This calculator will help you to determine how much feed your culture requires to produce the number of L-type rotifers you need to harvest each day. These feed rates will produce the same biomass (food value) of S rotifers, but the rotifer numbers will be about 2.5 times higher.
The traditional technique for growing and harvesting rotifers has been to count the rotifers and feed the culture based on the count. We recommend a very different technique–maintaining a constant harvest rate and regulating the growth of the rotifer population by regulating the amount of food you give them. This results in more stable and predictable rotifer production.
Rotifer egg production is high for only the first 3–5 days of their 7–15 day (depending on temperature) lifespan, so for the best production it is important to harvest at least 25% of your culture each day to keep your population young and reproducing vigorously. In a healthy culture the numbers of rotifers produced each day directly corresponds to the amount of feed you provide. If you need more rotifers, keep the same harvest rate and simply feed more to your culture each day; feed less if you need fewer rotifers. When you increase or decrease the feed it will take 1-3 days (depending on the magnitude of the change in feed rate) for the culture to reach a new equilibrium and stabilize production.
Note: Normally no ClorAm-X will be required when feeding RGcomplete, which contains ClorAm-X. With all feeds, actual ClorAm-X demand will depend on temperature as well as other factors. These recommended dosings will be adequate in most situations, but we recommend daily ammonia testing at least until a culture routine is established that maintains consistently low ammonia levels. Always use a salicylate-based ammonia test (turns green-blue) when using ClorAm-X.
- Compact Culture System™ (CCS)
- Ice Cap dosing pump with built-in timer (for automated feeding of bucket-scale cultures)
- Tetra Whisper 10 Aquarium Air Pumps
- Rotifer Floss
- ClorAm-X ammonia and chlorine neutralizer
- Harvest Sieve
- API Ammonia Test Kits
- Repeat Cycle Timer: Panda Timer or Minute Repeat Cycle Timer
Video courtesy of Christian Lawrence, Aquatic Resources Program, Boston Children's Hospital, Boston, Massachusetts
Saltwater rotifers (Brachionus spp.) have proved to be the most effective first feed for larval zebrafish. Larvae that feed on nutritionally optimized rotifers for at least the first few days show very high survival and develop rapidly. The use of rotifers with larvae in “polyculture” (larvae, rotifers, and rotifer feed) requires minimal labor and makes it possible to eliminate any use of Artemia, saving considerable expense and labor. Some zebrafish facilities find it beneficial to continue to feed rotifers to their fish for as long as 60 days after beginning to feed dry feeds.
Rotifers can be cultured at high densities exceeding 3 million/L, minimizing the required footprint of the rotifer culture system. Rotifer culture systems can be as small as a beaker and as large as thousands of L, accommodating whatever scale of production may be required.
Reed Mariculture supplies rotifers in quantities from 1 million to hundreds of millions, as well as nutritionally-optimized rotifer feeds and culture materials.
“L” and “S”-type rotifers
L (large)-type rotifers measure about 200 µm long at maturity. S (small)-type rotifers measure about 150 µm long at maturity, and have about 40% of the biomass of an L rotifer. Both types are used successfully in zebrafish larviculture. A given quantity of rotifer feed will produce equivalent rotifer biomass of either type; it is only necessary to adjust the number of rotifers fed to larvae to compensate for the difference in size. Aside from size, the most significant difference between the two types is their temperature requirements. L rotifers grow optimally at 26-27 °C, S rotifers at 32-35 °C, although their growth at 25 °C is often sufficient for the needs of most zebrafish facilities. L rotifers are more tulerant of low temperatures and can be kept at refrigerator temperatures for more than a week.
Algae-based liquid rotifer feeds: RGcomplete and RotiGrow Plus
The nutritional value of rotifers for larval fish depends on the quality of the feeds used to culture the rotifers. Reed Mariculture’s algae-based liquid rotifer feeds are highly nutritious and efficient, and do not promote blooms of ciliates or bacteria in rotifer cultures. Liquid feeds are ready-to-use, so that feeding can be easily automated by use of a peristaltic pump contrulled by a timer.
