ROTIFER PRODUCTION & MARINE LARVAL NUTRITION SOLUTIONS

Our Closest-to-Nature Microalgae Feeds Provide Superior Nutrition, Cost-Saving Productivity, and Cleanliness

Instant Algae™ RotiGrow Rotifer Products Uniquely Offer “Whole Cell – Whole Food” Nutrition

In every hatchery, healthy, enriched rotifers are essential to grow healthy and vigorous fish larvae, but serious problems can quickly arise.

Reed Mariculture Inc’s (RMI) Instant Algae products for rotifer growth, enrichment and greenwater enable you to avoid problems, and save time and money, by: providing a stable, consistent production base, a low-maintenance and extremely clean production environment, and the healthiest and most nutritious rotifers for your larvae.

Our unique growth and enrichment concentrates offer the most effective nutrition for rotifers because they are the closest to nature of any product on the market, consisting of minimally processed and intact, whole microalgae cells that have all of the benefits of live cells.

The Rotifer Compendium – a practical resource for fish growers and live feeds personnel

We have created a Compendium to share the latest information about the role of rotifers and microalgae in marine larval nutrition, including the most effective nutrition practices.

The Compendium represents RMI’s extensive experience growing microalgae and zooplankton. As the world’s largest producer of marine microalgae, and a company focused exclusively on marine larval nutrition, we have accumulated a large body of knowledge. We invite you to comment and give feedback to help us expand this resource to support the larviculture industry.

Feed rate is determined by Wet Weight Biomass produced (or more accurately by Dry Weight Biomass produced), the feed conversion ratio of the microalgal concentrate used and the efficiency of your grow-out protocols.

Rotifer Count is a useful proxy for rotifer mass once rotifer mass per million rotifers has been determined. Rotifers vary considerably by strain and growing conditions.

Rotifer mass (g/million rotifers) can change significantly over time in response to feed, growing conditions, and season. This can significantly affect production results when feeding according to rotifer count without taking these changes into account.

Continuous Systems Dose for Biomass Harvested Each Day

Table E.3: Microalgae Concentrate Feed per hour per 100g of rotifer harvest (wet weight). Also shown is the corresponding number of rotifers harvested at different rotifer sizes

Feed rate is determined by the Wet Weight Biomass of rotifers you need to harvest each day. For a stable system, do not adjust the feed rate every time rotifer density changes, the system will find its own equilibrium.

• The Wet Weight Harvest number should be used as an estimate of harvest per feed. Each hatchery is different and the feed conversion rate will vary from time to time and hatchery to hatchery. Let your system find it’s own equilibrium.
• It is possible to feed according to the number of rotifer to be harvested after adjusting for the size of the rotifer.
• It is also possible to feed according to the number of rotifers in the tank prior to harvest, after adjusting for rotifer size and desired harvest rate (growth rate). In a harvest range of 25% to 50%
  • Feed_Batch = Feed_Cont x Harvest (%), where:
  • Feed_Batch = Feed rate: ml per hour per 100 grams Wet Weight rotifers in the culture
  • Feed_Cont = Feed rate: ml per hour per 100 grams Wet Weight rotifers harvested (from chart 6.1 above)
  • Harvest (%) = percent of rotifers harvested per day (25-50%)

  * However, it is easiest to simply feed according to the Wet Weight Biomass you wish to harvest. Please see “Equilibrium Management” in section D.6.2. | Rotifer Production: methods.

Wet weight is easily obtained by screening the whole or a small percentage of your harvest and blotting the bottom of the screen with a paper towel to remove excess moisture. If you know the number of rotifers you need to harvest and their size, but not their Wet Weight Biomass, use the Lorica Length to Biomass Calculator (Table 6.2) below the feed table.

Table E.4: Rotifer length to mass conversion

Notes:

a. These numbers are mathematical approximations using the RMI “Mini-L 160” as the base data point. Actual numbers will vary depending on rotifer shape (thin vs. plump) and composition (density).

b. Rotifer length is measured as the distance between the anterior tip if the lorica with the corona retracted and the base of the foot. Rotifers retract their corona when fixed with formalin.

c. Better data can be obtained by screening a liter of rotifer culture and dividing the weight by the number of rotifers in the liter of culture.

Please note:

1.Harvest rate (% of culture harvested per day) does not affect harvested biomass at equilibrium. It affects the median age of rotifers in the rotifer culture by affecting feed per rotifer and the rate at which rotifers grow. Please see “Population Dynamics” inset Box and Table D.4 in D.6.1 | Rotifer Production, Continuous vs. Batch

2. Feed rate (Table 6.1 above) is determined by: (1) The biomass of rotifers needed for harvest together with (2) feed density and (3) the Net Biomass Conversion rate of the feed.

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Table F.4: Long Enrichment over 8 hours using N-Rich Standard with RotiGrow Plus as the Base Feed.

Table F.5: 2 hour “Quick” Enrichment with N-Rich Plus followed by 6 hours using a maintenance dose of N-Rich Standard with RotiGrow Plus as the Base Feed.

Table F.6: 3 hour “Quick” Enrichment with N-Rich Nanno followed by 5 hours using a maintenance dose of N-Rich Standard with RotiGrow Nanno as the Base Feed.

Table F.7

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Enrichment feed is calculated by the wet weight of the rotifer harvested and enriched, not by rotifer count, as rotifer vary considerably in size.

Screening the harvested rotifers prior to enrichment is not a recommended practice as it causes excessive rotifer stress. The preferred methods of determining wet weight are:

• Sample a portion of the culture to be harvested,
• Estimating wet rotifer weight from rotifer count using past experience or
• Estimated wet weight from rotifer count and the rotifer length to weight converter (Table F.2).

Table F.2: Lorica Length to Mass converter

Once you know the wet weight of the rotifers to be harvested, feed the following amounts of N-Rich every hour (depending on the protocol you choose). Please keep in mind the enrichment points listed below in the feeding table.

Table F.3: Enrichment Feeding Rates

1. Rotifers can be enriched at relatively high densities (4g wet weight per liter or 3000+ RMI “Mini-L160” rotifers per ml). N-Rich is a very clean feed and rotifers can be enriched at even higher densities, but Dissolved oxygen and ammonia stress can become an issue.
2. The enrichment culture will be extremely clean. Nonetheless, it is helpful to use a fully equipped culture tank with aeration and filter floss for enrichment.
3. Oxygen stress can severely compromise rotifers during enrichment. For Maximum rotifer health and delivery of nutrition to your larval fish, please monitor oxygen effectively.
4. The greater the density of the enrichment culture the greater the demand for oxygen. Watch oxygen closely until you have a proven protocol. Please note that aeration can drive oxygen and impact the effectiveness of oxygen delivery. Changes in the size of the oxygen bubble can also significantly affect oxygenation.
5. The enrichment culture will need increasing levels of oxygen after the first hour.
6. Ammonia may be an issue with enrichments lasting more than 1 or 2 hours. If ammonia exceeds 5.0ppm ionized ammonia becomes toxic even if lowered pH prevents un-ionized ammonia from being toxic. It is helpful to add ClorAmX to keep Ammonia from becoming a problem.

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How you choose to grow and enrich your rotifers depends on the level of enrichment that is needed and the enrichment constraints of your hatchery. Most hatcheries feed twice a day, in the morning and in the evening. Some hatcheries feed throughout the day and night with warm stored or cold stored rotifers.

The choices for enrichment are so varied that it is impossible to present them all. Below are a few grow-out and enrichment scenarios that are used including how RotiGrow feeds should be used.

