• Biology, A.P. Biology, Anatomy and Physiology students
• Integrated or Co-ordinated Science students
• Grade Level:  9-12
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Notes to the Teacher: to top
Ordering:
     Medaka eggs may be ordered from a biological supply house and observations can begin as soon as the eggs arrive.  In order to see the very earliest stages of development, a breeding set of fish may be ordered and placed into a prepared aquarium.  Breeding sets (approximate cost $30-35) usually include six females and four males.  We recommend ordering a breeding set of adults and removing the newly fertilized eggs yourself as oppossed to ordering the eggs (see aquarium set up that follows).  One breeding set usually suffices unless large quantities of eggs (several hundred) are needed within a specific time period.  Many supply companies include a very useful manual on the maintenance and care of adult Medaka when you purchase a breeding set.  Adult fish may  be induced to lay eggs by controlling the photoperiod (see culturing section below).  It takes approximately ten days to induce breeding, so order the fish to arrive a minimum of two weeks before the expected use date.  Students may begin their observations as soon as the eggs are removed from the female fish.
 
Aquarium Set Up:
     If a new aquarium (10 gallon tank is a good size to begin with) is to be set up, either follow the instructions from the equipment source or follow the general instructions below.  If an old aquarium is to be used, make certain that it is clean and free of disease and then follow the same general instructions:  Fill the aquarium with a 10% sodium chloride solution and allow it to stand for at least 24 hours.  Follow this treatment with several warm water rinses.  Rinse the gravel, also.   You will need a light source (flluorescent preferred) on a timer, but a heater is not usually needed in the average classroom.  Generally, the fewer plants and other items in the aquarium, the better, because it will then be easier to recapture the female fish for egg removal.  A simple filtration system and aeration are also recommended for your aquarium.

Culturing and Egg Removal:
     The best temperature range for breeding Medaka is between 21 and 26 degrees Celsius, but the fish are tolerant of a wider temperature range.  It is beneficial to have the temperature higher during the light phase, so it is a good idea to allow the light source to warm the water slightly.  For continual egg production, keep the water temperature at 25 to 28 degrees C, but do not have the temperature above 29 degrees.
     A sixteen hour light phase followed by an eight hour dark phase should induce egg laying within ten days. The fish usually lay their eggs between one and two hours after the start of the light phase.  Fertilization immediately follows.  Feeding will sometimes trigger egglaying within approximately one hour.
     Do not overfeed the fish during the induction phase.  Some sources recommend feeding sparingly three times a day during breeding.
     To safely remove the eggs from the female fish, remove a female carrying a clutch of eggs from the aquarium approximately 1 hour after the light phase of your photoperiod begins.  Use a small dip net.  The eggs should be easily visible attached to her abdomen.  During the first hour after the beginning of the light phase,  her eggs are normally fertilized.  Place the female in a petri dish.  Use the edge of a 3x5 index card to gently remove the eggs from the female. Immediately return the female to the aquarium. Follow the same procedure for other egg-bearing females.
 
Embryo Care, Rearing, and Observation:
      Separate the eggs with a pair of tweezers (plastic works well) or sterile dissecting needles and place them into small individual sterile plastic petri dishes filled with embryo rearing solution (see materials section below).  Keep the embryos at a temperature of between 20 and 25 degrees Celsius.  When the students observe their embryos, they should be careful not to overheat the embryos with the microscope light.  Compound microscopes seem to work well for observing embryos, but dissecting microscopes may be used.  Depending on the temperature of the classroom, eggs generally hatch  in 14 to 21 days.

Required of students - ability to use microscopes and follow directions
Preparation time needed - two weeks are needed to set up the aquarium and induce egg laying
Class time needed - An entire 45-55 minute class period should be allowed for the initial observations and explanation of the procedures.  Less time is required on subsequent days as students become familiar with making their observations.  Perhaps 15 to 20 minutes will be required for a period of two weeks. Observations should be taken every day during the first week as morphological changes are most rapid then.  During the second and third week of development, observations can be taken every other day.  One-two entire periods can be devoted to discussion and anaysis of the data collected, constructing graphs, etc, at the conclusion of the investigation.
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For student use
Each team of students will need:
*One petri dish with Medaka egg and embryo rearing solution
*Compound or dissecting microscope (scanning objective on compound scopes with cool fluorescent light works best for us)
*Study guide for reference to stages of development.  Obtain from biological supply company or from other websites.
click here to view developmental sequence diagrams
*small metric ruler for microscope measurements, metric ruler slide (cut and tape small section of plastic ruler onto a microscope slide - use only metric scale to avoid confusion)
*microscope and/or depression slide
*toothpicks
*paper and writing implement for recording observations
*additional materials for extensions
*teacher prepared data sheet (if available)

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UTILIZING JAPANESE MEDAKA TO OBSERVE AND INVESTIGATE EMBRYOLOGICAL DEVELOPMENT