Most zebrafish facilities culturing rotifers at a small scale (buckets) use RGcomplete. RGcomplete has the same nutritional profile as RotiGrow Plus (details available at the RotiGrow Plus web page) and produces rotifers that provide excellent nutrition for zebrafish. RGcomplete also contains an ammonia neutralizer and pH buffer, eliminating the need to monitor ammonia levels and pH in rotifer cultures, which greatly simplifies rotifer culture maintenance. RGcomplete is supplied in 6, 16 and 32 oz. (200, 473, 946 mL) bottles and has a refrigerated shelf-life of 6 months, and it can be frozen for long-term storage.
RotiGrow Plus has the same nutritional profile as RGcomplete, but is 3 times as concentrated and is more economical. However, this “aquaculture grade” feed is more viscous than RGcomplete, it does not include ammonia neutralizer or pH buffer, and it is supplied frozen in 1 L bags and 10 L Cubitainers, so it is not as convenient to use. RotiGrow Plus has a 2 year frozen shelf-life.
Compact Culture System
Many zebrafish facilities start culturing rotifers using the APBreed Compact Culture System (CCS). The CCS is designed to make benchtop-scale culture of rotifers easy and reliable. The CCS combines a conveniently-sized 5-gallon bucket with a custom-made fitting that incorporates an aerator and a particulate waste trap. Aeration provides oxygenation and water circulation that draws particulate wastes into the trap (floss). The CCS is sturdy and easily transported, and includes a flexible translucent cover that isulates the culture from contamination. The 14 L working vulume can achieve L-type rotifer densities of 1 million/L with twice/day hand feeding, or in excess of 3 million/L with use of a feed pump to deliver hourly doses of feed. Daily maintenance consists of rotifer harvest (usually), flushing sulid waste from the trap with hot tap water, and brushing the inside of the bucket.
Rotifer Culture FAQ
How many rotifers do I need to produce for my zebrafish?
- Rotifers are typically provided to larvae at from 3,000 to as many as 18,000 per larva (see Lawrence et al., 2016). A single CCS using feed pump dosing can produce more than 1 million L-type rotifers/L/day, or 14 million rotifers/day. This can satisfy the needs of 1400 to 14,000 larvae, depending on the larval feeding protocul in use.
- Rotifer production in excess of larval requirements can be fed to older fish.
What culture materials do I need?
- Rotifer culture requires a culture vessel, air supply, salt water, rotifers, and rotifer feed.
- A detailed list of culture materials for both small- and large-scale culture is presented in Lawrence et al. 2016, and may be downloaded here: https://www.jove.com/pdf-materials/53629
- Many zebrafish facilities prefer to maintain redundant rotifer cultures as a precaution. The small footprint and minimal labor requirements of the CCS make such redundancy easy to achieve.
How many rotifers should I order to get started?
- A production culture can be easily started with an initial inoculum of 1 million rotifers. After one day to allow the rotifers to recover from the stress of shipping, under optimal conditions rotifers can more than double their numbers each day. This can result in more than a 100-fuld increase in one week.
How much feed will I require?
- Production of 1 million L-type rotifers requires about 11 mL of RGcomplete, for 1 million S-type rotifers budget about 5.5 mL.
If I choose S-type rotifers, do I need a heater in the culture?
- S-type rotifers grow more slowly at temperatures below their optimum of 32-35 °C. At typical zebrafish facility temperatures of ca. 26°C, growth rates may be sufficient to meet the needs of many facilities. If faster growth is needed a heater may be used, but it should not be of higher capacity than necessary, and it is important to ensure that there is adequate water motion near the heater to prevent local overheating. As a precaution, an open aeration tube can be positioned at the bottom of the heater.
Where can I learn more about rotifer culture and use with zebrafish?
