenile life stage. Larvae are stocked in this
manner weekly, to provide a steady supply of juvenile fish for
testing. Random lots of juveniles not used in testing are stocked
at a density of 400-600 per 75 gallon tank and grown to maturity
(3-4 months)'for use as future breeding stock.
2.6 Feeding
Medaka have been maintained on a variety of foods: e.g.;
Tubifex and Tetra-min (Awaji and Hanyu, 1989), Tubifex. Daphnia.
and dried fish food (Robinson and Rugh, 1943), commercial tropical
fish food, mosquito larvae, Euchvtreaf and Artemia (Kirchen and
West, 1976). In the ERL-Duluth culture system, newly hatched
Artemia nauplii are the preferred full life-cycle food, with the
exception of the last few days before sacrifice for histological
analysis. Dried food (Cordon flakes) is offered for one week at
21
-------
the end of the grow out period. This clears the gut of Artemia
which interferes with histological sectioning.
All life stages of medaka are fed live Artemia ad libitum,
twice each week day, and once a day on weekends. Because the
density of the Artemia slurry can be variable, it is important to
carefully observe the amount fed. In general, the fish are fed the
amount they can consume in 20 - 30 minutes. Close observation is
critical for adjusting feeding rates. If food is rapidly
exhausted, more is supplied. If uneaten food is observed on the
bottom of the tank hours later, the ration is decreased at the next
feeding. In flow-through systems with large tanks it is not
necessary to rinse the brine shrimp. In static or low flow systems
rinsing is desirable to avoid salt build-up.
Since larvae begin to feed at approximately 24-48 hours post-
hatch, it is important that food be offered at this time. The gape
size of larval medaka is approximately .32 - .36 mm, while the
width of a newly hatched Artemia nauplius is about .20 - .24 mm.
The length of a newly hatched Artemia nauplius is .44 - .64 mm (T.
Roush, pers comm, 1989). Larval medaka therefore are able to
ingest first instar Artemia nauplii, but only in one orientation.
If Artemia are allowed to grow for too long post hatch (into second
instar), they become too large for the medaka larvae to ingest.
They also provide fewer nutrients to the fish, since they use up
the nutrient rich yolk sac in respiration and growth. For these
reasons, it is extremely important to harvest the Artemia and feed
the fish immediately after the Artemia hatch (first instar) as the
22
-------
fish are essentially getting their nutrients from the yolk sac of
the young Artemia.
2.7 Nutritional Content of Artemia
Different geographical strains and brands of Artemia have
widely varying nutrient and contaminant levels. It is critical
that the strain being used be analyzed for contaminants as well as
for proximate nutritional content. Parameters for the strain of
Artemia currently in use at ERL-D (Biomarine, Hawthorne, CA) are
given in the Appendix (Tables 2-6). The analyses were performed by
Woodson-Tenent Labs in Memphis, TN.
Mouse and rat carcinogenicity assays utilize synthetic diets
in which the levels of contaminants and nutrients are defined. The
National Institute of Health recommends an open formula diet where
contaminant levels and heat labile nutrients must meet acceptable
levels (National Toxicology Program, 1984). The Artemia used at
ERL-Duluth have been screened for a wide variety of chlorinated
organic contaminants, and have been found to be below those levels
recommended by the NTP (Table 2). Braun and Schoettger (1975)
compared the levels of selected contaminants in a variety of fish
foods. The levels of DDT, PCBs, HCB, dieldrin, and endrin were
much lower in the Artemia nauplii than in the commercial dry fish
diets.
The nutritional requirements of rats, mice, and other animals
have been elucidated through removal and substitution experiments
with various dietary components. Dietary requirements are known .
23
-------
for some freshwater fish species, particularly carp (Nose, 1979),
catfish (Wilson et al., 1978) and rainbow trout (Walton, et al.,
1984). Medaka, however, have not received the detailed attention
required to define their nutritional requirements.
Tables 3 and 4 provide the proximate analysis and cimino acid
profile for the lot of Artemia currently being used at ERL-Duluth.