1. Harvest at night and enrich throughout the night for fully enriched rotifers for use the next day
   • Grow with RotiGrow Nanno or Nanno 3600 and enrich with N-Rich (Nanno) for 2-3 hours (to get initial DHA boost) followed by N-Rich Extend at enrichment levels.
   • Or, Grow with RotiGrow Plus and enrich with N-Rich Plus for 1-2 hours (to get additional HUFA boost) followed by N-Rich Extend at enrichment levels.
2. Harvest in the morning and quickly enrich rotifers. Use some of the rotifers for morning feeding and cold bank the remaining rotifers for evening feeding.
   • Grow with RotiGrow Plus and enrich with N-Rich Plus for 1-2 hours (to get additional HUFA boost) followed by N-Rich Extend at Cold storage levels.
   • Grow with RotiGrow Nanno or Nanno 3600 and enrich with N-Rich (Nanno) for 2-3 hours (to get initial DHA boost) followed by N-Rich Extend at enrichment levels
3. Harvest in the morning and quickly (1 hr) enrich the morning rotifers continue enrichment for high HUFA rotifers for the evening feeding.

   • Grow with RotiGrow Plus and enrich with N-Rich Plus for 1-2 hours (to get additional HUFA boost) followed by N-Rich Extend at Cold storage levels.
4. Harvest in the Morning, feed enriched rotifers throughout the day, feed rotifers from the grow-out tank through the evening and night.

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General Information

N-Rich feeds are exceptionally clean, effective and easy to use rotifer enrichment feeds that offers full fatty acid enrichment without stressing your rotifers. N-Rich is so clean that rinsing of rotifers after enrichment is unnecessary. N-Rich is an algal blend composed primarily of Pavlova and Isochrysis.

With Whole Cell-Whole Food microalgal nutrition, Astaxanthin and Stay-C added, N-Rich products provide broad spectrum rotifer nutrition for rotifer tissue enrichment, cold and warm storage nutrition maintenance and gut loading nutrition.

All N-Rich biomass is from marine microalgae (except Stay-C).

The N-Rich Family of Enrichment Feeds includes:

(Please see Table 7.1 and Charts 7.1b for enrichment feed comparisons)

• N-Rich Extend – has very high protein content and appropriate levels of lipids and carbohydrates for optimal rotifer and larval fish health. Extend is used for:
  1. Extended enrichment (all day enrichment or overnight enrichment) with RotiGrow Plus as the base feed. Extend provides superior rotifer tissue enrichment with optimum DHA:EPA:ARA ratios and Lipid Class profile.
  2. Extend is used for two-stage extended enrichment with N-Rich Plus and N-Rich Nanno.
  3. N-Rich Extend is also ideal as a maintenance and gut-load feed for warm and cold stored rotifers. N-Rich is high in protein for long healthy rotifers and larval fish. Extend maintains rotifer health through proper protein nutrition.
• N-Rich Plus – Used for rapid, yet gentle full DHA:EPA:ARA/Lipid Class enrichment in two hours or less. N-Rich Plus is formulated for use with RotiGrow Plus as the based feed and pre-enrichment. N-Rich Plus has a higher lipid content than N-Rich Extend, yet contains substantial levels of protein and carbohydrates for optimal rotifer and larval fish health.
• N-Rich Nanno – Used for rapid, yet gentle full DHA:EPA:ARA/Lipid Class enrichment in three hours or less. N-Rich Plus is formulated for use with RotiGrow Nanno and Nanno 3600 as the based feed and pre-enrichment. N-Rich Nanno has a higher lipid content than N-Rich Extend, yet contains substantial levels of protein and carbohydrates for optimal rotifer and larval fish health.
• N-Rich Specialty blends – Custom blend for custom application. Reed Mariculture is happy to custom blend enrichment formulas to meet your hatcheries needs. Slow enrichment, fast enrichment and HUFA levels in excess of 8% of dry weigh rotifer biomass easily obtainable.

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There are two sources of nutrition for larval fish from enriched rotifers:

1. Digestible rotifer tissue – primarily the soft internal tissue of the rotifer, and

2. Gut-loaded feed released from the rotifer gut.

There are four stages (sources) of enrichment for the rotifer:

1. Pre-enrichment from the production feed

2. Primary enrichment to achieve:

a. Optimal soft tissue nutritional profile:

b. Optimal feed profile of feed in the rotifer gut

3. Cold and warm storage feeding to maintain rotifer gut and soft tissue nutritional profile.

4. Greenwater feeding to maintain rotifer gut and soft tissue nutritional profile.

Optimal enrichment

Optimal enrichment occurs when both rotifer soft tissue and the feed loaded gut match enrichment objectives for a specific species. In an ideal world, the typical profile for both soft tissue and the feed loaded gut would approximate the following table:

Table F.1 “Optimal” enrichment table of enriched rotifers for unspecified marine larva.

Numbers based on the best numbers available to RMI at this time. Intended for general discussion only. Optimum profile changes with species.

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Ammonia in water system reduces the excretion ammonia in the rotifer. Ammonia exists in marine systems in an “ionized” and “un-ionized” or “free” form. Both forms of ammonia are toxic, but un-ionized ammonia (NH3) is about 100 times more toxic that ionized ammonia (NH4). Unionized ammonia starts to become toxic to fish above 0.05mg/l; Ammonium starts to become toxic to fish above 5.0 mg/l

The percent of ammonia in the form of un-ionized ammonia is determined by pH and Temperature (see table below)

Table E.9: Un-ionized Ammonia as a Percent of Total Ammonia

• Any level of total ammonia above 5.0mg/lliter is toxic because Ionized ammonia is toxic at this level
• Given the pH and temperature range for optimal B. plicatilis culture growth, total ammonia needs to be kept below 1.0mg/liter or be chemically neutralized
• Given the pH and temperature range for optimal B. rotundiformis culture growth, total ammonia needs to be kept below 0.5mg/liter or be chemically neutralized

Neutralizing Ammonia – ClorAmX

Cleaning

[information coming]

Filter floss

[information coming]

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Rotifers can survive very low levels oxygen levels. However, a drop in DO, even for a short period, can significantly stress the rotifer. At RMI we routinely measure DO using the % oxygen setting (calibration setting). We use this setting because it is the “partial pressure” of oxygen that the rotifer experiences and is unaffected by temperature and salinity.

Rotifer stress is more visible during enrichment when the rotifers are exposed to very high levels of lipid rich feeds. At there time O2 must be set properly and monitored closely. If your enrichment protocol extends for several hours and O2 is not kept near saturation throughout the enrichment process, rotifer stress will be apparent in the form of a higher percentage dirty, shrunken (lorica is angular rather than round and plump) and “wasted” (clear lorica) rotifers.

Natural saturation of oxygen is 21% (equilibrium with the atmosphere). At RMI we maintain O2 levels at 17-27%. High levels of O2 seem to do no harm. Lower levels of O2 (<15%) seem to increase susceptibility to stress and reduce vigor and productivity

At RMI we consume O2 at a rate of 3 lpm per billion rotifers in culture (“RMI Mini-L 160” at 9000 rotifers per ml). This number is affected by the size of the O2 bubble, the amount of normal aeration and rotifer density and feed rate. Every system must be adjusted differently.

Salinity

Rotifers (Brachionus) are brackish water animals. Brachionus (both B. plicatilis and B. rotundiformis) can survive full salinity and even hyper-saline conditions (to at least 45ppt). Brachionus can also survive at very low salinity and have been acclimated to 2ppt for some freshwater applications. However, Brachionus do best in a range of 15-20ppt. Only at this range will they achieve maximum productivity.

pH

Brachionus rotifers prefer normal marine salinity 7.8-8.2 and may thrive at a slightly higher pH. Typically, feed and rotifer waste reduce pH below this level. If pH drops below 7.0, the low pH is mildly stressful and productivity.

It is a common practice to culture rotifer at a ph below 7.0 to reduce ammonia toxicity (see ammonia below). Below a pH of 7.0, ammonia is almost completely non-toxic. Above pH 8 controlling ammonia toxicity become critical.

Temperature

Brachionus plicatilis (L-Type) grow best at moderate temperature (20°C-24°C). They can be grown successfully at temperatures as low as 16°C and as high as 28°C. B. plicatilis will swim well at 10°C and will even swim (though slowly) at 4°C. The tolerance of B. plicatilis to cooler temperatures is especially useful for temperate and coldwater fish and cold storage of rotifers for later feeding.

