There is no evidence that rotifers and artemia possess the chemical toolkit needed to assemble astaxanthin from lessor carotenoids. They are, however, obligate filter feeders with a circa 8 micron mouth gape. Any zooplankton small enough to be taken in are rich in carotenoids including astaxanthin.
It is commercially not viable to replicate zooplankton as a first feed. Hatcheries are forced to make a choice and typically select rotifers or artemia as first live feeds. Rotifers in turn are fed marine algae. This practice effectively removes astaxanthin (and other micronutrients) from predator diets.
While replicating zooplankton is beyond reach, formulating rotifer and artemia feeds that “gut load” these animals with astaxanthin and micronutrients found in zooplankton prior to feeding to predators is economically viable.
Amplifeed(tm) Replete rotifer and artemia feed contains di-esterified 3S, 3’S astaxanthin as well as a variety of other micronutrients. It is used as a finishing feed during the last 24 hours prior to harvesting and an economically viable method of enhancing larval growth rates and health. The feed is milled to 8 microns to match the mouth gap of adult rotifers resulting in nearly 100% uptake into the gut and rotifer cells. We have measured astaxanthin concentrations in rotifer cells at a density of 200 ng/rotifer.
We have tested Amplifeed Replete in over 200 species/varieties of fish including marine ornamentals, shrimp, cod and other pelagic spawners. We observe larval prey fed enriched rotifers and artemia followed by astaxanthin enriched first dry feeds doubled growth rates at 42 days post hatch.
No surprise.
By protecting mitochondrial health using astaxanthin during larval development allows the predator to more efficiently process feeds into the power needed to accelerate growth and develop strong immune system function.
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Sustainable Aquatics Inc (SA) hatchery has been raising fish using rotifers as a first feed for over 15 years. In this article we compare the reliability, nutritional content, and cost of operating SA’s “Closed Rotifer System” vs. the “Open-Air Rotifer Systems” widely used throughout the aquaculture industry. Our conclusion is that SA’s closed rotifer production system outperforms open-air systems on all three accounts.
Reliability- SA’s Closed Rotifer System
Our rotifer production system is designed to produce about 500 million rotifers per day. Each day we inoculate a new 1000-liter bag with about a liter of live starter algae, either Tetraselmis or Isochrysis. These algae are grown in clean systems, using RO water and sub-micron filtered air, and we also use sub-micron ceramic filters to remove about 98% of the water from the algae and replace that with clean sterile artificial seawater.
We add algae growth nutrients to the bag, illuminate it, and blow sub-micron filtered air through the bag from the bottom to create strong turbulence.
After about 7 days, we inoculate the bag with rotifers at about 0.1 per ml that are free of contamination from other rotifer species and ciliates.
Both the rotifers and algae amplify in this culture, but soon the rotifers overtake the algae and the bag color goes from deep green to nearly clear.
At this point we add our patented rotifer feed, Amplifeed Replete, a 8-micron feed containing a lipid rich, nano-emulsified blend of micronutrients including astaxanthin, selenium, DHA, vitamins, minerals, and metals such as iron, zinc, and magnesium and marine proteins and fats.
Then on about day 14, we harvest the rotifers and at densities between 300 and 1000 rotifer per ml and feed them to fish larvae. Notice that we are striving for reliability, not maximum density. We want to avoid at all costs conditions that could lead to rotifer crashes and the loss of fish larvae. We typically operate our rotifer production systems crash-free for over 3 years, and limit crashes to single bags if one does occur.
Reliability- Open-Air Rotifer Systems
Open air rotifer production systems typically grow rotifers in large, uncovered tubs linked to protein skimmers and bacterial bioreactors. These rotifer cultures are fed algae biomass from brands such as RotiGrow™ or RGComplete™[1]. Other common algae feeds are called “Nano Pastes”
These systems often operate at rotifer densities of 2000 to 4000 per ml.
Regular crashes can be expected unless the cultures are isolated in a clean room which is operated to scrupulously to eliminate contamination.
The following are typical types of contaminants found in rotifer cultures:
Ciliates:
For instance, ciliates are common contaminants and are co-travelers with copepods, bacteria and other rotifers and are dispersed airborne. They often out-compete rotifers for food and have higher population growth rates, leading to suppression of rotifer harvests.