In general, 30%-55% crude protein is required for growth in fish
(Millikin, 1982; National Research Council, 1983). These Artemia
contain about 61% protein (Table 3). On a dry weight basis, the
sum of the amino acids equals about 59%, which approximates the
protein content.
Table 3 shows that these Artemia contain 5.42% crude fiber.
Leary and Lovell (1975) found that fish foods with more than 8%
fiber depressed fish growth. In practical diets, more than 3-5%
fiber will probably not benefit the fish, as it will only increase
waste production (National Research Council, 1983).
Artemia also contain vitamin E, vitamin C, and carotenoids
(Table 3) . Vitamin E promotes growth and prevents muscular
dystrophy. In addition, it prevents the formation of fatty livers.
This is important in medaka research, as the liver is a major
target organ for many carcinogens, and fatty livers can interfere
with histological sectioning and pathology analysis.
Vitamin C is essential for collagen metabolism. Since' fish
generally can't synthesize vitamin C and it is water soluble, a
constant supply is needed to prevent deficiency signs. Though the
24
-------
minimum level required in the diet of the catfish is only 60 mg/kg,
the Artemia diet far exceeds that with 3917 mg/kg.
The bright orange color of Artemia is imparted by the
carotenoids B-carotene (Czeczuga, 1980) and canthaxanthin (Soejima
et al., 1980). Poston et al, (1977) found that B-carotene has the
potential for conversion to vitamin A in fish. High levels of
canthaxanthin were found to hasten the onset of spawning and
increase the fertilization rate in rainbow trout (Deufel, 1965).
This suggests that the carotenoid content of Artemia could be
beneficial to the medaka.
Table 4 provides the percentages of the various amino acids.
When these values are compared to the recommended values for other
fish species (Table 5) , most essential amino acid levels are
comparable, as they fall within the pooled ranges. The level of
methionine (1.81%) in the Artemia was found to fall below the
average of the pooled range (2.99%), but the dietary need for this
amino acid can be compensated for by the high level of cystine
(0.72%) . This has been substantiated by studies that show that the
presence of dietary cystine in channel catfish and rainbow trout
decreases the amount of methionine needed for maximum growth
(Harding et al., 1977; Kim et al., 1983). In addition,
phenylalanine (1.99%) was found to be quite low, but tyrosine, a
non-essential amino acid that can replace 50% of the phenylalanine
requirement in channel catfish (Robinson et al., 1980), was found
to be high (2.35%) (Table 4).
25
-------
Table 6 lists the fatty acid composition of Biomarine Artemia.
Fish are incapable of de-novo synthesis of linoleic, linolenic,
eicosa-pentaenoic and docosahexaenoic fatty acids. Thus, these are
most likely to be the fatty acids essential for fish growth and
survival (Kanazawa, 1985). When the levels of these acids are
compared to the recommended levels in carp food (Takeuchi and
Watanabe, 1977) and rainbow trout food (Castell et al., 1972),
linolenic acid in Artemia is higher and linoleic acid is lower.
The diet analysis indicates that no carbohydrates are present
in the Biomarine brand Artemia. Although carnivorous fish have an
obvious need for protein and fat in their diet, they have
difficulty digesting carbohydrates (Cho and Kaushik, 1985).
Digestible protein can, in turn, provide much of the energy
yielding nutrients needed by the medaka.
Thiaminase, a thiamin destroying enzyme often found in raw
fish, is not found in Artemia (Greig and Gnaedinger, 1971). In
this respect, Artemia may be superior to commercial fish foods that
contain unpasteurized fish products. A thiamin deficiency has been
shown to produce dark coloration and mortality in catfish, as well
as fin congestion, nervousness, and a fading of body color in carp
(National Research Council, 1983).
2.8 Disease
Diseased fish, or fish that have been chemically treated for
disease cannot be used in testing. The best way to deal with
disease is to prevent it. This is best accomplished by using low
26
-------
stocking densities, keeping tanks clean, and using a nutritionally
adequate diet. Any lots of fish discovered to be diseased must be
discarded and the tanks disinfected. Methods for detecting fish
disease can be found in Warren (1981), and Post (1983).