Brachionus rotundiformis (S- and SS-Type) grow best between 27C and 32C. They can be grown successfully at temperatures as low as 20 °C and as high as 34°C.

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Rotifers do not like stress. They like stable conditions and do tremendously better when all system parameters remain stable for weeks on end.

Stress can be result from low oxygen, prolonged feed loss (several hours) salinity changes, pH shock, temperature, ammonia spikes and numerous other factors. These factors compound each other resulting in stress when a single variable alone does not appear to be off enough to cause stress. Stress results in a reduction of egg production and subtle changes in the appearance of the rotifer. Stress can occur without noticeable rotifer death and can occur a day or more before a problem is even noticed.

Rotifers are more sensitive to stress than many Live Feed Personnel realize, even though they can survive a lot of abuse and continue to produce some eggs. Stress often permanently damages rotifers, reducing reproduction, health and resistance to pathogens. Because the rotifers alive at the time of the stress are permanently damaged, the culture will only return to full strength when the old rotifers are removed and new rotifers hatch to replace them. This process can take many days. In the mean time, these stressed and minimally productive rotifers remain in your culture, consume food and look like they are healthy. See Table below:

Table E.8: The percent of old, damaged rotifers 4 days after a stress event at different harvest rates.

Rotifers recover slowly (see below). It is not uncommon for rotifer cultures to experience repeated mild stress that goes unnoticed by live feeds personnel. In these cases rotifer production continues and seems normal, but the rotifers do not reach their full potential and remain susceptible to crashes and pathogens.

It is not uncommon for rotifers to require a week or more to recover form a stress event. If your rotifers experience one stress event per week, even a mild stress event, your rotifers will never achieve their potential and their reduced productivity may come to be seen as normal.

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Feed rate is determined by the Wet Weight Biomass of rotifers in the tank at the time of feeding. For Batch Systems it is often more convenient to use rotifer count, standardized by the size of the rotifer, as a proxy for mass. Feed rate per gram (or million rotifers) can be adjusted to increase or decrease the rate at which the rotifers grow (doubling time).However, in the end, the mass of rotifers harvested is directly proportional to the amount of feed used.

Table E.5: Feed per hour per 100 million rotifer at a growth rates of 80% per day, Adjusted for rotifer Lorica length

Notes:

• Feed Rate is based on count (biomass) at the beginning of the day. To change the rate at which the rotifers grow, adjust feed proportionally. Example: to grow RMI Mini-L 160 rotifers on RotiGrow Plus at 60% growth per day instead of 80% growth per day, multiply the above feed rate (12.7 ml per 100 million rotifers per hours) X (60%/80%) = 8.5ml per 100 million rotifers per hour. You will use less feed per day, but it will take longer to produce the desires mass of rotifers. The change in feed rate is proportional because the net feed conversion rate is constant.

Table E.6: Typical feeding application using RotiGrow Plus and feeding at a rate that produces 80% growth for RMI

Wet weight is easily obtained by screening the whole or a small percentage of your culture and blotting the bottom of the screen with a paper towel to remove excess moisture. If you know the number of rotifers you need to harvest and their size, but not their wet weight biomass, use the Lorica Length to Biomass Calculator (Table 6.2) repeated below.

Table E.7: Rotifer length to mass conversion

Notes:
a. These numbers are mathematical approximations using the RMI “Mini-L 160” as the base data point. Actual numbers will vary depending on rotifer shape (thin vs. plump) and composition (density).
b. Rotifer length is measured as the distance between the anterior tip if the lorica with the corona retracted and the base of the foot. Rotifers retract their corona when fixed with formalin.
c. Better data can be obtained by screening a liter of rotifer culture and dividing the weight by the number of rotifers in the liter of culture.

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• Frozen microalgae concentrates (not including RotiGrow Chlorella) should be stored frozen for long-term storage. RotiGrow Chlorella is a refrigerated product and has a limited shelf life (4 weeks).
• To defrost the frozen concentrate, remove from the cardboard box and place in a refrigerator. 1 liter bags will defrost overnight. 10 liter Cubitainers® will defrost in 1-3 days
• Previously frozen Instant Algae concentrates will remain good for at least 3 weeks when kept refrigerated.
• When Using a pump or drip system for continuous feeding, the feed reservoir for the microalgal concentrate should always be kept cold. A Styrofoam box with ice or a counter top refrigerator with holes drilled for feed line work well. It is helpful to keep the microalgae reservoir as close as possible to the microalgae pump and rotifer system.
• Microalgal concentrates work best when fed continuously using a peristaltic pump or other injection system.
  The microalgal concentrate can be hand fed with periodic dosing, but the more regular the feeding is, the better the results will be.
• It is recommended that you do not thin the microalgae concentrate with water. Thinning the concentrate creates the possibility for spoilage and settling of the concentrate and can negatively impact the rotifer culture and larval fish.
  • Adjustment of the feed rate is best accomplished using a cycle timer to turn the pump on and off.
  • If it is necessary to thin the paste to control feed flow, It is suggested that the thinned feed be kept cold in a refrigerator or ice chest, and that mix be stirred or very lightly aerated.
    

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General information

Using Instant Algae microalgae concentrates in an existing system

• There is no need to modify you rotifer grow-out system configuration to use Instant Algae feeds (RotiGrow Plus, RotiGrow Nanno, Nanno 3600, RotiGrow Chlorella or Rotifer Diet)
• General system parameters and Useful tips are provided after the feeding protocols below

Choosing the Right Rotifer Grow-out Feed

Each microalgal concentrate feed has different advantages.

• Provides high phospholipid DHA, EPA and ARA pre-enrichment
• All-in-one grow-out, enrichment and gut pack for applications where HUFA enrichment requirements are less than 40mg/g dw. and DHA:EPA is no more than 1.6:1
• Very clean and effective
• High level of pre-enrichment allows use of premium enrichment feeds that are more nutritionally balanced
• Allows for extremely high DHA enrichment — if desired
• Microalgal blend
• Frozen for long shelf life and easy logistics

RotiGrow Nanno

• Highest biomass conversion rate – least organic waste in your rotifer tank
• Very clean and effective
• Produces phospholipid rich rotifers
• Provides a high EPA and ARA pre-enrichment boost for use with high DHA enrichment protocols
• Single species – Nannochloropsis. A very tough microalgae and a robust microalgal concentrate feed
• Frozen for long shelf life and easy logistics

• Similar to existing Chlorella feeds but with a slightly extended shelf life. Extended shelf life offers easier and less expensive logistics
• Provides moderate DHA, EPA and ARA enrichment (less than 25mg/d d.w. HUFA)
• Orders must be arraigned in advance
• Refrigerate. 4 week shelf life

• Original Instant Algae Nannochloropsis feed
• Very effective with established protocols
• Provides a EPA and ARA pre-enrichment boost for use with high DHA enrichment protocols
• Frozen for long shelf life and easy logistics

• Original blended Nannochloropsis / Tetraselmis feed
• Very effective with established protocols
• Provides a EPA and ARA pre-enrichment boost for use with high DHA enrichment protocols
• Frozen for long shelf life and easy logistics

Feed Comparison charts

Table E.1: Production efficiency – Rotifer harvest per liter of feed

* cod fatty acid numbers from Sargent and Toucher 1984

¹ mg/g dry weight

² percent of dry weight

³ Percent of lipids

Table E.2: Typical HUFA pre-enrichment of rotifers harvested from Grow-out tank

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Because Continuous Systems and Batch Systems are simply two ways of looking at and managing the same rotifer population dynamic, you can use batch feeding protocols for a continuous systems. For example: If you are growing RMI “Mini-L 160” rotifers and you want the rotifer culture to grow at 67% per day, you will want to feed 0.063ml RotiGrow plus per hour per million rotifers. The next day you should harvest 40% of the culture which should bring the count back to where it was.

Metrics: Rotifer Biomass or Rotifer Count (Why rotifer counts don’t add up)

Microscopic observation and counting of rotifers is the traditional method of monitoring rotifer cultures and determining feed and enrichment requirements. For a specific strain of rotifers this can be an acceptable method.