Bacteria:
Of course, bacteria are easily dispersed airborne and are between 200 nm and 2 microns. Some are pathogens, suppressing rotifer growth and infecting fish larvae.
Copepods and Rotifers:
Some copepods and rotifers produce eggs that survive desiccation. These eggs are about 30-50 microns in diameter and can be dispersed by air currents.
What are the culture conditions in the open vs closed approaches?
The SA approach uses closed sterile plastic bags that likely exceed a Class 1 clean room standards using submicron filtration of all air and water.
The open approach normally places rotifer mass cultures in an open room sharing common access and air flow with the entire hatchery. The air is often rich in airborne by-products of the aquaculture environment and full of contaminants.
Conclusion: If reliability is the highest value in the management of producing rotifers in a hatchery, it is clear that the SA method is a prerequisite for long term, consistent success.
Nutritional Content: All rotifers are not equal
Most fish larvae developing in the wild consume a diet of rotifers and copepods, which provide marine proteins and fats plus a rich array of micronutrients, including xanthophyll carotenoids such as astaxanthin, DHA, folate, selenium, iron, magnesium, zinc, and vitamins.
We developed Amplifeed Replete to gut-load rotifers with the micronutrients that fish larvae typically consume in the wild, but are absent in rotifers raised on algae-only diets.
The nutritional content of all cultured rotifers is 100% reflective of their diet. In the wild brachionid rotifers are indiscriminate filter feeders, ingesting all particles from 2-15 µm. They are therefore not obligate algivores. Feeding rotifers to fish larvae lacking the full spectrum of what they normally consume in the wild will short-change the larvae and compromise their health, immune systems, and growth and maturation rates.
At SA we have performed experiments to explore the difference in larvae survival, growth, health and maturation rate based on supplementing rotifers with Amplifeed Replete.
In the SA approach, we add Amplifeed Replete 1-2 days prior to harvest.
This practice increases eggs per female, color, and nutritional value of the rotifers, as well as their density. The figure below illustrates what happens when larvae are fed these rotifers enriched with Amplifeed Replete compared to no Amplifeed Replete:
In addition to its novel composition, Amplifeed Replete is a slow sinking feed, milled to sub-8 micron particles, so that they can be consumed by brachionid rotifers. Amplifeed Replete remains in the water column for a few hours and is nearly 100% consumed, lowering feed cost, and improving water quality.
Cost Comparison
The following compares the cost of operating a closed vs open rotifer mass culture production system. We have not considered the costs of periodic culture crashes and subsequent loss of fish larvae in this analysis, even though they are more common in open systems.
There is a cost in the SA method for cultivating algae, but this cost is far less than the cost of buying algae pastes at USD 90 per liter. Our analysis shows that the SA closed system of rotifer production is about 2.4 times less expensive to produce 1 billion rotifers than an open system using algae paste.
Summary:
SA’s closed system approach to mass culturing rotifers is superior on each of these metrics:
[1] Rotigrow™ is a trademark of Reed Mariculture, RGComplete™ is a trademark of ReefNutrition.
[2] Cloram-X™ is a trademark of Reed Mariculture
]]>Hatcheries targeting cold water species such as Cod and Ballan wrasse typically hatch larvae in water temperatures of 8-10°C and salinity of 35 ppt, mimicking the hatching temperatures and salinity they observe in nature.
Both species suffer from very poor yields (as low as 5% hatch to harvest). The majority of the failures are tied to poor larval survival rates while on live rotifer feeds. After discussions with Cod and Ballan wrasse hatcheries, we have concluded rotifer cultures and practices are the likely culprit behind the poor yields.
Four Observations:
Species Selection
There are some marine Brachionus species that grow well at 8-10 °C, but little is known about their mobility and nutritional profile at low temperature, or their suitability for mass culture.
We offer cysts of 15 brachionid species from fresh, brackish and salt-water sources from around the world. Our rotifer cysts are free of contamination from other rotifer species and ciliates and are well characterized as adults for body size and population growth rate under standard conditions. This allows hatchery mangers to match the type of rotifer cultured to the mouth gape of their larval predator. All of these rotifer species have been domesticated for aquaculture and have superior mass culture performance as compared to rotifers collected from wild populations.