A dilution of 3.8 ml/liter of household bleach is an
effective, cheap disinfectant for tanks and tools. This provides
a concentration of 200 ppm free chlorine. Contact time should be
1 hour. Disinfection should not be overused, in order to avoid the
production of chlorinated organic residues. , Rather, it should be
used to prevent the spread of disease, or at major junctures in the
culture scheme (e.g.: sterilizing tanks to set up new spawners).
Chlorine can either be neutralized with sodium thiosulfate, or
allowed to dissipate for 24 - 48 hours. Test kits are available to
check for the presence of free chlorine. Any tanks or tools that
have been chlorinated must be rinsed thoroughly with culture water
before being used with fish.
Fungus is ubiquitous in the aquatic environment. Its presence
should not be considered a valid reason to discard all associated
embryos unless the labor involved in picking and sorting out the
bad embryos is not worth the return at hatch.
2.9 Record Keeping
Records are kept on the numbers of embryos collected each
week, so that depleted spawners can be identified and replaced.
All mortalities in the culture unit should be recorded, and any
fish showing evidence of tumors or other deformities should also be
27
-------
recorded and preserved. Records must be maintained on the age of
spawning fish, and the dates on which rearing tanks are stocked
with larvae. Recording embryo deposition dates and lairval hatch
dates is critical to wise stock management. For example, the
embryo collection date is recorded, and a date label is placed on
the incubation pan. Ten days later is the larval hcJttch date.
Thirty days post-hatch is the "juvenile" date. At 60 - 90 days
post-hatch fish begin to reach sexual maturity. Recording and
tracking the ages of all fish in culture will allow the most
efficient use of different life stages for testing. Fish should be
observed daily for abnormal appearance and behavior.
2.10 Summary
Though many aspects of medaka biology have received intensive
scrutiny, the use of this species as a toxicological tool has
opened numerous new areas for inquiry. Definition of nutritional
requirements, as has been done for trout or catfish, will allow for
development of refined, formulated diets. This will be critical in
metabolism, comparative toxicology, and carcinogenicity testing.
Application of genetic information to the development of specific
strains for specific research applications is also fertile ground
for new research. Development of disease free cultures, such as
those for other fish species or mammalian research models is
another issue that has not been addressed. The need for research
in these areas will become more urgent as use of the raedaka model
becomes more widespread.
28
-------
APPENDIX
Nutritional and contaminant tables for Artemia
Table 2 . National Institute of Health ' s Limits for
Contaminants in NIH-07 Diet for Mice, Compared to the
Pesticide Screen of Biomarine Artemia Nauplii
Pesticide
Hexachl orobenz ene
BHC
Lindane
Heptachlor
Aldrin
Heptachlor Epoxide
DDE
Dieldrin
Endrin
ODD
DDT
Mirex
Methoxychlor
Chlordane
Toxaphene
PCB (total)
Diazinon
Methyl Parathion
Malathion
Ethyl Parathion
Ethion
Ronnel
NIH-07 Diet for
Mice (PPM)
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.02
0.05
0.05
0.10
0.20
0.20
0.02
0.50
0.02
0.02
0.02
Artemia
Nauplii
(PPM)
<0.01
<0.01
<0.01
<0.01
0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
29
-------
Table 3. Nutritional Analysis of Biomarine brand Artemia
Nauplii
Test
Moisture
Fat
Protein - Kjedahl
Fiber, crude
Ash
Calories (bomb calorimeter)
Beta Carotene
Vitamin C
Alpha Tocopherol (Vit E)
Iodine - low levels
Dry Basis
94. 46%*
37.36%
61.19%
5.42%
5.23%
578 cal/lOOg
3.97 mg/kg
3917 mg/kg
954 lU/kg
3.61 mg/kg
* wet weight basis
30
-------
Table 4. Amino Acid Profile of Biomarine Brand Artemia
Nauplii
Test
Tryptophan
Aspartic Acid
Threonine
Serine
Glutamic Acid
Proline
Glycine
Alanine
Cystine
Valine
Methionine
Isoleucine
Leucine
Tyros ine
Phenylalanine
Histidine
Lysine (total)
Arginine
Percent (as % of total dry
weight)
0.90
5.78
3.61
4.33
9.21
3.97
3.25
2.53
0.72
1.99
1.81
2.35
4.15
2.35
1.99
1.44
3.97
4.87
31
-------
0
(A
P
Q)
13
0)
H
3
&
H
Q)
&
CO
^
to
H
Pn
Other
id
H
g
0)
.p
(3
H
00
Sp
13
id
S
S?