However, rotifers have great variability in size and biomass, and therefore in feed and enrichment requirements per rotifer. Counts are very misleading and of little value for establishing protocols or comparing across strains. Even within strains different temperatures and reproduction rates can affect mean rotifer mass. As a result, feeding by rotifer count can lead to massive underfeeding or overfeeding of production feeds and enrichment feeds. This disconnect between rotifer feed and rotifer size (and biological requirements) is a significant source of culture stress and confusion for live feeds management and staff.

Examples:

1. A 120 µm S-type rotifer, by simple geometry has ¼ the volume, mass and feed and enrichment requirements of a 190 µm S-Type rotifer and 1/8^th the mass and requirements of a 240 µm L-Type rotifer.
2. If you feed a continuous rotifer system 1000 ml of RotiGrow Plus per day and harvest 35% per day, your system equilibrium biomass will be 135 g dry weight and your harvest will be 47 g dry weight per day. This will be true regardless of whether your system contains 730 million “Mini-L” 160 rotifers at 185 ng d.w./rotifer or 365 million larger rotifers at 370 ng d.w./rotifer.

Note: If you now increase the harvest rate to 45% per day your final system equilibrium rotifer biomass will fall to105 g dry weight but your harvest will still be 47 g dry weight (the ratio of feed input to rotifer output remains constant). Feed biomass per rotifer biomass will have increased from 5.8% per hour.to 8.35% per hour. The yield in terms of rotifer biomass per volume of feed used remains unchanged.

Switching between Rotifer Count Metrics and Rotifer Biomass Metrics

To switch between management by rotifer count and management by rotifer mass requires knowledge of the relationship between the two. The following table is a useful first approximation of the relationship between rotifer length and rotifer biomass:

Table D.7

Note: These numbers are mathematical approximations the RMI “Mini-L 160” as the base data point. Actual numbers will vary depending on rotifer shape (thin vs. plump) and composition. Rotifer length is measured as the distance between the anterior tip if the lorica with the corona retracted and the base of the foot. Rotifers retract their corona when fixed with formalin.

This second point above and the attached note underscore one of the basic tenants of the Instant Algae method for growing rotifers – Don’t Worry About The Count. Calculate the biomass you want to feed to your fish. Then, based on the feed conversion rate and biomass yield for each feed, choose the correct amount of feed for your system and harvest at the rate that works best for you. You will get an equilibrium rotifer count that satisfies your larval needs. What that rotifer count is will depend on the size of your rotifer strain.

Now, once you have established a baseline equilibrium, rotifer counts are useful for monitoring your system.

Of course you will need a starting point based on the size of your rotifers and your history of how many rotifer you use. The following table provides a rough guide for converting lorica length to dry weigh and wet weight biomass:

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Batch Rotifer Systems start with a fixed inoculum and are allowed to grow in density over a period of 2 to 5 days. Batch systems are not equilibrium systems and values of all parameters from feed requirements and oxygen demand to ammonia increase over time.

Batch rotifer systems start with a small inoculum and are fed a proportionally small amount of feed. As rotifer density increases feed is also increased, as are other inputs such as oxygen. With large doses of a good microalgal feed, rotifer density can increase at a rate in excess of 100% per day. However, a lower growth rate of 50%-80% per day is often considered more practical.

The primary drawback of a batch system is also its primary advantage. A batch system requires more monitoring and more labor to ensure the rotifers are progressing properly and to make adjustments to feed and other inputs. A batch system requires a more hands-on approach and, depending on the hatchery, this may be more or less desirable.

Because Net Biomass Conversion is constant, increasing the feed rate per million rotifers does not waste the feed (within reason). Instead, the culture will grow faster. In the end you will have more rotifers with more feed used; microalgal biomass to rotifer biomass conversion will be constant.

In a batch system the feed rate per million rotifers only determines growth rate and rotifer age. The total feed delivered determines the final biomass of the rotifer harvest.

Quick comparison of Continuous and Batch Protocol Differences

Table D.5: Protocol comparison for determining feed rates

Table D.6: Feed Rate Comparison (Continuous System vs. Batch System) at different harvest/growth rates

(For rotifers with known dry weight biomass per rotifer. The following chart is for the RMI “Mini-L160” with a dry weight of 180ng/rotifer

Note: you cannot estimate feed per million rotifers without knowing the size of your rotifers. Even then it is a poor practice (even if it is established protocol). See the next section.

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In a Continuous Rotifer System rotifers are harvested daily and the culture continues growing in the same tank. Continuous rotifer systems are equilibrium systems, and it can take some time to find the right balance. However, these systems can produce tremendous numbers of rotifers in a very small space. With proper protocols for cleaning-in-place and transfer of the culture to new vessels, continuous cultures can remain extremely stable for years. A good rotifer system with a good microalgal feed can be harvested at a rate of 50% per day, although 30%-45% is more practical.

Continuous rotifer systems are chemostats. That is to say, they are steady-state or semi-steady-state bioreactors with a single limiting factor (feed) that is added at a constant rate. Optimally, feed rate per hour and harvest rate per day are fixed at constant rates and all other variables (notably rotifer count) float to equilibrium.

Note: It is common for culturists to try to adjust feed and harvest rates as rotifer counts change. Sometimes these adjustments are necessary. However, this kind of tinkering invites chaos because there are no fixed parameters to anchor the equilibrium point of the system.

Because Net Biomass Conversion is constant, increasing the harvest rate does not increase the harvest. Instead, the culture will thin, the ratio of feed to rotifer will increase, and the culture will grow faster. In a couple of days the culture establishes a new equilibrium at a lower density, higher harvest rate and an identical net harvest. In order to increase (or decrease) the rotifer harvest, the feed rate must be increased (or decreased).

In Continuous Systems at equilibrium, the harvest rate only determines rotifer age. Feed rate determines the biomass of the rotifer harvest.

The primary benefit of a continuous system is that can provide a consistent and reliable supply of rotifers with minimal monitoring and labor. Continuous rotifer systems are usually fairly automated, with a continuous-feed peristaltic pump and water exchange. Because they usually have a high density of rotifers they often need supplemental Dissolved Oxygen (DO). It is somewhat common for continuous systems to include automated DO monitoring and supplementation and sometimes pH and temperature monitoring as well. However, daily logs* with manual entries are often sufficient. In higher pH systems (pH>7.8) a common automated input is the addition of ClorAm-X or other ammonia detoxifiers.

* An example of the rotifer production log used at RMI is included in the protocol section

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(equilibrium feed chemostat vs. progressively increasing density and feeding)

Continuous and batch rotifer system are simply two methods of managing the same rotifer population dynamic. In continuous systems you harvest 25% to 45% of the culture every day and “re-inoculate” with the remaining 55-75% of your culture volume. In batch system you harvest every 3-5 days and re-inoculate with 10-30% of your culture volume. While rotifer growth dynamics may be the same, there are significant differences in how you manage continuous and batch systems.

Table D.4: Population Dynamics with Continuous and Batch Harvest systems equivalencies for different harvest rates from a continuous system:

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General protocols and instructions for growing rotifers using the Instant Algae rotifer method will be discussed in the next section (E).

Best Production Methods

The variety of methods employed to grow rotifers is extensive, to say the least. Here are the basic choices that we see:

• High density vs. low density
• Continuous vs. batch culture (Equilibrium management vs. progressive density management)
• Monitor and feed by rotifer biomass vs. rotifer count

High density vs. low density

Aquaculture is always site and situation specific. There is no single right answer for every application. The same holds true for rotifers. In some tropical settlings rotifers are sometimes grown most cost-effectively in 100 metric ton tanks with densities of 100 to 200 per ml. In laboratory and intensive hatchery situations, where space and resources are expensive, rotifers are sometimes grown at densities exceeding 15,000 per ml. Most intensive hatcheries choose to grow rotifers at a density between 1,500 and 6,000 rotifers per ml.

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Microalgal feeds reduce all rotifer-related costs except the feed cost. Labor, space, management and other resource demands are all reduced. When all factors are considered most hatcheries find that the cost of using microalgal feeds is less, often far less, than alternative feeds.

