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By Vladimir S. Apekin /Sive Lab
Introduction
The small size of the X. tropicalis makes
the use of the traditional X. laevis methods of embryo collection
very difficult. Therefore, we have developed a new method of embryo collection
for this species, which is based on the peculiarities of the behavior
of X. tropicalis during spawning.
In artificial settings, spawning of the X.
tropicalis occurs in two stages:
1) The first stage is calm. It begins 1.5-2 hour after final injection
and takes place over 15-60 minutes. The behaviors are very similar to
the spawning habits of the X.laevis. The male grabs the female
and the couple starts to move around dispersing embryos. Periodically
they stop for rest.
2) The first step gradually escalates into the
second - ecstasy step, which is characterized by the frogs periodic frenzied
and quick movements, and by their turning over onto their backs (Fig.
7 and 8). The male holds onto the female tightly, so it is easy to move
the couple into a different container for short period of time. In most
cases, this can be done without interrupting the spawning process.
The intensity and consistency of the spawning depends on the health of
the frogs and on the amount of hormone injected.
Method for Collecting Embryos
A. Injecting Hormone
1) Dilute Chorionic Gonadotropin with distilled
water so that the final concentration is 1500 IU/ml. Fill a 1 ml syringe
with the hormone, and use a 26-gauge needle.
2) Take the frog in your hand so that the dorsal side is against your
palm and the back legs are placed between your index finger (Fig. 1).
Hold the back right leg firmly between the index and middle fingers, which
will temporarily stop the frog from moving (Fig. 2). Using your thumb
move the back left leg so as to clear a space for the injection (Fig.
3). Be careful not to stretch the leg too tightly.
3) Inject hormone into the dorsal lymph sac (Fig. 4 and 5). Make sure
that only half the length of the needle is forced into the frog’s
body. Inject females with 0.1ml and males with 0.05ml. Leave the needle
in the body for a second after the injection, so as to prevent the fluid
from leaking out.
This entire procedure must be done quickly, and due to the small size
of the X. tropicalis, requires practice.
B. Collecting the embryos
1) Inject two females and three males and place
them together into an adequately sized aquarium contain of frog system
water (conductivity = 1.5 + /- 0.2 siemens, Temp. = 26 +/ -1°C) with
the height of the water approximately 2 inches from the bottom. Cover
the aquarium with a perforated lid.
2) 1.5 to 2 hours later, the male will grab the female, which marks the
beginning of spawning (Fig. 6). At this time transfer the linked couple
into a different aquarium with the same conditions and give them another
15-60 minutes to let the process increase in intensity.
3) After the frogs start to regularly turn over (Fig. 7), carefully move
the coupled frogs into a 1 L glass or Nalgene beaker containing 400 ml
of 0.1 x MBS (Fig. 8). In this beaker the frogs will deposit the embryos.
In the majority of the cases, the couple will not separate and will continue
spawning, however, in the cases where the male leaves the female you might
have to wait for them to reunite in order for the spawning process to
continue.
4) The above method was used for collection of
3-4 sets of embryos. If the frogs spawn intensely, 10-20 min is enough
to collect 200-400 embryos, which is usually enough for one experiment.
You can then transfer the couple into another glass beaker and collect
additional embryos so that each beaker contains a set of embryos that
are at approximately the same stage. One couple or pair of frogs can produce
up to 3000-5000 embryos during one whole spawning process.
It is important to note that the eggs in the glass/beaker are clumped
together because of the frogs’ movement. The embryos should be placed
into Petri dishes and the clumps removed soon after the embryos are deposited.
This will allow equivalent oxygenation to each embryo, which will permit
equal development time and prevent suffocation.
Discussion
The Method’s Benefits
- High
percentage of fertilization and development (80-90%)
- Simplicity
- No
need to sacrifice the males
The
Method’s Drawbacks
- The
process of spawning continues for 2-4 hours and the optimal collection
time is limited to 1-2 hours.
- The
collected egg samples should be processed immediately after they have
been collected, as the embryos are in clumps and may suffocate.
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Figure
1.
Hold the frog with it's back against your palm and your index finger
between it's legs.
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Figure
2.
Hold the right leg between the index and middle fingers.
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Figure
3.
Move left leg under your thumb.
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Figure
4.
The field of injection.
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Figure 5.
Injection
of hormone.
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Figure 6.
Frogs coupled together.
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Figure 7.
Couple flip over to back.
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Figure
8.
Spawning in the beaker.
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If you have any questions about this method or how we care for frogs here
at the Sive Lab, feel free to forward them to Vladimir Apekin, at apekin@wi.mit.edu.
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