Temperature and Salinity Transitions
Rotifer mass cultures are typically amplified at 25°C and salinity of 15-20 ppt. Little is known about what happens to rotifers when they are transferred to larval rearing tanks at 8-10°C and 35 ppt salinity levels. We know that rotifers typically survive this transfer, but not how long their mobility is suppressed or how these conditions affect rotifer gut evacuation rates. Rotifer mobility in larval tanks is critical because it affects larval predator-rotifer prey encounter rate and gut evacuation rate affects the nutritional quality of ingested rotifers.
We have observed that brachionid rotifers can be transferred to very low salinity water with minimal osmotic shock as long as the receiving water has high alkalinity (>300 PPM). Here we have seen that rotifers continue to swim vigorously for about two hours, by which time they are mostly consumed by larval predators.
We also find that we can ship and store rotifers chilled in water as low as 3°C, where they can survive for a few weeks. Further guidance on managing temperature and salinity transitions can be found at: Fielder DS, Purser GJ, Battaglene SC (2000) Effect of rapid changes in temperature and salinity on availability of the rotifers Brachionus rotundiformis and Brachionus plicatilis. Aquaculture 189:85–99.
Toss out open rotifer mass culture systems
Operating open rotifer mass culture systems with common air flows with the main hatchery is a recipe for contamination. Best practice is to either isolate your rotifer mass cultures into a clean room or to use closed sterile bags in an open room. We do the latter, growing algae and rotifers in sterile 1000L bags, prepared with RO water with commercial sea salts, passed through UV treatment, and aerated with blowers using Heppa filters. Either way, clean algae and rotifer innoculants are essential for producing reliable rotifer mass cultures. You can’t deliver consistent rotifer production if you are growing rotifers in contaminated cultures.
Shift from Algae only to Omnivore Diets
Since brachionids are indiscriminate filter feeders, they should be considered omnivores rather than herbivores. They are capable of ingesting particles of about 10 microns diameter. We patented and use a proprietary process to make a highly nutritious rotifer feed, Amplifeed Replete, which is less than 8 microns and very slow sinking. It includes lipid rich, nano-emulsified astaxanthin, DHA, folate, selenium, and several vitamins, as well as a variety of plant and animal proteins. Experiments have shown that Brachionus fed a combination of the green algae Tetraselmis plus Amplifeed Replete make more eggs, reproduce faster, and, when fed to clownfish larvae, produce 40% larger larvae at 42 days compared to rotifers fed without Amplifeed Replete enrichment.
]]>Aquaculture is heavily reliant on live rotifer feeds for the larval rearing of many marine and some freshwater fish. Sustainable Nutrition recommends using rotifers as a first feed for zebrafish fry with small mouth size. In any hatchery operation, the main objective is to provide healthy, nutritionally enriched rotifers on demand. This is more important than the marginal cost reduction that would be achieved growing compromised rotifers at very high densities.
Did you know? Rotifer cultures are quite sensitive to water quality. If rotifers are suffering from poor water quality in their culture, they are stressed and stop eating. Feeding fish larvae starving rotifers does not supply them with the nutrients necessary for robust growth. Additionally, as a rotifer density surpasses 500 per ml, the rotifer culture accumulates metabolites and water quality declines. Eventually, once a rotifer culture reaches a density of 1000 to 2000 per ml, it becomes prone to periodic crashes! After years of struggling with culturing rotifers at high density in open systems, our pilot customer, Sustainable Aquatics Inc converted to a closed, batch culture system which has been very consistent, with no crashes. They converted to our culture system using closed bags, a sub-micron filtered water and air, and clean algae and rotifer cysts for inoculation.
Let’s talk about algae! We also believe that the best, most consistent results are obtained when rotifers are cultured on a diet of live algae. We suggest the motile green alga Tetraselmis suecica: https://snextracts.com/collections/for-rotifers-and-artemia/products/sterile-tetraselmis-algae. It grows very well in low salinity, high density cultures and resists invasion by other unwanted algae species.
But…don’t forget about the salinity of your water! We recommend culturing your rotifers at 6ppt, and enriching at 2ppt, in order to minimize osmotic shock when you transfer the rotifers to feed larval fish.
