CHAPTER 3 - EMBRYONIC AND LARVAL CULTURE

Raising Larvae in a Nursery

Raising babies under "nursery" conditions is relatively labor-intensive, but ensures that they receive lots of hands-on attention for maximal survival.

General procedures

Fish must be raised in the Nursery from fertilization until they are big enough to live in regular tanks. They enter the Nursery in beakers and graduate to larger containers, like mouse cages, as they grow. Similarly, they require a changing diet as they grow. Throughout this period they require daily cleaning and feeding.

The person in charge of the Nursery has the job of caring for the baby fish from the time they begin eating paramecia until they graduate to young adulthood. This means instructing new people on the current methods of placing babies into the Nursery, caring for the babies when they arrive, placing the babies into the main facility, and feeding the babies in the main facility until they become adults.

Zebrafish embryos must be prepared properly before they are transferred into the Nursery. The user should separate the fertile embryos from the infertile ones and then meticulously clean the fertile embryos. If the eggs are not too dirty, several rinses with system water are sufficient. For extremely dirty eggs or when infection is suspected, bleaching the eggs may be necessary.

A solution of bleach water is made by placing 0.1 ml of 5.25% sodium hypochlorite (bleach) and 170 ml of system water into a 250 ml beaker. To bleach the eggs, first rinse them with system water and then place 25 to 30 into the beaker of bleach solution. Swirl the beaker and allow the eggs to separate and the entire surface of each chorion to come into contact with the water. After one to two minutes in the solution, remove the eggs with a clean pipette and place them into a beaker of clean water. Again, swirl the eggs and allow each one to receive a thorough rinse. Repeat the rinse procedure and then place the eggs into 250 ml beakers at 25 to 30 eggs each.

A tough chorion can often be a by-product of the bleaching procedure. Thus, if the young fish do not hatch on their own, remove the chorions manually (See Removing Embryos from Their Chorions, Chapter 4).

To avoid problems with contamination that will arise if eggs are incorrectly prepared, the Nursery cannot accept beakers of embryos that have been improperly sorted or inadequately cleaned. In addition, a beaker of over-crowded embryos (30 or fewer is the accepted number) cannot be allowed in the Nursery.

Baby zebrafish should be placed in the Nursery before the fourth day of life when they need to begin eating food. (They grow using the nutrients in the

yolk from day one to day four.) If a beaker is placed in the Nursery before day four, it is the responsibility of the owner of the fish to clean the beakers and remove any dead babies. After day four, the nursery person begins daily cleaning and feeding.

It is important that a proper label accompany every beaker of fish brought into the Nursery. Each label should contain the name of the person who is responsible for the fish, the date the fish were fertilized, the name of the fish (strain), and the stock number of the fish. If the fish are to be euthanized before they would normally go into a tank (e.g. to be used for an assay or histology) the number of days after fertilization that they are to be killed should be marked on the label (e.g. "14 dx").

After placing new babies in the Nursery, the person responsible for them should locate the Tank Sign-Up book and record the number(s) of the tanks(s) the fish will occupy in the main room. Failure to do so could result in the loss of that group of baby fish. The user should completely fill in all the information required in the Tank Sign-Up book.

The daily Nursery duties can be divided into three main categories:

• cleaning the baby fish containers
• feeding the baby fish
• growing brine shrimp

Cleaning the baby fish containers

Both types of baby fish containers, 250 ml beakers and mouse cages, need to be cleaned on a daily basis.

Cleaning the 250 ml beakers

Baby fish that are between four and nine days old are kept in 250 ml beakers at 25-30 fish per beaker. These fish are fed paramecia, but sometimes fish die and need to be removed. Use a pipette (5 3/4" disposable Pasteur pipette) to remove dead fish. Look into the beakers with baby fish and identify the dead fish. The paramecia will eat the dead fish, so a fish to be removed will be lying on the bottom of the beaker and will appear rough or fuzzy around the edges because it is covered with paramecia. Use the pipette to remove the dead fish and record the death (on a piece of tape located on the side of the beaker). Use only one pipette per stock number (type of fish) to minimize the chance of contaminating the various strains with a disease or protozoan infestation. Follow this procedure once every day.

Cleaning mouse cages

The baby fish are placed in a mouse cage nine days after fertilization when they become old enough to eat baby brine shrimp (small artemia). The baby fish eat most of the brine shrimp they are fed, but some portion is always left and these brine shrimp die in the normal fish water. Twice a day the fish are fed brine shrimp and twice a day the containers must be cleaned of old, dead shrimp. This serves not only to remove the decaying brine shrimp, fecal material left by the fish, and any baby fish that might have died, but it also provides us with an opportunity to give the fish fresh water.