Further, the nutritional quality of the rotifer and rotifer enrichment are higher, which in turn often translates into higher larval survival. Given the tremendous cost of running a hatchery, even a small increase in larval survival can have a major impact on total costs.

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Pre-enrichment is the process of pre-loading HUFA (DHA, EPA and ARA) lipids into the rotifer during production. Providing pre-enrichment is an extremely valuable quality for a grow-out feed.

1. Pre-enrichment in the production stage increases the final level of enriched rotifer HUFA lipids and increases the proportion of these lipids in the form of membrane lipids (phospholipids, glycolipids and sterols). This is because the rotifer has time to incorporate the HUFAs throughout its tissue as membrane lipids instead of concentrating the HUFAs as triglyceride energy stores. Membrane HUFA enrichment is further enhanced by the fact that rotifers emerge from the egg with high levels of HUFAs already incorporated in their membranes.
2. The higher the level of polar HUFA enrichment at the production stage, the shorter and less severe the secondary enrichment process becomes. This in turn dramatically improves speed and ease of enrichment, reduces rotifer stress, improves rotifer health, and increases the value of the rotifers to both fish and hatchery.
3. By reducing the need for extreme secondary HUFA enrichment, pre-enrichment allows for a “whole food” approach to be applied. “Whole food” enrichment allows protein, carbohydrates, carotenoids, vitamins, minerals and other nutrients to be added to the enrichment mix, improving the nutritional value and health of the rotifer, the nutritional profile of the gut-load of the rotifer, and ultimately the nutrition of the larval fish.

Table D.3: Typical HUFA pre-enrichment of rotifers grown on the following feeds and harvested from production tanks

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(feed productivity and waste production)

Net biomass conversion is the rate at which feed biomass is converted into rotifer biomass and is excellent measure of the quality of a feed. The higher the net biomass conversion, the closer the feed matches the metabolic needs of the rotifer, making the rotifer culture healthier.

With higher net biomass conversion, less feed is used and significantly less “waste” is produced, to be consumed by protozoans and bacteria. Increasing net biomass conversion can increase production, rotifer and fish health, reduce fouling, and reduce labor cost.

Table D.2

Notes on Biomass Conversion:

1. We measure productivity in terms of grams biomass of rotifers produced per gram biomass of feed added. We find that conversion rates are consistent across all methods of production. A continuous production system with a 25% harvest rate will have the same net biomass output per liter of feed as a continuous production system at a 45% harvest rate using the same feeds, and the same net biomass output as a batch system at 70% growth per day. Thus net biomass conversion is a very useful tool for comparing the productivity of feeds.
2. The numbers above are average feed conversion rates from 2 years of testing at RMI. Not all rotifers will respond to the feeds in the same way all the time. Our cultures sometimes experience minor (10%) swings in productivity for a few weeks at a time. Some of our customers have reported better total productivity from RotiGrow Plus than from Nanno 3600, even though our numbers show the contrary. As with all of aquaculture, results will vary with conditions. Choose the microalgal feed that works best for you.

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Quality feed is an essential (and probably the most important) requirement for quality rotifers and clean and productive culture systems. The best all-around feeds are liquid microalgal feeds. Nannochloropsis- and Chlorella- based microalgal grow-out feeds are outstanding. Liquid microalgal feeds:

1. Are exceptionally clean and easy to use.
2. Allow high-density, high-yield production.
3. Promote rotifer health, vigor, and cleanliness.
4. Produce stable and reliable rotifer cultures.
5. Reduce cost by reducing requirements for labor, space, and other resources.
6. Through improved rotifers, increase larval survival and hatchery productivity.

Microalgae are naturally microencapsulated feeds and effectively deliver many critical nutrients, both obvious (protein, lipids) and non obvious (vitamins, carotenoids, minerals, etc.) that are critical for maximum rotifer production and larval nutrition.

Key Qualities of Effective Liquid Microalgal Feeds

• Biologically Clean – The Synergistic Effect of Microalgae
• Net Biomass Conversion
• Pre-enrichment
• Cost

Biologically Clean

The Synergistic Effect of Microalgae

Liquid microalgal feeds are exceptionally clean feeds. There are a number of factors that come together when microalgal feeds are used to produce such clean and healthy rotifer cultures.

Microalgal feeds:

1. Are nutritionally balanced and promote vigorous, healthy rotifers that grow rapidly, aggressively consume feed and resist harmful bacteria.
2. Support very high rotifer growth rates, resulting in cultures with a high proportion of young rotifers. These rotifers are by their very nature healthy and clean. This is because rotifers are free from fouling bacteria and are healthy when they hatch. Internal and external fouling of the rotifer takes time, and rotifers produced a high growth rate can have a median age of 28 hours or less. (see Growth rate and Age chart below).

Table D.1









3. Are very efficiently consumed and produce relatively little waste (see the last row in the table in the “Net Biomass conversion and optimum fed rate section” below).
4. Are naturally microencapsulated and do not release their nutrients prior to being ingested by the rotifers.
5. Promote the presence of beneficial bacteria that displace harmful and pathogenic bacteria.

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A dirty system is a nightmare: foam, protozoans, dirty rotifers, and clogged screens and more. Fouling reduces capacity, reduces rotifer value, increases labor cost and makes harvesting difficult. Worse, fouling can lead to unhealthy bacterial flora. Dirty systems are usually the result of dirty feed or poorly set up culture systems.

Healthy (probiotic) bacteria

Bacterial counts in rotifer systems are always high; the higher the density and feed rate, the more bacteria.

Most bacteria are benefit the rotifer culture by metabolizing waste such as ammonia, producing vitamins and by consuming nutrients that could otherwise feed harmful and pathogenic bacteria. This benefit is known as the Probiotic Effect. A high population of strong beneficial bacteria will displace pathogenic bacteria to the point where they are not a problem. Most systems will develop their own bacteria populations without intervention. However, the addition of cultured probiotic bacteria is seen by many as an effective way to stabilize cultures.

Harmful but non-pathogenic bacteria

Bacteria are a critical element of all rotifer systems and most of the bacteria are helpful bacteria. However, it is not uncommon for rotifer systems to lose productivity with no clear indication as to why. Sometimes it is a leak or other mechanical problem but sometimes it is bacteria. When the wrong bacteria take up residence rotifer production can be cut by a third or more. Check the smell. It might smell different. Sometimes when a system is left alone to run normally the system will spontaneously re-invigorate itself weeks later. However, it never hurts to transfer the culture to a new system and sanitize the old one.

Vibrio

Vibrio species are ubiquitous. Most vibrio bacteria are harmless. A few vibrio species are pathogenic and cause identifiable disease. Often vibrio will cause digestive problems that do not rise to the level of “disease,” but result in diminished rotifer production and health and, when fed to the fish, poor growth and development of your larvae. If you think you have harmful bacteria and test for vibrio, be sure to use a protease-specific test for harmful vibrio bacteria. A simple vibrio test will almost always test positive.

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• The culture needs to be healthy and strong to remain stable.
• The rotifers must be healthy enough to tolerate aggressive enrichment. Enrichment is often a source of significant stress for the rotifers and can result in significant numbers of rotifers that are dead, dirty, atrophied or have low mobility.
• After enrichment, rotifers are often placed in storage for many hours. Only the most vigorous rotifers can withstand the stress of both enrichment and storage.
• After all these stresses, the rotifers need to be strong and motile so that they can be of value in the larval rearing tank.
• Further, although it is hard to document, it is plausible that strong, plump, healthy rotifers pass more nutrition, with less stress, to larval fish.

The qualities of healthy and strong rotifers are:

• A strong, fairly rapid swimming motion
• A healthy egg count (25-40% egg:rotifer ratio)
• Little of no lorica fouling
• Plump, round rotifers. The signs of rotifer wasting can be subtle, but when the lorica edges seem straight with angular transitions instead of being fully plump and smooth, this is a sign of rotifer stress.
• Full lorica cavity with minimal “clear lorica” area.