Which rotifer is for you? The mouth gape sizes of larval fish determine the size of rotifer prey that can be ingested, so it is important to provide an appropriately sized rotifer to facilitate successful larval rearing [1]. We stock more than a dozen brachionid species, ranging in size from 120 to 300 µm in length, each with different physiological optima. [3].
Proales similis is one of the smallest rotifer species measured, averaging only 88µm in length, and inhabit low salinity waters. With 63 species documented, most of which occupy freshwater, Brachionus is the oldest known genus among the monogonont class of rotifer [3]. B. rotundiformis are the smallest of the brachionid species, measuring approximately 120 µm long, and prefer higher salinities and temperatures. B. manjavacas and B. plicatilis are larger, averaging approximately 247µm and 261µm in length, respectively. B. plicatilis also grow better at lower salinities and temperatures than other brachionid species, and inhabit inland salt lakes and coastal marine habitats [1,3]. One additional way to optimize rotifer production and culture stability is to select among the brachionid species best suited for growth under the specific environmental conditions of the hatchery, and also those that are best suited for the specific geographic region of the world [1].
Moreover, using an appropriate size rotifer for larviculture largely contributes to its overall success. A 2007 study reported that 15 days post-hatch larval grunt Parapristipoma trilineatum fed B. rotundiformis grew much better for the first 7 days than larvae fed B. plicatilis, exclusively. However, after day 7 the same larvae grew better when fed the larger B. plicatilis [2]. Maintaining mass cultures of two different size rotifers may benefit hatcheries by increasing the overall success of larviculture.
What happens if your rotifer culture crashes? For this reason, we suggest always keeping a backup of rotifer cysts in case of emergencies. Rotifer cysts can be hatched in 24 hours into thousands of young rotifers capable of rapid asexual reproduction to inoculate mass cultures. We offer types of L-strain, including Brachionus plicatilis and B. manjavacas, and multiple types of S-strain Brachionus rotundiformis. For those that love to experiment, we even offer a sample pack of 15 different rotifer strains that you can use to optimize rotifer prey to your larval predator.
Rotifer cysts can be found on our website:
We also conveniently offer shipping of enriched live rotifers as one other option, which ship within 1-2 days of ordering: https://snextracts.com/collections/live-amplifeed-enriched-rotifers. Hatcheries often find it difficult to maintain pure-rotifer cultures with no cross-contamination. Periodically re-inoculating a rotifer culture using rotifer cysts, allows hatchery managers to maintain tighter control over the genetic composition of their rotifer species. Sustainable Nutrition’s benchtop rotifer culture system is designed for ease of quickly re-starting cultures genetically characterized rotifers [1].
With that in mind, check out our benchtop rotifer production system! Available in both 30L and 50L sizes, and convenient for a zebrafish or small aquaculture lab. One two-foot system is designed to produce enough rotifers to meet the needs of most small to medium sized labs. It also offers built-in redundancy to minimize the impacts of inopportune rotifer culture crashes: https://snextracts.com/collections/zebrafish/products/complete-benchtop-rotifer-production-system-30l
What is our secret to success? Amplifeed Replete! We have observed a tremendous benefit to enriching rotifers in the last 24-36 hours prior to feeding to fish larvae. Rotifer enrichment significantly enhances the rate of growth, maturation, and survival of larval fish. We observe that optimizing larval nutrition is as important as egg yolk quantity and quality, and is the most important factor in the long-term health and productivity of fish. Formulated with our nano-emulsified astaxanthin, we believe that Amplifeed Replete is the best rotifer enrichment on the market.
We are always happy to offer suggestions, or answer any questions that you may have!
Sincerely,
The Sustainable Nutrition Team
[1] Snell T.W., Johnston R.K., Matthews A.B. Utilizing Brachionus biodiversity in marine finfish larviculture. 2018. Hydrobiologica. 844: 149-162.
[2] Hagiwara A., Suga K., Akazawa A., Kotani T., Sakakura Y. 2007. Development of rotifer strains with useful traits for rearing fish larvae. Aquaculture. 268: 44-52.
[3] Snell T.W., Genetic Resources of Rotifers in the Genus Brachionus. 2019. The Biological Resources of Model Organisms. 55-67.
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