For the first three days after the fish are placed into baby fish containers (e.g. mouse cages), add only a few drops of brine shrimp and do not clean the container. Beginning on the fourth day, use a clean egg siphon and a 5-gallon bucket and "vacuum" the debris off the bottom of the container. Place one hand on the glass part of the siphon to direct it and the other on the hose part ready to pinch it off if a fish is inadvertently sucked up the tube. A moderate speed is required so the maximum amount of debris is removed, while at the same time leaving the fish in a comfortable volume of water. When one mouse cage is finished, move on to the next one using the same siphon and bucket.

A wise technique for a novice cleaner is to check the bucket before going on to the next container. If a live fish has been sucked up, it can be rescued and returned to its proper mouse cage. If many mouse cages have been cleaned and a live baby is noticed in the bucket, it should be euthanized rather than returned to a mouse cage. This will avoid the possibility of mixing different strains of fish.

After all the mouse cages have been cleaned, fill them back up with system water. Keep a large tank of clean system water in the Nursery for filling the mouse cages.

Feeding the baby fish

The baby fish are fed two types of food. The smallest baby fish get paramecia and the bigger ones get brine shrimp. The intermediate baby fish eat both kinds of food.

Paramecia

Baby fish four to fifteen days old should be fed paramecia. The paramecia are cultured in finger bowls on a rack in the Nursery (see methods for growing paramecia in following sections of this chapter). To collect paramecia, take a cloth handkerchief and place it in a 1-liter funnel. Place the funnel in a ring stand on top of the paramecia rack and place a 1-liter beaker under the funnel. Choose a stack of fingerbowls from the rack with the oldest brine shrimp and pour the contents of each fingerbowl through the handkerchief. The empty fingerbowls should be rinsed and placed in the tub of dirty dishes to be washed before reuse. The handkerchief should be rinsed out and hung up to dry. The 1-liter beaker contains the paramecia that are ready to be used for feeding the babies in mouse cages.

If the fish are receiving their first feeding in a mouse cage, they should get 50 ml of paramecia per mouse cage. For every feeding after that, they should get 25 ml. It should be noted that some strains are weaker than others and may require more paramecia per feeding. In addition, some of the weaker strains may not grow as fast as others and the number of days they are fed paramecia should be lengthened accordingly.

To feed younger babies in the 250 ml beakers, the paramecia need to be filtered. Start by following the procedure for collecting paramecia as outlined above. Take one piece of filter paper (P8 qualitative; Fisher) and fold it 8 times so it becomes accordion-like. Place the folded paper in a 1-liter funnel and place an empty 1-liter beaker beneath it. Pour the beakers of collected paramecia into the funnel with the filter paper in it. The waste water will collect in the beaker at the bottom and the paramecia will become concentrated in the top. When approximately 500 ml of waste water has been collected, take a pipette or other long, clean object, and while holding the funnel over a clean, empty beaker, poke the bottom of the paper. The concentrated paramecia will pour into this beaker. This beaker of paramecia is ready to be fed to the babies in the 250 ml beakers.

Use a clean 10 ml glass pipette to give each beaker approximately 5 ml of the paramecia suspension. After all the beakers have been fed, check to make sure they are covered (e.g. with plexiglass) and then clean the paramecia-collecting equipment with tap water. Discard the used filter papers. Feed the babies in the 250 ml beakers once each day.

Brine shrimp

The brine shrimp are hatched in 5-gallon tanks. To collect brine shrimp, choose the tank with the oldest shrimp and turn off the air lines that are used to aerate the shrimp culture. Place a lamp in front of the tank and turn it on. The brine shrimp will move toward the light and in five to ten minutes, they can be siphoned out.

To siphon the brine shrimp for a morning feeding, place a 2-liter flask with a funnel and a handkerchief on the floor in front of the tank. Remove the lid from the tank and slowly lower the siphon into the water. (A layer of unhatched eggs always covers the surface of the water and a small puff of air as the end of the siphon passes through this layer can clear the unhatched eggs so they do not go into the siphon.) Position the end of the siphon directly in the thickest concentration of brine shrimp and start the siphon. The shrimp will collect on the handkerchief and the salt water will collect in the flask.