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Rotifer systems can be susceptible to “crashes”. Crashes are sometime due to environmental changes or changes in bacteria, but are often due to an unforeseen and unknown problem in the management of the rotifer culture. Crashes often catch a Live Feeds Manager by surprise. Rotifers don’t make mistakes, but equipment breaks, input water can change, and people sometimes do make mistakes. However, systems can be reliable. At RMI we continuously run 3-4 systems and it has been a couple of years since we had a real “crash” in any system.

Your best guarantees: redundant systems and good clean food

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• Qualities of a good culture system
• Qualities of a good rotifer including the characteristics of rotifer cultures that produce good feed rotifers
• The importance of the choice of feed used to grow rotifers

Some basic principles of managing rotifers using high-productivity microalgal feeds.

Section Summary

• Strong, Stable, Reliable Systems
• Healthy Vigorous Rotifers
• Clean Culture and Healthy Bacteria
• Liquid Microalgal Feeds
• Key Qualities of Effective Liquid Microalgal Feeds
• Methods

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Larval Gape

Considerable attention has been given to obtaining small rotifers. This is because the small mouth size (gape) of many marine fish larvae requires a small prey. While the relationship between measured mouth gape and prey size would seem to be straightforward, there are significant difficulties in relating measured gape to larval food preference. Studies have shown that copepods are consumed even when they are much longer than larval gape, as long as the copepods are not as wide as the larval gape. Other studies have shown that larvae prefer prey organisms significantly smaller than their gape.

Table C.1

1. Lorica length and width data for 70 strains of L, S and SS-Type rotifers from Hagiwara and Kuwada in The Second Hatchery Feeds and Technology Workshop, Sydney, September 30-October 1, 2004, p. 28
2. Additional data points were included for the RMI “Mini-L 160” and UNCW 120 µm SS-Type rotifer available from Aquatic Ecosystems Inc.
3. The orange line shows rotifer mass, derived from lorica length. It is calculated by simple volumetric projection from the RMI “Mini-L 160” length to mass ratio. The curve represents L-Type, not S-Type rotifers (the two strains have different body shapes).

Rotifer Shape

Brachionus plicatilis tend to be longer than they are wide whereas B. rotundiformis is more nearly round. If larval prey capture is limited by the width of the prey, then a longer body shape (L-Type) potentially delivers more biomass per rotifer. If however, prey capture is limited by prey length, then a rounder body shape delivers more biomass for the same rotifer length.

Some additional size and shape information:

Puvanendran et al.^[1] measured first feeding Atlantic cod at 4.3 mm with a gape of about 160 µm. Larvae were fed small (192 X 150 µm) and large (242 X 181 µm) rotifers under a number of different conditions. First-feeding cod seemed not to ingest the large rotifers until day 8, and preferred the small rotifers until day 20, when the rotifers were 5.7 mm long with a gape of about 290 µm. Significantly, larvae were able to ingest rotifers longer than their gape, presumably because rotifer width was less than the larval gape and/or because the rotifers are deformable.

Data on other species are scanty. First-feeding Red Drum have been shown to consume relatively large prey so long as the width is less than the gape (220 µm).^[2] Hamasaki et al. demonstrated that Amberjack (a particularly small-mouthed fish) prefer 140 µm rotifers at first feeding, but will consume rotifers over 200 µm when offered larger rotifers.^[3]

Metabolic Cost of Rotifer Size for Larval Fish

However, smaller rotifers come at a metabolic cost associated with feeding. To obtain comparable nutrition, a larval fish must consume four 120 µm rotifers to equal the value of each 190 µm rotifer they consume. A recent publication by SINTEF in Norway found higher survival and specific growth rate when cod larvae were fed larger (270 µm) instead of smaller (180 µm) rotifers.^[4]

Rotifer Size and Feeding and Enrichment Protocols

It is standard practice to state feeding and enrichment protocols as if all rotifers are the same. Yet clearly, if you change rotifer strains to a strain twice is long as your are accustomed to, the you can expect the dry weight biomass to increase by 2 cubed (8-fold). Naturally you should expect feed and enrichment requirements to expand 8-fold as well.

The effect of Rotifer size on feeding and enrichment protocols is discussed more fully in the section on production.

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[2] Justin M. Krebs & Ralph G. Turingan (2003). Intraspecific variation in gape–prey size relationships and feeding success during early ontogeny in red drum, Sciaenops ocellatus. Environmental Biology of Fishes 66: 75–84,.

[3] K. Hamasaki et al. (2009) Aquaculture 288:216–225

[4] Gunvor Øie, Ingrid Overrein, Sunniva Wannebo Kui, Yngvar Olsen, Kjell Inge. SINTEF Fisheries and Aquaculture AS, Trondheim, Norway http://www.sintef.no/upload/Fiskeri_og_havbruk/Faktaark/Different%20rotifer%20(Brachionus)%20size%20in%20firstfeeding%20of%20cod%20(Gadus%20Morhua).pdf

Brachionus plicatilis is most easily distinguished by the blunt tips of the anterior spines of the lorica, easily seen when the corona is retracted. The most important attributes of L-Type rotifers for aquaculture are that they:

1. Culture best at moderate temperatures (20-25 ºC).
2. Easily tolerate cold storage. Cold storage allows rotifer enrichment to be carried out once per day and a portion of the rotifers stored for feeding later in the day.
3. Are active at the low temperatures used to cultivate cold-water fish (7-10 ºC).

Brachionus plicatilis grows best at a moderate salinity (15-20 ppt) but tolerates full seawater salinity and even slightly hyper-saline conditions. Brachionus plicatilis is a broad species complex with considerable size variation. Mean lorica length has been reported to range from 150 to 280+ µm. Corresponding dry weight rotifer biomass ranges from 160 ng to over 900 ng per individual

Culture Sources:

A very small L-Type Brachionus plicatilis, the RMI “Mini-L 160” is commercially available from Reed Mariculture. Other culture strains are exchanged among hatcheries and universities. Live cultures and cysts are available from some suppliers.

S-Type, SS-Type – Brachionus rotundiformis

Brachionus rotundiformis is most easily distinguished by the pointed tips of the anterior lorica spines. Its most important attributes for aquaculture are that it:

1. Cultures best at higher temperatures (28-35ºC).
2. Is smaller (some strains)

B. rotundiformis grows best at 15-20 ppt salinity but tolerates full seawater salinity.

B. rotundiformis also constitutes a broad species complex with considerable size variation. Mean lorica length has been reported to range from 120 to 180+ µm. Corresponding dry weight rotifer biomass ranges from 90 to over 200 ng per individual.

Culture Sources:

Culture strains are exchanged among hatcheries and universities. A very small S-Type B. rotundiformis is commercially available from Aquatic Eco-Systems Inc. (Apopka, Florida, USA).

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Understanding of rotifer strains and the differences among them has expanded greatly in the past two decades. Originally only one species of rotifer, Brachionus plicatilis, was recognized in marine aquaculture. People recognized that there were differences among strains and began to denote rotifers as L-Type (large), S-Type (small) and SS –Type (super small). In the 1990’s it became apparent that there are in fact at least two distinct species, B. plicatilis (L-Type) and B. rotundiformis (S-Type and SS-Type). More recently, with application of genetic analysis, we have discovered and begun to identify a diverse complex of species with a wide range of strains.

Section Summary

• Rotifer Types
• Rotifer Size
• Rotifer Shape

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Lipids are remarkably diverse and their functions are diverse as well. While the phospholipid and triglyceride classes make up the bulk of lipids, the other classes are critical as well. It is important that there be a rich nutritional balance of all these lipids in your rotifer feed and enriched rotifers to assure healthy larval fish.

Vitamins

Vitamins are critically important, but information on the vitamin requirements of larval marine fish is scattered and difficult to pull together. John Halver has an excellent chapter in Fish Nutrition where he discusses the vitamins one by one; there is an excellent chart on p. 69 that lists the known vitamin deficiency effects for marine fish.

Using a fish egg standard and comparing the vitamins of enriched rotifers to eggs seems an excellent approach, but it is difficult to find comprehensive data on the vitamin content of different fish eggs.