*"
(1)
H1
w
*
to
a)
H
O
Q)
CO
r]
10
&4
d
H
O
o
G
H
5
(d
H
P
C
0)
to
to
W
o
*
r«.
^
o
vo
CO
CO
tjl
ll 1
d)
T3
o
43
I
"*
H
m
H
H
(N
in
H
0>
o
o
43
id
to
H
vo
CO
0
CO
o
^
1
0
CM
CO
I
CO
CO
r-i
CD
(d
1
O
CM
«
CO
o
^J,
1
CNJ
n
a)
TJ
o
id
i-H
(0
o
^J-
o
(d
0)
0
^
o
^
H
1
CO
0
a)
73
S
EH
8
H
73
-H
G
H
0)
4->
O
rH
id
-P
O
O
p
c
Q)
O
nook salmon
-H
g
1
id
o salmon
43
O
o
43
&
id
o
o
g
o
o
o
anese eel
nnel catfish
nbow trout
f}| id, **H
-------
H
H
H
§*
10
5?
rd
H
E
0)
4->
V>J
rt<
0)
C
H
fd
e
o
H
CQ
O
C
o
-H
-P
H
(0
O
D
e
o
o
d
o
-P
rd
r* i
vo
0)
rH
(d
EH
4J
H
EC
^j
Q)
EC
<*>
-d
H
0
<
^i
-P
4J
(d
fa
X!
H
a?
s*
4J
O
d
H
O
rfj
4J
«d
^
vo
VO
» »
O
CO
rH
^-*
d
H
o
0
rH
rH
fd
0)
w
Q
< -
0
vo
>-
"
o
H
O
rij
O
rH
O
a
rd
U
vo
in
o
n
* ».
rH
CO
rH
< *
d
H
O
<
O
H
O
rH
O
Q
55
. >
0
CO
V--
T3
-H
O
O
-H
-H
a
O
CO
n
VO
**
CM
CO
H
N.*
d
H
O
o
a)
H
o
c
H
Q
^~.
o
..
o
rH
"*^
d
H
O
rtj
o
H
a
rd
U
t^
^
"*
CM
>*,
n
.
CO
H
^
d
rH
0
0
H
C
0)
i-H
O
c
rH
Q
«-^.
O
*
H
H
X^1
d
H
o
^J
0
rH
0
c
(d
U
Q)
d
c
CO
n
CO
13
rH
O
rij
0
H
O
c
a>
rd
M
-p
0)
4J
fd
o
CD ^->
^3 ^
4J CO
D H
Q
5?
x-^.
0
CM
rH
*-*
H
O
KjJ
O
H
3
Q
55
X >
o
0.
CT>
rH
s
d
H
O
o
anoi
o
0)
d
cd
G
O
55
i
*»>
o
«
f)
rH
>-<
d
H
O
H
O
o
VO
0
^^
rH
*sj*
H
*
T3
H
O
rtj
O
H
0)
rH
O
-P
CO
H
*
Q
^^
CM
O
CM
v_*>
d
H
o
rfj
o
H
ieno
d
u
o
o
H
w
Q
55
O
*
in
H
*^
d
rH
O
o
H
o
c
(d
o
U
d
id
P
c
Q)
Q
rT
0
*-*
TJ
H
O
O
H
O
C
0)
-H
-P
w
o
o
H
Q
55
rH
in
rH
^m
13
H
O
U
-H
O
c
0)
o
0)
d
rd
0)
Q
^^
*$f
,,
O
CM
-d
-H
O
o
H
O
T3
H
JG
O
fd
rH
<
VO
CM
in
rH
^.^
0
VO
rH
^
-d
H
O
H
4J
H
e
rH
&
CO
O
r>
,_,
in
*
o
CM
S ^
d
H
o
0
H
o
c
0)
4J
c
0)
G
w
o
o
H
CM
O
in
^^
rH
VO
rH
*d
H
o
o
H
0)
rH
O
P
-H
rH
rd
0<
Q
55
^.^
0
CM
CM
d
H
O
0
-rH
c
Q>
X!