Introduction:
    Japanese Medaka are small fish similar in size to guppies.  Originally from Southeast Asia, these freshwater fish have also been named the Geisha-girl fish, top minnow, and ricefish.  In their natural habitat, medaka fish commonly feed on mosquito larvae.  Medaka can be easily maintained in a simple aquarium.  Under the right conditions with the proper light-dark cycle (photoperiod) and food, female medaka lay clutches of 10-30 eggs.  These eggs remain attached to the abdomen of the female for a short period of time. The male medaka fertilizes the eggs with its sperm while the clutch is still attached to the female.  After fertilization, which usually occurs 1-2 hours after the female releases its eggs, the egg clutch can be gently removed from the female, the eggs separated, and observed under a microscope.
    During sexual reproduction, a sperm fertilizes an egg cell or ovum to form a zygote.  Fertilization in itself is a complex process that involves chemical cues between the sperm and ovum. Once a single sperm fertilizes the ovum penetrating the outer membrane, chemical changes occur in the membrane of the ovum to prevent any addditional sperm from fertilizing the ovum.  The sperm nucleus which is haploid (N) then fuses with the nucleus of the ovum which is haploid (N) to produce a diploid cell (2N) or zygote.  This new cell has all the genetic material to become a complete, complex multicellular organism such as a fish, rabbit, dog, pine tree, or human being.  The processes and changes that occur between fertilization and hatching or birth are generally referred to as embryonic development and take various amounts of time depending upon the species.   Many significant and interesting events occur during development.  The major processes involved include cell division or cleavage, cell differentiation and migration, morphogenesis, and organogenesis.
    The zygote divides by cell division to become two cells.  Two cells become four cells.  Four cells become eight cells and so forth.  These early cells are called blastomeres.  The process of cell division is termed cleavage.  In fish eggs with a large yolk such as the medaka, these cell divisions occur on one side of the egg in an area termed the blastodisc.  A ball or mass of cells called the morula is soon formed.  As the number of cells increases to a thousand or more a blastula is formed.  Cells in the blastula soon begin to differentiate and migrate to different areas of the embryo.  This process is termed gastrulation and the mass of cells a gastrula.  Eventually the yolk is almost entirely covered by a thin layer of cells.  The beginnings of the embryo body apear as a streak or shield concentrated in one area on the top of the yolk.  The cells in the gastrula differentiate into the three main embryonic layers (endoderm, mesoderm, and ectoderm) that will eventually form complex tissues, organs, and organ systems.  The ectoderm forms the nervous system and body covering.  The endoderm forms the lining of the digestive system and associated organs.  Many internal organs such as the heart and muscles form from the mesoderm.  The major organs of the nervous system  (brain and spinal cord) form early from a folding together of ectodermal cells into a neural tube along the midline of the embryonic streak in a process termed neurulation.   The primitive gut or digestive tract forms soon after the neural tube along with the notochord that eventually gives rise to the backbone.  Other organs and organ systems then develop internally from the three cell layers through morphogenesis (development of  form) and organogenesis.  Limb buds give rise to legs and other appendages in animals that have these structures.

Purpose:  In this activity you will observe, monitor, document, and measure changes that occur in the embryonic development of medaka fish eggs from fertilization to hatching of eggs into small fish or fry.

Equipment:  Medaka egg/s, culture or petri dish, embryo rearing solution, medicine dropper, toothpicks, compound microscope and/or dissection microscope, depression slides, metric ruler slide.

Procedure:

DAY ONE
    1.  Obtain a metric ruler slide, toothpick, depression slide, and medaka egg in a petri dish or another small container from your teacher.
    2. Using a medicine dropper, place the egg in the depression on your slide with several drops of embryo rearing solution. Rotate the egg gently with a toothpick to view different areas of the egg.
    3.  Examine your egg under low magnification with a compound microscope. Draw an accurate     sketch of the egg in the data sheet provided by your teacher or on your own paper.  Label any identifiable structures including the chorionic filaments, egg membrane, and oil droplets. Consult a guidebook (if available) to help you identify these structures.   Depending upon the approximate age of your egg (time after fertilization) you may be able to see several cells in early cell division or cleavage (2,4,8, 16 cell stage) or a blastula (mass of cells) at the pole opposite where most of the oil droplets are congregated (vegetal pole).  Label the dividing cells (blastomeres) or blastula if visible.  Using your ruler slide, measure the field of view under low magnification (40x).  You can then use the ruler slide and the distance from one side of the field of view to the other to estimate the size of structures while viewing them under you microscope. One millimeter on your slide equals 1000 microns or micrometers.  Most measurements of cells and cell structures should be made in microns.  Later, you may measure the length of your developing fish in millimeters.
    4.  Remove the egg from the depression slide and place it back in the container provided by your instructor.
    5.  If eggs are available at different stages or ages of development, obtain a second egg from your teacher and repeat steps #1-5 above.

