6. LARVAL FEED

Under natural conditions, penaeid shrimps are either omnivorous scavengers or detritus feeders. In general, shrimp larvae feed on phytoplankton, detritus, polychaete and small crustaceans and their food preference changes with age. They start feeding at protozoea stage. Protozoea and early mysis stages prefer phytoplankton (although the digestive system is not yet fully developed). At mysis and early postlarvae, food preference changes to zooplankton such as rotifer or brine shrimp. As the larvae grow older than P₆, feeding habit changes again to that of a bottom feeder. Polychaetes, chopped mussels or cockle meat are fed during these stages.

Large-scale production of phytoplankton for larval rearing can be obtained in two ways: by direct fertilization of seawater in the rearing tanks or from pure culture. Many workers in the field rely partly on mixed populations of phytoplankton present in rearing water. In the past, fertilization of tank water was done directly with the resultant algal population from the “blooms” utilized for feeding purposes. The method, however, has its disadvantages. Using direct fertilization techniques, heavy “blooms” of undesirable species often occur. Such circumstances have led to the development of techniques for screening different algal species suitable for larval culture and isolation techniques for pure culture of specific types of phytoplankton.

In addition, many other types of feed have been developed and tested as part of penaeid shrimp larval rearing strategies. These feed may either be frozen or dry material from molluscs, crustacean tissue, soy bean cake, soy bean curd, egg mustard and fertilized eggs or oyster. Other types are available as formulated diet or the newly-developed microparticulate and microencapsulated diets. However, the choice of a particular feed used should be properly evaluated based on the following criteria:

1. availability and ease of handling (including technical support);

2. feed performance; and

3. feed cost and rate of return on capital.

The basic feeding strategies regarding the type of larval feed in penaeid shrimp hatchery employed to date are summarized as follows:

1. use of mixed diatoms through direct fertilization in combination with dry or fresh feed material such as soybean curd, soy bean cake, fertilized oyster eggs followed by live food organisms such as rotifer, artemia nauplii.

2. use of mixed diatoms through direct fertilization and/or pure culture diatom followed by rotifer and Artemia nauplii.

3. use of mixed diatoms through fertilization and/or pure culture of diatoms in conjunction with fresh/frozen mollusc and crustacean tissue.

4. use of mixed diatom through direct fertilization and/or pure culture of diatoms in conjunction with other dry feed materials or formulated diet.

5. exclusive use of microencapsulated or microparticulate larval feed.

6. exclusive use of wet or dry product of crustacean tissue.

6.1 Preparation of larval feed

6.1.1 Phytoplankton culture

The more popular phytoplankton species found to be suitable as food for the early larval stages of shrimp are Chaetoceros calcitrans, Skeletonema costatum, Tetraselmis sp. These plant component of the oceans usually require certain environmental conditions for growth. In the wild, phytoplankton abundance is easily affected by fluctuations in temperatures, day length, presence of grazers, availability of nutrients, water depth and turbidity as well as the seasons of the year. Manipulations, however, of the above-mentioned factors is possible under laboratory conditions or controlled rooms.

Phytoplankton culture is usually carried out by subjecting a known amount of phytoplankton to an environment suitable for its growth.

6.1.1.1 Water

Primary consideration must be given to water quality which serve as base of the culture media. The water must be clear and free of any toxic material. Offshore water is ideal because it is unpolluted and contains less sediments. This type of water can be filtered easily.

6.1.1.2 Glassware

Absolute care must be taken to ensure that all glassware are clean and sterilized in autoclaves or drying ovens prior to use.

All culture vessels must be covered after sterilization. Non-absorbent cotton plugs or sterile surgical gauze can be used to plug sterilized test tube or bottles. Dilute acid solutions can be used to disinfect plastic carboys or other large culture containers as sterilization may be omitted for mass-scale production.

6.1.1.3 Nutrients

Phytoplankton require certain nutrients for growth. Reagent grade chemicals must be used for stock cultures while technical and agricultural grade fertilizers may be used for mass-scale propagation of algae. These nutrients enrich the sea water media thereby allowing faster growth of phytoplankton in shorter periods of time.

Chemicals must be kept in a cool dry room and containers closed tightly after use.

6.1.1.4 Nutrient media

There are two types of nutrient media for marine algal culture: (a) enriched seawater media and (b) artificial or synthetic seawater media. Appendix 1 provides a list of the more commonly used media in phycology laboratories while Appendix 2 describes the steps involved in the preparation of said media. The use of enriched seawater media which has been fopund to be very practical is relatively simpler and involves lesser cost in terms of the amount of chemicals or nutrients required.

