The overall goal of the Rotifer Polyculture Method is to harness the natural productivity and biological characteristics of salt water rotifers to create an environment that promotes rapid growth, and the maximum survival of first-feeding larval zebrafish. The primary advantage of this technique is that it dramatically decreases the amount of time and space required to successfully rear large numbers of zebrafish larvae for use in research experiments. Begin rotifer culture maintenance by filling the feed-out culture vessel, or FCV, to 90 percent capacity with clean, dechlorinated fresh water dosed with 10 grams per liter of aquarium salts.
Ensure that the water is thoroughly mixed and that all of the salt is fully dissolved. Then, set the airflow into the vessel so that it maintains a rolling boil. Measure the salinity of the liquid with a refractometer.
Next, sample the rotifers in the culture vessel, or CV.Make sure that the culture is well mixed. Then, collect three two-to-three milliliter samples using a transfer pipette from different parts of the culture and combine the samples in a tube. Transfer one-to-two milliliters of the combined sample onto a petri dish.
Visualize the sample under a dissecting microscope, and check the quality of the culture. Add 100 microliters of 50 percent Lugol's iodine solution to the sample to immobilize the rotifers and count them. Immediately take a two milliliter sub-sample with a plastic pipette and dispense one milliliter into a Sedgewick Rafter counting slide.
Using the dissecting microscope, count as much of the slide area as necessary to count up to 100 intact rotifers and the total number of eggs attached to the rotifers. Then, calculate the number of rotifers per milliliter for later use. Initiate harvest by removing the air supply and floss filter.
Slowly open the valve at the bottom of the CV and allow the water to flow gently into a plankton collector with a 53 micron mesh screen. Collect the water flowing out of the bottom of the collector after filtering into a bucket or draining. Next, fill a bottle with 10 to 15 grams per liter of clean salt water.
Invert the screen over the FCV and wash the rotifers into the FCV with a gentle stream of salt water. Start the programmable metering timer or PMT to deliver feed to the FCV. Then, scrub the entire inside of the CV with a clean scrub pad.
Create a new 15 grams per liter mix in a five gallon bucket to replace the volume of water lost to harvest. Stir the solution vigorously until the mixture is completely dissolved. Then, add the solution to the CV.Next, rinse the floss filter with a high-pressure spray.
When the floss filter is free of debris, return it to the CV.Adjust the feed rates of algae delivered to the CV by changing the duration of each dosing event. After 24 hours, repeat the process, starting the harvest of the rotifers remaining in the FCV. Concentrate the rotifers in two liters of fresh, clean, dechlorinated water with five grams per liter of salt.
These can be stored at four degrees Celsius as a back-up supply, or used to feed later stages of fish beyond what is described in this protocol. Collect zebrafish embryos from a spawning event by pouring spawned embryos through a tea strainer. And then, gently rinsing with sterile fish water from a wash bottle into petri dishes.
Once all the embryos are collected, incubate them. Begin the polyculture phase when more than 90 percent of the hatched larvae are actively swimming up in the water column. Add 500 milliliters of the rotifer culture directly from the FCV to a 3.5 liter nursery tank.
Next, gently pour the larvae from one petri dish into the nursery tank. Ensure that no larvae remain in the dish. Add 500 milliliters of clean, conditioned fish water from a dedicated water source to the tank to a final volume of one liter, and 5 grams per liter final salinity.
Observe the polyculture tank at least once per day to ensure that the rotifers and fish are present and growing. Check that the rotifers are visible throughout the water column. Ensure that the fish are also visible within the water column and that they are swimming among the rotifers.
It's critical to asses the status of each tank on a daily basis to make sure that they're alive and swimming with rotifers available for all fish larvae. Upon insurance that the rotifers and fish are growing, start normal water flow through the tank. Finally, place a screen over the drain port to ensure that the larvae are not flushed out of the tank.
The typical performance of a rotifer culture was monitored over a 30 day period, and yielded a mean culture density of 932 rotifers per milliliter. And a maximum of 1, 330 rotifers per milliliter. The rotifer population in a polyculture tank with zebrafish has a mean rotifer density of 589.4 rotifers per milliliter.
The minimum value of 481 rotifers per milliliter is 10 times better than previously published work. Zebrafish showed steady growth each day over the five day period from a mean total length of 3.9 millimeters to 5.4 millimeters. After watching this video, you should have a good understanding of how to culture rotifers for use in a polyculture system that's devised to meet the nutritional demands of first-feeding zebrafish.