(see nutrition chart in Section B.5.0 for egg and feed vitamin levels and levels in rotifers)

Closing statement – A Farmer’s approach to Nutrition

The complexity of nutrition is why we believe in a farmer’s approach to nutrition: Start with what nature gives you and what nature intended. If it works and is cost affective, stick with it. Fish and zooplankton have adapted over hundreds of millions of years to be what they are, to need what they need, and to get their nutrition from microalgae.

Microalgae are the basis of the marine food chain. Everything that is in zooplankton (in nature) and is transmitted to fish ultimately comes from microalgae. The key to good nutrition is to appropriately use high-quality microalgae, microalgae that have been the backbone of successful marine aquaculture for over 30 years.

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Given that our focus needs to be providing limiting nutrients in enriched rotifers, it must be said that in the past Highly Unsaturated Fatty Acids (HUFA) lipids and especially in Lipid Membrane form have been very difficult and critical to manage nutrients.

Most marine fish larvae need very high levels of the essential fatty acids DHA and EPA and just the right amount of ARA. It is important to provide these critical levels without developing an excess of DPA or other less critical HUFAs. Further, it is very important that the majority of these fatty acids be Membrane Lipids (polar phospholipids and glycolipids and non-polar sterols), as opposed to triglyceride oils.

Why? Larval fish build tissue, and especially neural tissue, very rapidly. Fish cell membranes are mostly made up of polar phospholipids and glycolipids constructed from DHA and EPA, and non-polar sterols. These lipids are especially important for neural and eye development. Fish larvae must have an abundance of these lipids to grow well and without deformities. While larval fish can easily form their HUFA membrane lipids from other HUFA membrane lipids, they have a very limited ability to produce phospholipid and glycolipid HUFAs from triglyceride (oil) HUFAs. Further, marine larvae lack the ability to generate HUFAs (DHA, EPA and ARA) from more plentiful shorter-chain fatty acids.

Why? The details involve enzymes (elongase, desaturase, reductase and more). These enzymes are deficient in larvae that have evolved to consume phospholipid-rich live prey (the details make my head spin). Suffice it to say that HUFA phospholipids and other membrane HUFAs are the form of nutrition found in most species of marine larval fish eggs. That is what nature offers and that is what nature demands. Unfortunately, when rotifers are enriched with the wrong feeds or protocols, they can accumulate triglyceride (oil) HUFAs and still be quite deficient in membrane lipid HUFAs.

If you want to know more I would suggest starting with Sarget, Toucher and Bell, in Fish Nutrition edited by Halvar and Hardy. From there go on to Sargent et al. (1999), Aquaculture 179 217-229. Good Luck!

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The critical nutrients that require special attention are limiting or deficient nutrients. Individual amino acids, for example, are usually not limiting when a feed contains high levels of high quality protein (50%+). However, they can become limiting and critical when protein nutrition is poor.

Lipids, proteins, carbohydrates, minerals, and vitamins can all become limiting nutrients in the absence of a complex, well-balanced diet. Unfortunately, we have limited knowledge of the nutritional needs of most larval organisms and how these needs compare to formulated feeds, their uptake.

Lipids and their importance relative to other nutrients

Much is made of the lipid class profile and fatty acid profile of enriched rotifers, especially HUFAs. This is with good cause. However, before going on I would like to quote Sargent, Toucher and Bell in Fish Nutrition edited by Halvar and Hardy. There is “…the impression that because publications on lipids often dominate research journals and conference proceedings on fish nutrition, lipids are the most important nutrients for fish. They are not. Lipids are neither more nor less important than any other group of nutrients — proteins, carbohydrates, vitamins, or inorganic elements.” They go on to say that we know less about the lipid nutritional requirements of fish than for any other class of nutrients, in part because the can be so complex.

Reed Mariculture’s Nutrition Philosophy

Reed Mariculture’s approach to nutrition is to start with proven natural microalgal feeds and to provide them in as unrefined a state as possible. Thus, our “Whole Cell, Whole Food” approach to nutrition.

Next, Reed Mariculture combines our different feed microalgae to create feeds that produce rotifers that most closely match the needs of the larval fish, as estimated primarily from the content of healthy fish eggs as well as the content of common natural prey organisms. We follow this approach for fatty acids, lipid class, carotenoids, sterols, vitamins and any other nutrient we can identify.

Below is a chart of some of the nutritional parameters of some fish eggs, enriched rotifers grown on RotiGrow Plus and enriched with N-Rich, and Production Rotifers grown on RotiGrow Nanno and RotiGrow Plus. The information is incomplete, but nevertheless instructive.

Table B.1 Nutritional Parameters

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The digestive systems of larval marine fish are very immature when the larva fish first begins feeding. Digestive bacteria present in live feeds are critical for establishing the digestive bacteria of the larval fish. Thus, rotifers have a probiotic effect for larval fish.

This probiotic effect is connected to but should not be confused with probiotics used for culturing rotifers. Having the right probiotic bacteria is an important aspect of culturing rotifers and keeping rotifer cultures clean and healthy. Undoubtedly, clean and healthy rotifers with beneficial pro-biotic bacteria have a critical impact on the cleanliness and health of larval fish. This topic is addressed further in the section on production.

Digestive enzyme deficiencies and anabolic enzyme deficiencies

Larval fish can be deficient in digestive enzymes, so the digestive enzymes in live feeds can provide a critical boost for nutritional uptake, growth and survival.

Yet, digestion (catabolism), the breakdown of food, is not the only critical enzymatic function. Enzymes are critical for the creation of new tissue from digested nutrients (anabolism).

It has been strongly suggested by Sargent and Touch and other researchers that enzyme deficiency in larval fish can significantly effect tissue creation. In particular, larval fish can lack sufficient enzymes to fully utilize oily feeds for the creation of cell membranes. It is likely that enzyme depletion is a problem for other nutritional factors as well. Enzyme depletion in the context of lipid synthesis is addressed in the next section.

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Nutrition from rotifers can be divided into four functional areas:

1. Lorica
2. Egg
3. Gut load
4. Internal soft tissue

Digestibility and nutritional significance of each of these components is a matter of debate; here is what we believe to be true:

• The Lorica is composed primarily of protein similar to Keratin and is largely indigestible. The Lorica is thin and amounts to no more than 10% of biomass.
• Rotifer eggs are similar in composition to rotifer biomass (15-20% lipid, 50-60% protein) and are also, by-and-large, indigestible. The volume of a rotifer egg is typically 15-20% of the volume of the parent, but egg biomass density is higher (30%) than rotifer tissue biomass (15%). In a culture with an egg to adult ratio of 1:3, 12-15% of biomass is indigestible egg.
• Together, indigestible egg and lorica amount to roughly 25% of biomass. Digestible soft tissue and gut load account for the remaining 75% of rotifer biomass
• Gut-Load: Rotifers can, with ease, continuously consume 10% of their biomass as microalgae every hour^[1]. The residence time of feed in the gut can be up to 2 hours. Their consumption rate can double during enrichment. If mean residence time of feed in the gut is one hour, gut load can amount to 15-20% of biomass and 20-25% of digestible tissue. It is possible that this number is higher. Thus, simply in terms of biomass, the composition of the feed in the gut significantly affects the nutritional value of a rotifer.
• Internal soft tissue account for the remaining 50-60% of biomass, and 75%-80% of digestible biomass.

Larval Digestion

One of the key challenges for larval fish, especially first-feeding larval fish, is that their digestive tracts are very poorly developed and digestion and nutrient uptake are difficult. Most larval fish must begin feeding soon after hatching and cannot rely solely on the egg yolk for nutrition more than 2 to 3 days post hatch (dph).

Larval fish have delicate digestive tracts and poor digestion. When a rotifer is ingested by a larva it is often squeezed and the internal organs and partially digested rotifer feed are forced out of the rotifer lorica into the larval gut. Often the lorica and attached eggs pass through the larva undigested. It is for this reason the most nutritionally valuable parts of a rotifer are its soft internal tissues and the food-loaded gut.

