Collecting eggs and sperms and fertilization

1) Transferring jellyfish into dish

Ovulation of eggs occurs 2 hours (110~135 minutes) after the light is turned on, after minimum 3 hours of dark. Sperm release may occur a bit earlier and mechanical shock may induce sperm release earlier. Transfer jellyfish to 10 cm dish to collect eggs and sperms.

Sperm: transfer male jellyfish (5~10) into a deep 10 cm dish with 200 ~ 300 ml sea water within 1 hour after the light is on.

Eggs: transfer female jellyfish (max 30/dish) just before the ovulation. (90~100 min after the light is on.) This is not to leave jellyfishes for long time in a dish at high density.

  • Max 30/dish for 1 hour
  • Max 12/dish up to several hours
  • 5~8/dish for overnight incubation in a dish

2) Collecting eggs (for microinjection purpose)

For microinjection, you may transfer eggs to smaller dish (2 cm ~ 3 cm diameter) using a mouse pipette or a pasteur pipette. This step can be done before or after transferring jellyfish back to culture tank. Please remember that rate of irregular development increases if eggs are fertilized > 60~70 minutes after spawning. It is therefore often necessary to collect eggs as quick as possible if microinjection (or other manipulation before the fertilization) is planned. Procedure (a) is recommended for this purpose. It is also used to collect as many eggs in the dish as possible. Procedure (b) is easier if you don't do microinjection and if you don't need to collect all eggs in the dish. Procedure (c) is mixture of (a) and (b); begin with procedure (a) first. Then once you get enough eggs to inject in 15~20 minutes, switch to strategy (b) and go for injection while you are waiting to concentrate eggs on the shaker.

a) Collecting eggs during ovulation (jellyfishes are still in the dish).

This can be done to collect as many eggs as possible or as quicker as possible in order to microinject into a lot of eggs. Jellyfish swimming keep eggs suspended in sea water, making egg collection a bit painful. Start egg collection under binocular as soon as you see the first several eggs released into the dish. Normally we use mouth pipette to collect if allowed. Try not to pick jellyfish with the pipette.

b) Collecting eggs after taking out jellyfish from the dish back to the culture tank.

Once ovulation is over (or there is enough eggs released into sea water), you can take out jellyfish from the dish to culture tank. Then collect eggs into other dishes quietly. If there is no manipulation/microinjection is necessary, you can fertilize eggs in this dish (without collection eggs) by adding male sea water (see step (3).) In this case, you can collect fertilized eggs/embryos after confirming the first cleavage.
You can concentrate eggs at the center of the dish by leave the dish on a rotary shaker for 10~15 minutes. The speed of rotation to efficiency concentrate eggs depends on dishes and volume of sea water. The concentrated eggs are ready to transfer into an injection chamber.

c) Mixture of a) and b): for a lot of injection (600 or higher)

Use (a) approach to collect eggs just enough for microinjection in the next 15~20 minutes (3~400 if you are experienced). Then switch to (b) approach, meanwhile you perform the first series of injections . When you finish the injection to these eggs (15~20 minutes later), there are concentrated eggs in the center of the dish ready to transfer to the injection chamber, to continue microinjection. The point of this mixed approach is to save time to collect eggs to spend more time to do microinjection.

3) Fertilization

Fertilization has to be done with in 60~70 minutes after spawning. (3 hours ~ 3 hours 10 minutes after the light ON.) Rate of cleavage error goes higher if you wait longer.)

Take out male jellyfish from dish and quantify approximately the amount of sperm with dark-field illumination with the dissecting microscope. Take sea water (male seawater) from the bottom of the dish and put it into egg dishes. Use plastic transfer pipette for cell culture.

Amount of sperm is OK when you estimate 20~40 spermatozoids are swimming around an egg. Amount of sperm will be dependent on the number of eggs, size of the dish etc. Usually 3~5 ml of sea water taken from the bottom of 10 cm dish with 10~12 male jellyfish is optimal for 300~400 eggs in 3 cm dish. Too much spermatozoid results in polyspermea and cleavage error. (Eggs from old jellyfish (= 3 weeks after maturation or older) tend to have polyspermea.)

4) If you simply need to get fertilized eggs.

Put 10 female + 1~2 male in a 10 cm dish and leave it on the shaker. This approach is also useful if you need to fertilize midnight or early in the morning using timer-controlled lighting.