A clean empty beaker should be standing by to receive the brine shrimp. A 600 ml beaker works nicely; however, if there are a lot of brine shrimp, a 1-liter beaker is required. The brine shrimp should be rinsed with fish water to remove any excess salt water before feeding. Keep a squirt bottle near the clean water tank for this purpose. To transfer the brine shrimp to the beaker, place one hand in the shape of a cup under the handkerchief and lift the shrimp out of the funnel. With the other hand, use the squirt bottle to rinse them off the cloth and into the beaker. If the concentration in the beaker is too thick, add more clean system water to thin it. Take the flask of salt water and pour it back into the brine shrimp tank. Locate the air lines and turn them on so the water is bubbling again.

Use a pipette (5 3/4" disposable Pasteur pipette) with a bulb to feed the baby fish in the Nursery. Fish that are 9-11 days old need only a couple of drops of the brine shrimp. Fish that are 12-17 days old can eat at least one full pipette and if they are between 17 and 21 days old, they will eat 1.5 to 2 pipettes full.

In addition to the fish in mouse cages in the Nursery, babies in regular tanks (in the regular, non-nursery facility) also need to be fed brine shrimp. All the babies in the main facility should be labeled (e.g. with pink pieces of tape on their tanks that have the word "babies" written on them). Simply start at one end of the main room and walk down each aisle looking for tanks with "baby stickers" on them. A general rule for feeding is one pipette full per 10 fish, so a tank with 50 fish will get 5 pipettes of brine shrimp.

Some adult fish may also need a supplement of baby brine shrimp to help them grow better. These tanks should be marked (e.g. indicated by round, green stickers) and should get one pipette per 20 fish.

After all the fish with either pink baby stickers or round green stickers have been fed, any extra food can be given to other adult fish (e.g. the fish in the largest stock tanks). The beaker and the pipette can be rinsed with regular tap water and put on a paper towel to dry.

Afternoon feedings

To collect brine shrimp for the afternoon feeding, obtain a clean, empty mouse cage and place the flask that will hold the salt water in the mouse cage. Put the mouse cage and flask on the floor and siphon almost all the brine shrimp from the tank using the procedures outlined for the morning feeding. Use two 1-liter beakers to hold the brine shrimp and pour the collected salt water back into the tank. A larger amount of brine shrimp is required because the afternoon feeding includes a supplement of baby brine shrimp for all the fish in the facility. The general rule for feeding adults labeled with green dots (see above) can be used as a guideline for feeding the adult supplement in the afternoon. After all the fish have been fed, the beakers and pipette are cleaned as in the morning.

Growing brine shrimp

New tanks of baby brine shrimp should be started on a daily basis. Use a clean 5-gallon tank for the new batch of brine shrimp. Begin by removing the old tank from its place by disconnecting its air lines from the metal valves and moving it aside. Place the clean tank on the counter and connect its air lines to the valves. Use clean system water to fill the tank almost full (approximately 4 gallons), add 500 g of rock salt, and turn on the air valves to begin stirring the water. Add 15 teaspoons (75 ml) of brine shrimp eggs to the tank. Cover the tank with a piece of plexiglass. Label the tank with the present day of the week.

The dirty tank should be taken into the kitchen and cleaned using tap water. First, remove the bubbling apparatus and rinse it inside and out. Second, dump the old brine shrimp and salt water down the drain, and third, rinse out the tank. After the tank has been cleaned, place it in the Nursery to air dry.

Transferring fish out of the Nursery

When the baby fish have reached 21 days of age, they are ready to be transferred out of the Nursery into a full-sized tank in the main zebrafish facility. To transfer fish, first update the nursery record book. In the book, all entries are organized by stock number. Find the stock number of the fish to be transferred out of the Nursery and fill in the columns for total number of live fish remaining and total number of dead fish recorded on the label. Up to 50 baby fish can be placed in a 10-gallon tank. Divide the total number of babies so that each mouse cage has approximately 50 fish. Pour the babies from each mouse cage into a separate 10-gallon tank making sure all the babies have been transferred and none remain on the sides or bottom of the mouse cage. Remove the label with the name of the fish from the mouse cage and place it in the upper left corner on the front of the tank. All other labels on the mouse cage can be thrown away. Make a label showing the total number of fish in the tank and place it so it can be seen easily. Replace the lid on the tank and check to make sure the fresh water is dripping, place a piece of netting over the drain to keep the babies from escaping, and start the air bubbler. The dirty mouse cage should be washed before reuse. Enter the new fish information into the computer database. One form should be filled out for each stock number.