Rotifer culture protocol

This protocol presents and describes elements to consider for rotifer culture used to feed zebrafish larvae.

Typical setup

• The culture vessel should be filled with 50 L of culture water at 15 ppt salinity.

• The culture is first inoculated with rotifers, which may be purchased from a commercial supplier (eg, Reed Mariculture Inc., supplied in ‘‘breatheable’’ plastic bags). A population of 5–10 million rotifers is suitable to start a 100 L culture. Upon receipt, each bag of rotifers is temperature-equilibrated (the rotifers are shipped chilled) by floating it in the culture water (or water at culture temperature) for 20–30 minutes.

• After temperature equilibration, the bags are opened and the contents transferred to the culture vessel.

• Aeration is started and feeding begins.

• The rotifers will require about a day to recover fully from the chilling they are subjected to during shipping, so their feeding rates are initially depressed. At first, feed algae concentrate, for example, 0.5mL RotiGrow Nanno_ per million rotifers every 6–8 hours, or enough to impart a distinct green tint to the water.

Rq : Rotifer feeding should noticeably reduce the green tint after a few hours. If there is no reduction in the green color, examine the rotifers to confirm that they are swimming actively and that there is green coloration in their guts, and continue feeding only when the green tint of the water is reduced.

• If feeding has recovered, after 24 hours the volume of water in the culture vessel can be increased to 100 L, and feeding continued. ClorAm-X_ should be added at 0.11 g mL- 1 of RotiGrow Nanno_ used. No need to add ClorAm-X if rotifers are fed with RGcomplete that includes ClorAm-X.

• At 48 hours post-inoculation, routine operation of the culture may commence.

• The culture is fed automatically with a timer-controlled peristaltic pump, which draws from a container of algal concentrate kept at 4°C. The timer should be set to deliver feed at least hourly, and 2–4 times/hour is still better. This setup should be checked daily to ensure that the supply of algal concentrate is not exhausted and that the supply lines are not clogged.

• To determine feed dosing rate, it is not enough to know how many rotifers are in the culture, because it is the harvest rate that determines the production required, and so how much feed is needed. Here are few examples of feeds (in mL) required per million rotifers harvested :

Routine operation

• Approximately 25%–50% of the culture should be harvested each day. Aeration should be maintained in the culture vessel during collection to ensure that that the rotifers are uniformly distributed in the water column.

• The drain valve is opened to permit a convenient flow rate, and the desired volume of culture is allowed to pass through the tubing and into a 40-60 µm strainer.

• The collected rotifers can be immediately transferred into 1–4 L of pre-mixed 3–5 ppt salinity water by gentle rinsing, distributed into squeeze bottles (Fisher 03-409-22D), and allowed 10–20 min for osmotic acclimation. This sub-stock of rotifers is then used for feeding larval fish.

• After the rotifers are harvested from the vessel, the floss filter should be removed and rinsed vigorously with a jet of fresh water until the rinse water runs clean. The entire inside surface of the vessel should then be swept with a designated brush.

• The culture vessel should then be replenished with new culture water. Finally, the clean floss filter is then re-suspended back inside the vessel and normal operation resumes.

• Rotifer density should be checked daily. A simple way to do this is to collect a sample of 100 L directly from the culture vessel and examine it under a dissecting microscope. The rotifers in this sample can be easily counted and assessed for health. A Sedgewick-Rafter plankton counting slide with grid (Aquatic Eco-Systems, Item No. M415) makes counting high densities easier.

Tips for a successful harvest 

• Discerning whether the rotifers in the sample are carrying eggs is also important. Egg ratios (egg count/rotifer count) typically range from 0.25 to 0.5, but an egg ratio of 0.25 in a heavily harvested culture is sufficient to double the population daily (because a rotifer can lay more than one egg per day). A low egg ratio or more than occasional detached eggs indicate adverse conditions.

• Unless rotifer densities are extremely high ( > 2000mL- 1), cultures will usually tolerate a day or two of neglect with no problem. Nevertheless, on weekends/holidays the culture system should be checked daily to ensure that it is operating properly (i.e., ensuring the algae feed supply is adequate, aeration is still on, etc.).

• Backup cultures of rotifers should be kept on hand in case of emergencies. Once every 1–2 weeks, a small portion (e.g., 3–5mL of concentrated, harvested rotifers, or other appropriate quantity) should be collected and placed in a clean 500mL flask containing 15 ppt water and a few drops of algae concentrate. Refrigerated rotifers will slow their metabolism, and can be kept at 4_C for up to 2 weeks. They can be used as a backup to re-start or bolster the culture should the need arise.

• With highly concentrated algae pastes such as RG complete, in use, the tubing used to feed the rotifer culture tends to become clogged and the algae end up clogging the tubing. This disrupts the pumping rate and thus the amount of algae delivered by the peristaltic pump with each pump action. Thus the quantity of algae delivered to the culture is no longer the same as the quantity required from the pump. To limit this type of problem and avoid errors in the algae supply to the rotifers, we recommend diluting the algae solution used. To do this, use water with 30ppt salinity and dilute the complete RG 10 times. This also allows a more precise distribution of the algae for rotifer culture throughout the day (24/24h). Remember that even when diluted, the algae solution should be stored away from light and at 4°C, ideally in a refrigerator.

• Check the flow rate of your peristaltic pump as well as the cleanliness of the tubing used as often as possible. To do this, calibrate the pump and change the tubing regularly.

Sources and references

• C.Lawrence et al., Zebrafish, vol 9 (3), 2012. Methods for Culturing Saltwater Rotifers (Brachionus plicatilis) for Rearing Larval Zebrafish

• Food and Agriculture Organization of the United Nations : http://www.fao.org/docrep/003/w3732E/w3732e00.htm#Contents

• http://reedmariculture.com/index.php