0)
m
CO
rH
H
fO
-H
O
O
rH
O
c
0)
H
d
o
0) ^s
d CM
rd
X vo
0) rH
Q
^^,
rH
CM
CM
H
O
o
H
O
W
vo
CO
0
^ ^
o
^
rH
d
H
O
o
H
rd
s
Q
^^
CM
9i
CM
CM
d
-H
o
o
H
o
c
0)
H
d
tn
o
o
o
Q
Q
55
^_^
i-l
^
rH
'd
H
o
o
-H
0)
rH
o
rd
Cr
rd
S
33
-------
References
Allison, D.T., and R.O. Hermanutz. 1977. Toxicity of diazinon to
brook trout and fathead minnows. U.S. Environmental Protection
Agency. EPA 600/3-77-060.
ASTM, 1990. Annual book of ASTM standards, Water and Environmental
Technology: Pesticides, resource recovery, hazardous substances and
oil spill response, waste disposal, biological effects. Volume II.
04 designation E-1241-88. American Society of Testing and
Materials, Philadelphia, PA 19103-1187.
Awaji, M., and I. Hanyu. 1989. Temperature-photoperiod conditions
necessary to begin the spawning season in wild type medaka. Nippon
Suisan Gakkaishi (formerly Bull. Japan. Soc. Sci. Fish.) 55 (4),
747.
Bardach, J.E., et al. 1972. Aquaculture: the farming and husbandry
of freshwater and marine organisms. Wiley-Interscience Press. 868
PP-
Benoit, D.A. 1982. User's guide for conducting life cycle chronic
toxicity tests with fathead minnows, (Pimephales promelas). U.S.
Environmental Protection Agency. EPA 600/8-81-011. 17 pp.
Benoit, D.A., et al. 1991. User's guide for conducting early life
stage toxicity tests with Japanese medaka (Orvzias latipes) draft.
US EPA Environmental Research Laboratory, Duluth, MN 55804.
Biesinger, K. E., and G. M. Christensen. 1972. Effects of various
metals on survival, growth, reproduction, and metabolism of
Daphnia magna. Jour. Fish. Res. Bd. Can. 29: 1691-1700.
Brauhn, J. L., and R. A. Schoettger. 1975. Acguisition and culture
of research fish: rainbow trout, fathead minnows, channel catfish,
and bluegills. US Env. Prot. Agcy. EPA-660/3-75-011. 45pp.
Castell, J. D. et al. 1972. Essential fatty acids in the diet of
rainbow trout (Salmo gairdneri) : Growth, feed conversion, and some
gross deficiency symptoms. J. Nutr. 102: 77-86.
Cho, C. Y. and S.J. Kaushik. 1985. Effects of protein intake on
metabolizable and net energy values in fish diets. 95-117. in: C.
B. Cowey, A. M. Mackie, and J. G. Bell, eds. Nutrition and feeding
in fish. Academic Press, Harcourt Brace Jovanovich. 489pp.
Czeczuga, B. 1980. Carotenoid content of Artemia eggs and vitality
of young specimens of this crustacean. In: G. Persoone et al. eds.
The brine shrimp Artemia. Vol. 2. Physiology, Biochemistry, and
Molecular Biology. Universa Press, Wetteren, Belgium. 636 p.
34
-------
Denny, J.S. 1987. Guidelines for the culture of fathead minnows,
Pimephales promelas. for use in toxicity tests. U.S. Environmental
Protection Agency. EPA 600/3-87/001. 42 pp.
Deufel, J. 1965. Pigmentierungsversuche mit canathanxin bei
Regenbogenforellen. Arch. Fischerewiss. 16: 125-132.