Gut-Loading and Digestive Function

Many hatchery managers strongly believe that the food-loaded gut of the rotifer is critical to larval fish nutrition. There are many reasons why this may be true.

1. The rotifer gut can contain a large portion of total rotifer biomass. A microalgae-fed rotifer can ingest more than 10% of its biomass per hour and much of that algal biomass can remain in the gut for up to 2 hours (see footnote below). During enrichment the feed rate can be even higher. This adds up to a lot of biomass.
2. When high-value microalgae are fed to the rotifer, this dense, highly nutritious and nutritionally balanced feed is concentrated and delivered straight to the larval gut.
3. Much of the gut content of the rotifer is partially digested, as evidenced by the significant free fatty acid portion of an algae-enriched rotifer’s total lipid content (ca. 5%). Partially digested proteins, carbohydrates and other nutrients are also present in rotifers fed microalgal and other nutritionally-balanced diets.
4. Rotifer guts contain essential digestive enzymes and acids that can aid the larval fish in digesting and assimilating nutrition.
5. Active bacteria in the rotifer act as digestive probiotics to help establish the gut bacterial flora of the larval fish.

Much has been made of the fact that rotifer eggs and lorica are not digested by many marine fish. Some suggest this demonstrates the poor value of rotifers. Instead, it should be viewed as a testimony to the challenges that first-feeding marine fish have digesting their food and taking up nutrition; there can be no doubt about of the high value of rotifers and other live feeds.

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[1] In a continuous rotifer system fed RotiGrow Plus and harvested at 45% per day, the average feed rate is 8.25% dry microalgal biomass/dry rotifer biomass per hour. Just after the daily harvest the feed rate exceeds 10.5%. And yet, feed conversion is just as efficient at the higher harvest and feed rate as it is at lower harvest and feed rates. This suggests complete nutritional uptake at feed rates in excess of 10% per hour.

…we can make educated guesses about the needs of a larval fish by examining what nature provides… the nutritional profile of healthy eggs of each species…

It is clear that the nutritional profile of healthy eggs is the intended nutrition larval fish need before first feeding. However, it is a bit harder to argue optimal nutrition at first feeding and thereafter.

The nutritional needs of larval fish are a matter of much research and debate. There is considerable understanding of the metabolic pathways for uptake of nutrients, but there is still much that is not understood. Fortunately we can make educated guesses about the needs of a larval fish by examining what nature provides.

Many studies of individual species have analyzed the nutritional profile of the egg, gonad, newly hatched larva, and wild prey organisms. Fortunately, for most species there seems to be a convergence of the nutritional profiles of each of these sources. This allows the nutritional profile of healthy eggs of each species to be used as a simple proxy for the nutritional goals for feed rotifers for that species.

This approach has been used to assess the fatty acid and lipid class requirements of larval fish, and can probably be used to assess other nutritional needs such as vitamins, carotenoids, protein, carbohydrates and minerals. This is the approach we use at Reed Mariculture in formulating our feeds, and the approach advocated by this document.

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It is often said that rotifers are simply nutrition delivery devices that mirror the nutritional profile of what the rotifers themselves are fed. In many respects this is true. However, this view underestimates both nutrition and rotifers.

The nutritional needs of first-feeding and early-stage marine fish larvae are very specific and critical. While some aspects of larval nutrition are well understood, many others remain obscure.

Moreover, rotifers are not simply passive carriers. They break down and reassemble lipids, proteins and other nutrients. This means they are biochemically active and contain active enzymes, co-enzymes, partially digested lipids and proteins, and a host of more complex nutritional factors. Ultimately, hatcheries use rotifers because rotifers work, and because artificial diets have not yet been formulated that can replace rotifers.

It is important to evaluate rotifer feeds and enrichments by the final nutritional profile of the digestible portion of the rotifers fed to fish larvae.

This profile is clearly different from the nutritional profile of the feed and enrichments. However, the nutritional profile of rotifer feeds is critical for the health and value of cultured rotifers and how the rotifers segment the nutrition they take up. A classic example is HUFA lipids. HUFAs that are consumed over time tend to be incorporated as phospholipid tissue while HUFAs that are taken up rapidly are more likely to be segmented to triglyceride energy stores and less digestible egg tissue.

Section Summary

• The Fish Egg Standard
• Digestion
• Enzymes and probiotics
• Fatty acids
• Lipid class
• Proteins
• Carbohydrates
• Vitamins
• Minerals and more

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Who uses rotifers?

• Finfish hatcheries
• Ornamental fish breeders
• Shrimp hatcheries
• Medical and toxicology research laboratories using larval fish

Rotifers are used as a first feed. The traditional feed progression for larval fish rearing is rotifer, artemia, weaning pelletized feeds and finally grow-out feeds. Many fish require rotifers until 20-30 days post hatch (dph), some fish only require rotifers for 2-5 dph, though they often do better when fed on rotifers for a longer period of time. A number of fish species do not require rotifers at all and progress straight to artemia or pelletized feeds.

One recent trend is eliminating artemia from the traditional process by extending feeding with rotifers and starting earlier with weaning diets. The advantage of this trend is that it simplifies the production process and reduces larval stress by reducing the number of feed transitions.

Why rotifers are important

Rotifers are important because they work!
Specific reasons why this is so can be broken down into three aspects:

Nutrition

Nutrition is ultimately the reason why live feeds managers use rotifers and it is a HUGE subject. See the next section B–Nutrition for more detail.

Mass production and ease of use

Rotifers can be grown at very high densities and generate enormous biomass using little space or other resources. A continuous culture system growing Brachionus plicatilis rotifers at a density of 5,500 per ml (an easy number to achieve) with a dry weight biomass of 185 ng per rotifer will have a rotifer density in excess of 1 gram dry weight (or 6.5 g wet weight) per liter of culture^[1]. At a 40% harvest rate a 10 ton system can churn out 25 Kg of screened rotifers (4 Kg dry weight) per day.

Rotifers can consume feed equivalent to 10% of their biomass per hour, and when using high-quality microalgal feeds, convert the algal biomass to rotifer biomass at an efficiency exceeding 30%. No other live feed can compete with rotifer production on a biomass basis.

Feed stimulation

Rotifers swim at a steady speed, following a straight or sinuous path, rapidly deflecting or reversing course when an obstruction is encountered. These swimming motions attract the attention of larval fish and stimulate feeding and are a key advantage of this live prey. However, the movement of rotifers is not ideal; larval fish feed on copepods (which swim with a jerking movement) much more actively. But for most fish the movement of rotifers is sufficient. Many fish, in fact, will feed on preserved rotifers and other prey if stimulated by the presence of live, swimming rotifers.

There are other feed stimulation factors that are not so well understood. Fish larvae have been reported to respond to rotifer extract. This is not surprising but research remains limited.

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Including comprehensive systems for growing and enriching rotifers, and for greenwater maintenance of rotifers with RotiGrow products from Instant Algae™ with emphasis on how we grow and enrich rotifers at Reed Mariculture.

This document has been written for fish growers and live feeds personnel to offer the current extent of our experience growing microalgae and zooplankton. Our goals are:

1. To communicate our present understanding of larval fish nutrition, the importance of rotifers, and the importance of high-quality microalgal diets for rotifers fed to larval fish.
2. To provide practical and concise information about growing rotifers including the best methods, (simple protocols) and the best feeds for these methods.
3. To provide practical and concise information about enriching rotifers and maintaining enriched rotifers, during storage and in larval tank greenwater situations including concise protocols for enrichment.

This document does not seek to portray itself as a scholarly document. Rather, it is an attempt to provide practical information to live feed producers based on the knowledge and experience of the staff at Reed Mariculture.

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We consider this document to be a work in progress. We welcome contributions to help supply missing information on larval fish nutrition, the nutritional profile of fish eggs and wild prey organisms, culture protocols, and any other information that would be helpful to live feed producers and hatchery personnel in the marine larviculture community. Comments may be added after each section of the compendium or general information may be sent as a message from the Contacts page.

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