1. Maintain broodstock zebra fish in clear plastic aquariums (20 L) containing 10-15 adults aged about 3-4 mo in each tank. Use tap water that has been aerated and kept overnight to remove the chlorine.
2. Spontaneously spawn the zebra fish, and collect the fertilized eggs at the one-celled stage prior to the first cleavage, as follows. Prepare the broodstocks for spawning by feeding with frozen "bloodworms" and live tubifex for a week prior to spawning. The temperature of the water in the aquariums should be maintained between 25 and 32°C. The light regime is 12 h light and 12 h dark.
3. Select ripe females from the stock tanks the evening (4 to 5 pm) before microinjection is planned, and place them in a tank of fresh, standing water with some aquatic plants. Select suitable gravid females (with enlarged bellies), and with slight pressure on the abdomen, examine the oocytes that emerge from the oviduct out to the ovipore. Select females with eggs that are translucent. Examination for ripe eggs must be swift and gentle so as not to stress or harm the fish. In the morning, more than 80% of the selected females should be ready for natural spawning. A female can produce about 200-400 eggs, and fertilizability is usually high.
4. Keep the selected males and females in separate tanks prior to fertilization. Place each female with two males in a breeding trap composed of an enclosure of nettings with apertures >1 mm that will allow fertilized eggs to pass through and sink to the bottom of the aquarium, but small enough to prevent the parent fish from gaining access to and eating the eggs (Fig. 2) Stuart et al. (6) use marbles to cover the floor of the tank to protect the eggs from being eaten without using a breeding enclosure. If the fish are ready, they will spawn within 5-10 mm after the opposite sexes are placed together.
5. The eggs persist in the one-cell stage only for 10-30 min after fertilization. Hence, microinjection at the one-cell stage has to be conducted within this time period. Spawn the fish during the early morning between 7 and 9 am, because eggs spawned after 11 am are often overripe and not viable. There is, therefore, a time limitation that determines the number of eggs that can be microinjected per day by one operator.
6. Collect the recently fertilized and water-hardened eggs with wide-mouthed pipeters or droppers, and carefully arrange the eggs into the holes of a preferred injection chamber. Each egg is rolled into and pressed lightly into a hole, where it will be held firmly and will not roll around when it is injected.
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