Egami, N. 1954. Appearance of the male character in the
regenerating and transplanted rays of the anal fin in females of
the fish, Oryzias latipes. following treatment with
methyldihydrotestosterone. J. Fac. Sci, Imp. Univ. Tokyo. 4. 271-
280.
Grady, et al. 1991. Laboratory management and husbandry of the
Japanese medaka. Lab Animal. 20(3). 22-28.
Greig, R. A. and R. H. Gnaedinger. 1971. Occurrence of thiaminase
in some common aquatic animals of the U.S. and Canada. Spec. Sci.
Rep. Fish., U.S. Dep. Commer. Natl. Mar. Fish. Serv. 631: 1-7.
Harding, D.E., O.W. Allen, and R.P. Wilson. 1977. Sulfur amino acid
requirement of channel catfish: 1-methionine and 1-cysteine. J.
Nutr. 107. 2031-2035.
Hawkins, A.D.* ed. 1981. Aquarium Systems. Academic Press. 452 pp.
Hyodo-Taguchi, Y., and N. Egami. 1985. Establishment of inbred
strains of the medaka, Oryzias latipes. and the usefulness of the
strains for biomedical research. Zoo. Sci. 2(3). 305-316.
Ishikawa, T., P. Masahito, and S. Takayama. 1984. Usefulness of the
medaka, Oryzias latipes. as a test animal: DNA repair processes in
medaka exposed to carcinogens. 35-43. in: K.L. Hoover, ed. Use of
small fish species in carcinogenicity testing. Nat'l. Inst. Hlth.
Monograph #65.
Kanazawa, A. 1985. Essential fatty acid and lipid requirement of
fish. 281-298. in: C.B Cowey, A.M. Mackie, and J.G. Bell, eds.
Nutrition and feeding in fish. Academic press. 489pp.
Kim, K.I., T.B. Kayes, and C.H. Amundson. 1983. Protein and
arginine requirements of rainbow trout. Fed. Proc. 43(3): 667.
Kirchen, R.V. 1990. Carolina Biological Supply, Burlington, NC.
letter dated 1/11/90.
Kirchen, R.V., and W.R. West. 1976. The Japanese medaka, its care
and development. Carolina Biological Supply Company, Burlington,
NC. 36pp.
Klaunig. J.E., B.A. Barut, and P.J. Goldblatt. 1984. Preliminary
studies on the usefulness of medaka, Oryzias latipesf embryos in
35
-------
carcinogenicity testing. 155-173. in: K.L. Hoover, ed. Use of small
fish species in carcinogenicity testing. Natl. Inst. Hlth.
Monograph #65.
Leary, D. F., and R. T. Lovell. 1975. Value of fiber in production
type diet for channel catfish. Trans. Am. Fish. Soc. 104: 328-332.
MCCormick, J.H., and R.F.Syrett. 1970. A controlled temperature
apparatus for fish egg incubation and fry rearing, manuscript. U.S.
Environmental Protection Agency, Duluth, MN.
Millikin, M.R. 1982. Qualitative and quantitative nutrient
requirements of fishes: a review. Fishery Bulletin. 80: 655-686.
Montesano, R., et al., eds. 1986. Long term and short term assays
for carcinogens: a critical appraisal. IARC Sci. Pub. |83. Oxford
Press, Oxford, UK.
Mount, D.I. 1971. Tentative plans for the design and operation of
a fathead minnow stock culture unit. U.S. Environmental Protection
Agency, Duluth, MN 55804. 7 pp.
National Research Council, 1983. Nutrient requirements of warmwater
fishes and shellfishes. National Academy Press, Wash, DC. 102pp.
National Toxicology Program. 1984. General statement of work for
the conduct of acute, fourteen day repeated dose, 90 day
subchronic, and two year chronic studies in laboratory animals.
National Toxicology Program, P.O. Box 12233, Research Triangle
Park, NC 27709.
Nose, T. 1979. In: J.E. Halver and K. Tiews, eds. Finfish Nutrition
and Fishfeed Technology". 145-156. Heenemann GmbH and Co., Berlin.