DAY TW0-FOURTEEN (Hatching)
    1.  Obtain your egg in the container assigned to you by your teacher.  Repeat your observations as in day #1.  Look carefully for changes in the egg.  Again, rotate the egg slowly and carefully with a toothpick to view different sides and locations in the egg. Carefully draw and label any visible structures of your egg.  By the beginning of the second full day of development, you should be able to see a streak or embryonic shield lying on the surface of the yolk.  This is the beginnings of the body of your embryonic fish.  .  Ectodermal cells from this area will form the nervous system of your embryo (brain, spinal cord).  Optic buds may also be visible projecting from the primitive brain.  These form an optic cup from which the parts of the eye develop.  As development progresses during the second day, the embryo lies over the surface of and curves around the yolk.  Various numbers of somites may also be visible in the tail or caudal region of your embryo.  Somites form blocks of muscle tissue and bone tissue.   A primitive heart may be visible below the head region by the end of the second day of development. By the end of the second day of development the three main regions of the brain should be visible.   Use your ruler slide to measure the length of your embryo and estimate the size of identifiable organs such as the optic bud or optic up.
    2.  Consult your guidebook or other sources available from your teacher to help you identify visible structures.
    3.  On each subsequent day, carefully observe, draw, label, measure, and identify observable structures. Rotate and examine your egg at different angles and from different views to try and observe all structures.  A possible sequence of events follows although the times at which structures appear vary with a number of environmental conditions.
        day 3-5:    The heart should now be clearly visible and beating.  Count and record the number of heartbeats during each day that you observe your embryo.    More somites are formed.  Count and record the number of somites.  Blood vessels should be visible over the yolk sack. Make sure you include these on your drawings. Carefully observe the eyes.  Draw and identify new structures that you can observe as they form such as the lens or cornea.  Pigmentation of the eye will also increase.  The embryo is actively growing.  The gut or digestive tube should be visible below the somites.  Otoliths forming internal ear structures are present behind the area of the brain.   Fins will begin to appear at the end of this period.  Measure and record the length of the entire embryo.
    day 6-14:  Organogenesis or formation of major organs and systems is now occurring.  Make sure to observe the heart and count the heartrate each day.  Two major divisions of the heart (atrium and ventricle) should be distinguishable.  Blood becomes red in color.  Melanophores or chromatophores containing pigment appear over the body of the fish.  There is increased blood flow and branching of vessels over the yolk.  More fins develop.  The embryo beings moving.  Record the exact day when you first observe movement of the embryo. A green organ will become visible.  This is the urinary bladder.  A bright red organ is the spleen.  The digestive tract should appear visible in the middle of the body of the embryo.  The liver lies over the green colored urinary bladder.  Your embryo should look more like a fish with each additional da of development.. Measure the length of your embryo each day.  As the body curls inside the egg, try and estimate its length.
    By day fourteen, the embryo should be reading for hatching.  Time of hatching may vary and be as long as three weeks, however.  Slowly swirl the fluid in the dish or container your egg is in to stimulate hatching.  Record the day on which your embryo hatches.  Carefully remove the small fish (fry) with a large plastic pipette into a depression slide with ample rearing solution.  Quickly measure its length, make and record any other appropriate observations.   Within 1-2 minutes,  transfer the small fish to the container provided by your teacher.  Small fry can be raised in a special environment separate from the adults.  Provided with the correct type of food, fry mature into sexually mature adults in a period of 2-3 months.

Observations:  Draw, label, identify, and measure all observable structures on each day or at each time you observe your egg.  Use the student data sheet if provided by your teacher or your own table.  If using your own paper, construct a data table to record your observations.  Record major events or stages such as cleavage, blastula (blastodisc), gastrula, embryonic body, formation or appearance of new organs or structures.  Make sure to keep careful records of the heartrate, number of somites, length of your embryo as well as any other parameters your teacher asks you to observe and record.
    Construct one graph, or three separate graphs, that show how heartrate, number of somites, and embryo length change versus time during your period of observations.
 
 
 
 
 
 
 

 Student Data Table:  Observations of the Embryological Development of Medaka Fish Eggs (construct a data table with three columns and at least 10 rows similar to the following)
 

Conclusions:  Write a two page conclusion addressing the following questions.  Describe the major events that occurred in the development of your medaka fish egg from fertilization to hatching while relating these to a specific time sequence of  hours and days.  Include in your answer an explanation of the processes of fertilization, cleavage, gastrulation, neurulation, and organogenesis as they relate to your actual observations.  Relate specific organs you saw to their respective embryonic layers (ectoderm, mesoderm, and endoderm) and possible origins.  Consult your textbook or other references for additional background information.   Discuss possible mechanisms (gene control and regulation, embryonic induction, chemical gradients, etc) that might regulate the developmental processes, events you observed, and morphogenesis of your egg.
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