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    • CommentAuthoraldeal
    • CommentTimeFeb 2nd 2012

    This protocol explains how to: obtain eggs by inducing in vivo fertilization by hCG injection (setup a "love bucket")

    Protocol revised: 2/2/2012

    Protocol written by: Alex Deal


    This protocol is to explain the process of setting up a breeding container (“love bucket”) to obtain eggs from Xenopus laevis fertilized in vivo, rather than using the in vitro method of “squeezing.”  Because this is a two day process, the sections have been split into two sections (DAY 1 and DAY 2).


    DAY 1
    ·    gloves
    ·    lab coat (recommended, but not required; water will splash out of the container)
    ·    1 mL syringes (3; likely already in the frog room)
    ·    26 ½ gauge needles (3; likely already in the frog room)
    ·    human chorionic gonadotropin (hCG; currently located in fridge of rat perfusion room)

    DAY 2
    ·    gloves
    ·    lab coat (recommended, but not required; water splashing)
    ·    medium Petri dishes (number depends on clutch size)
    ·    plastic spoon
    ·    20% Steinberg’s solution (tadpole rearing solution)


    DAY 1
    1.    Obtain a clean, large plastic container from the frog room or the stock room.

    2.    Fill the container with cold tap water in the frog room.

               ·    It is recommended that you add a quick squirt of dechlorinator to the water as the container fills.

    3.    Once the container is filled almost half-full with water set the container on a cart (one should be located in the room).  Also located in the frog room, get a white, plastic grid and beanbag to cover the top of the container.

    4.    Check the chart on the large water container beside the sink (see pic below).  Check for the row with the most recent date marked and look to see which female pair was used.  Move down one row to the next female pair, move over to the column of the current month, and write the date on the chart (see note under pic).

    Note: The columns are months and rows are bin letters.  The date is marked by writing the day in the proper month column and in the first open row.  For example, if the next breeding were to take place on May 20, 2011, the date (“20”) would be written in the “May ‘11” column in the bin letter row “DM.”  The females in bin DM would then be used for the procedure.

    5.    Roll the cart to the box of the female pair you just marked on the chart and, wearing gloves, transfer the females to the container on the cart.  Make sure the container the frogs are in now is covered by the white grid and beanbag.

    6.    Place the cart in an out of the way place as you are now going to get the male (see pic below of female and male X. laevis).


                                                 X. laevis female                                                                       X. laevis male (note: male is smaller than female; pic is enlarged)

    7.    Go to the male frog room and obtain a clean, small plastic container either in the male frog room or the stock room.

    8.    Fill this container with cold tap water (dechlorinator is not necessary as this container is only temporary).

    9.    Find a bin that does not say “injected” and remove a frog.

              ·    When selecting a male, look for darker pigmentation on the ventral side of the front phalanges, or if the male is mounting other males in the container.

    10.    Return to the female frogs with the male frog in his small container.

    11.    Place a white grid over the male frog container, though a beanbag is not necessary.

    12.    Ready the syringe by filling it with the appropriate amount of hCG.

              ·    Females—0.5 mL (500 μL)

              ·    Males—0.25 mL (250 μL)

    13.    Only fill and use one syringe at a time as you do not want extra needles of hCG lying uncapped in your work area (DO NOT fill all three and recap those you are not currently using as it is very unsafe to recap needles).

    14.    To inject, make sure the needle is bevel side up and insert the needle just under the skin (this is a subcutaneous injection, so no blood should be seen).  Depress the plunger and remove the needle at the same angle at which you inserted it.  When injecting the females make sure you are aware of which female has been injected.  The order of injections does not matter (see pic below for possible injection sites; inject using the injection site that is most comfortable for you and how you hold the frog). 

              ·    Be careful of the females’ cloaca as you are injecting.  Try to avoid holding the frog in such a way that you are touching the cloaca. (see pic above of female)

              ·    I will usually move the first female to the male container after injecting her to ensure I do not inject the same female twice.

              ·    If the frog kicks as you are injecting, remove the needle quickly so the frog does not hurt itself.

    15.    Once each needle has been used, discard it in the sharps container.

    16.    After all three frogs have been injected, place them all in the large container, reset the plastic grid and beanbag, and roll the cart to the corner of the room.  Get a small laminated card from on top of the shelves beside the sink and write on it the bin letters from which the females were obtained.  Also, place the label that reads “INJECTED; WAITING TO COLLECT” on top of the white grid with the box identification card you just made.

    17.    Remove any trash that may have accumulated.

    18.    This procedure is usually done later in the day and the frogs are left over-night.  Return the next morning to collect the eggs (see DAY 2 of this protocol for egg collection and returning the frogs to their proper containers).

    DAY 2
    1.    Roll the cart with the frogs from the frog room to the lab.

    2.    Using the spoon and Petri dishes, remove the eggs from the container.

    3.    Once a Petri dish is full, drain the water from the dish (the spoon can help hold the eggs in the dish), and replace with 20% Steinberg’s solution.

              ·    Be sure all of the eggs are submerged in the Steinberg’s (you can use the spoon to gently push the eggs under the surface as some eggs will float)

    4.    After transferring all of the eggs to Petri dishes, place the dishes in a safe place for cysteining later.

    5.    Roll the cart back to the frog room.

    6.    Fill a small container with cold tap water to hold the male (dechlorinator is not needed).

    7.    Remove the male from the female and place him in the small container.

    8.    Making sure the females are covered by the plastic grid, take the male to the male frog room.

    9.    A future procedure to return a male may differ, but the current system is as follows:

              1)    Locate the container labeled “INJECTED” and note the number of frogs in the container or the number of dates on the label (should be the same number).

              2)    If there are less than 5 frogs in the container, place your male in the “INJECTED” container and write the date.  If you introduce the fifth frog, write “CLOSED” on the label and date it.

              3)    If there are 5 frogs already in the “INJECTED” container and it has been closed, start a new “INJECTED” container by obtaining a new container filling it with cold tap water and a squirt of dechlorinator.  Place your male in this container, cover it with a white grid and beanbag, and label it as “INJECTED” with the date.

    10.    Return to the female frogs.

    11.    Place the females in the small container that held the male, partially filled with cold tap water.

    12.    Empty the large container and refill it with cold tap water (a small amount of dechlorinator can be used, but it is not needed).

    13.    Move the females back to the large container and cover with a plastic grid and beanbag.

    14.    Replace the sign “INJECTED; WAITING TO COLLECT” with “COLLECTED; RETURN TO SYSTEM” on top of the grid.

               ·    Make sure the box identification card you made yesterday is still on top of the grid as well.

    15.    Push the cart back to the corner of the room and remove any trash.