Oka, T. B. 1931. On the processes of the fin rays of the male of
Oryzias latipes and other sex characters of this fish..J. Fac. Sci.
Imp. Univ. Tokyo 4(2). 209-218.
Post, G.W. 1983. Textbook of fish health. TFH Publications. Neptune
City, NJ. 288 pp.
Poston, H.A., et al. 1977. The effect of supplemental dietary amino
acids, minerals, and vitamins on salmonids fed cataractogenic
diets. Cornell. Vet. 67: 472-509.
Robinson, E.J., and R. Rugh. 1943. The reproductive processes of
the fish, Oryzias latipes. Biol. Bull. 84. 115-125.
Robinson, E.H., R.P. Wilson, and W.E. Poe. 1980. Total aromatic
amino acid requirement, phenylalanine requirement, and tyrosine
replacement value for fingerling channel catfish. J. Nutr. 110.
1805-1812.
36
-------
Roush, T. 1989. Personal communication. U.S. EPA Monticello
Environmental Research Station, Monticello, MN 55362.
Shimkin, M. B. 1974. Species and strain selection, in: L. Goldberg,
ed. Carcinogenesis testing of chemicals. CRC Press, Cleveland, OH.
Soejima, T., T. Katayama, and L. Simpson. 1980. International study
on Artemia XII. The carotenoid composition of eight geographical
strains of Artemia and the effect of diet on the carotenoid
composition of Artemia. p. 613-622. In: The brine shrimp Artemia.
Vol 2. Physiology, Biochemistry, Molecular Biology. Persoone, G.,
P. Sorgeloos, O. Roels, and E Jaspars, eds. Univera Press,
Wetteren, Belgium. 636 p.
Spotte, S.H. 1970. Fish and Invertebrate Culture. Wiley-
Interscience Press. 145 pp.
Syrett, R.F., and W.F. Dawson. 1972. An inexpensive electronic
relay for precise water temperature control. Prog. Fish. Cult.
34(4). 241-242.
Syrett, R.F., and W.F. Dawson. 1975. An inexpensive solid state
temperature controller. Prog. Fish. Cult. 37(3). 171-172.
Tadakoro, Hiroshi. 1989. personal communication. Chemical
Inspection and Testing Institute. 19-14 Chuo-Machi, Kurume,
Fukuoka, Japan 830.
Takeuchi, T., and T. Watanabe. 1977. Requirement of carp for
essential fatty acids. Bull. Jpn. Soc. Fish. 43: 541-551.
Thurston, R. V., et al. 1986. Chronic toxicity of ammonia to
fathead minnows. Trans. Am. Fish. Soc. 115. 196-207.
Walker, William. 1987. personal communication. Gulf Coast Research
Laboratory, Ocean Springs, MS.
Walton, M.J., et al. 1984. The effects of dietary tryptophan levels
on growth and metabolism of rainbow trout, Salmo gairdneri. Br. J.
Nutr. 51. 279-287.
Warren, J.W. 1981. Diseases of hatchery fish. U.S. Fish and
Wildlife Service, Region 3. Federal Building. Ft. Snelling. Twin
Cities, MN. 91 pp.
Wilson, R.P. et al. 1978. Tryptophan and threonine requirement of
fingerling channel catfish. J. Nutr. 108. 1595-1599.
Yamamoto, T. 1975. Series of stock culture in biological field.
Medaka (Killifish) biology and strains. Keigaku Publishing Company,
Tokyo, Japan. 365pp.
37
-------
Yoshioka, H. 1963. On the effects of environmental factors upon the
reproduction of fishes. Bull. Fac. Fish. Hokkaido Univ. 137-151.
38
&U.S. GOVERNMENT PRINTING OFFICE: 1991 - 648-003W662
-------
-------
m
o
o
§
T) O
CD 3,
D ^
01 O
|
CD
CO
O
o
> m i
co 3
§
3
CD
TJ
I
O
Q5T
II
3 Q)
» =r.
=. o
01
ro
en
oo
-o
O
CO
ro
:=i m m i-
2:
p
CD
m
o
> -n
m 2
83m
------- |