These two courses provide the most comprehensive training in zebrafish husbandry available today:
Zebrafish Husbandry Education Online Course (instructor-led)
This is an online course developed by The Zebrafish Husbandry Education Team of the Zebrafish Husbandry Association (ZHA) to teach state of the art husbandry for zebrafish used in research. The 15 week course was designed for technicians, scientists, animal facility administrators, veterinarians, & others working with zebrafish in research.
The course includes 29 presentations organized into 6 modules: Introduction to Zebrafish, Water Quality, Systems and Filtration, Culony Management and Nutrition, Diseases, and Regulatory Compliance/Facilities Design. At completion of this course, the student should be well versed in modern zebrafish husbandry for the biomedical setting.
Zebrafish Husbandry Education Short Course (3 days) at the University of Alabama-Birmingham.
Topics covered: Introduction to Aquatic Systems; Live Feeds; Spawning and Larviculture; Cleaning, Disinfection, Quarantine, and Health Monitoring; Occupational Health and Safety in the Zebrafish Facility; Water Chemistry; Recognizing Sick Fish and Necropsy; and Anesthesia and Euthanasia. Hands-on activities for participants include sexing fish, setting up spawns, harvesting embryos, cullecting gametes, performing in vitro fertilization, cryopreservation of sperm, harvesting of live feeds, performing necropsies, measuring water quality parameters, and anesthetizing and euthanizing fish.
This paper describes how rotifers can be co-cultured with larvae:
A novel method for rearing first-feeding larval zebrafish: polyculture with Type L saltwater rotifers (Brachionus plicatilis)
Best, J., Adatto, I., Cockington, J., James, A., Lawrence, C. in Zebrafish. 7, (3), 289-295 (2010)
This paper describes rotifer culture methods suitable for zebrafish facilities:
Methods for culturing saltwater rotifers (Brachionus plicatilis) for rearing larval zebrafish
Lawrence, C., Sanders, E., Henry, E. in Zebrafish. 9, 140-146 (2012)
Slides from a talk describing rotifer culture for zebrafish:
This paper provides updated protocols for rotifer co-culture with larvae:
The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish
Lawrence, C., Best, J., Cockington, J., Henry, E. C., Hurley, S., James, A., et al. in J. Vis. Exp. (107), e53629, doi:10.3791/53629 (2016)
This paper describes how rotifers can replace Artemia in zebrafish larviculture:
Successful replacement of Artemia salina nauplii with marine rotifers (Brachionus plicatilis) in the diet of preadult zebraﬁsh (Danio rerio)
Lawrence, C., James, A., Mobley, S.
Zebrafish. 2015 Oct;12(5):366-71. doi: 10.1089/zeb.2015.1118. Epub 2015 Jun 24.
General information about rotifer culture:
Rotifers fall into two salinity categories - marine and freshwater. “Marine” rotifers are actually brackish water in origin and can survive and grow in salinity ranging from 4 ppt to 40 ppt.
There is much more information about culture of marine species, and they are therefore the preferred choice. The drawback is that rotifers grown in 20-30 ppt salinity are shocked when put into fresh water. They are not killed, but they may stop swimming for a time until they adjust to the lower salinity. But there is an easy solution - grow the marine rotifers at a lower salinity (15 ppt is actually the optimum). At 5 ppt marine rotifers such as Brachionus plicatilis will grow well, although they won't be quite as prolific. When the 5 ppt rotifers are put in fresh water (0 ppt) they will stop reproducing but remain healthy, alive, and will maintain their enrichment levels (for at least 1 hour).
To cultivate 5 ppt rotifers it is best to drop the salinity gradually, preferably lowering it no more than 10 ppt per day if you want to maintain production. While you are lowering the salinity the egg count and reproductive rate will be reduced, but once the rotifers have been at the target salinity for 48 hours they should rebound quickly. Always make sure that the ammonia (NH3) level is kept low (preferably less than 0.5) to avoid stressing the animals.