6.1.1.5 Inoculum

The quality and quantity of inoculum (seed or starter) added to the culture medium is one of the key factors for a successful algal culture.

The inoculum must be routinely examined under a microscope to check for the presence of contaminants. This will determine its “fitness”. The inoculum, once subjected to favourable growth conditions will increase in population size.

6.1.1.6 Incubation period

Incubation period is the length of time at which an inoculated medium has been subjected to controlled conditions until it is ready for harvest.

Aeration is provided throughout the culture period to prevent stratification of cells, allow gas and heat transfer, light penetration, disperse or dissolve materials and prevent adherence of cells to the walls of the culture vessel.

6.1.1.7 Maintenance/harvest

The conventional method for maintenance of culture is to allow the original inoculum to grow until high population densities are reached. Knowledge of the growth patterns of algal material will guide the culturist as to harvest time. A portion of the harvested culture may serve as a source of inoculum for future batches of culture.

A major difficulty encountered in continuous culture operations is susceptible to contaminants due to faulty installation of filters, rapid pressure changes, moisture in filters, unreliable plastic tubings stretched over glass or cracked or distorted rubber tubings. Possible sources of contamination include the culture medium, stock solutions, inoculum and filtered air or CO₂ gas (if present).

Size of culture vessels are gradually increased for mass production purposes. The use of 20-liter carboys, 100-liter glass aquaria, 1-ton and even 40-ton culture tanks are part of the scaling up operations (Fig. 21).

A few things have to be considered during mass production operations:

1. Illumination must be provided by sunlight, since artificial light will be too costly. Position culture tanks in areas where light intensity is sufficient for algal growth anytime during the year.

2. The use of expensive media should be minimized. Commercial grade or agricultural fertilizers may be used instead.

3. A cheap method of filtering the water must be devised (sand filter is a practical substitute) as water sterilization is no longer economical at this point.

4. Shorten incubation periods to prevent contamination (24–48 hours) or rapid biological succession.

6.1.2 Rotifer culture

The rotifer, Brachionus plicatilis, is the most important zooplanktonutilized as live food for various cultivable marine animals. Culture of rotifers usuallyinvolves the use of various algae like Chlorella and Tetraselmis as well as other food items like baker's and marine yeast.

Fig. 21. Flow chart for mass culture of Phytoplankton

6.1.2.1 Culture procedure

Culture vessels for rotifers vary from 20-liter tanks to 1.5-ton tanks. Initially, vessels are disinfected after which filtered seawater (75%) and freshwater (25%) is introduced to about half the tank volume. Chlorella and/or Tetraselmis is then added added at densities of 50,000–100,000 cells/ml respectively. The rotifers (10–30 individuals/ml initial density) are normally cultured outdoors where temperature range is from 28–35°C. Baker's yeast (1–5 million cells/ml) is added as supplemental food starting on the third day of culture. Brachionus populations are then allowed to increase about 100–200 individuals/ml covering a period of about 6–7 days. If partial harvest of the culture is done, 75% of the total volume is drained off or harvested for feed and the remaining 25% is then utilized as “starter” for subsequent cultures. The culture vessel is again filled up with filtered seawater and the same feeding scheme is then employed. For the total harvest, the whole tank is emptied and the harvested rotifers are then transferred to the newly prepared culture tanks.

To continually maintain the stock, regular monitoring of algal cultures should be carried out to check for presence of contaminants. Sufficient algal food supply is one of the key factors of successful rotifer culture (Fig. 22).

A flow diagram of rotifer is shown in (Fig. 23).

6.1.3 Brine shrimps as larval feed

Many hatcheries depend largely on brine shrimps to feed their growing shrimp and fish larvae. In most cases, it is the nauplii that are used to feed the larvae probably because of its small size and relatively slow moving habits. They can be easily preyed upon by the predatory fish or shrimp larvae.

Brine shrimp (Artemia) eggs are sold commercially but the quality varies with the trade brand representing different strains, geographical origin and processing methods. In purchasing brine shrimp eggs, it is essential to know the trade brand so that the quality of eggs or cysts can be assessed. Percentage of hatching of eggs or cysts varies with the brand of eggs (Table 9). However, the method of incubation and hatching is the same.

6.1.3.1 Incubation and hatching of Artemia cysts (eggs)

Incubation of Artemia eggs or cysts takes about 24–36 hours depending on the source of the eggs. The eggs should be thoroughly washed with clean fresh seawater for about 15–20 minutes before incubation. This is to remove dirt and other minute particles attached to the eggs. Often times, the eggs have a certain peculiar odor. By washing the eggs, this smell can be removed.

Fig. 22. Flow chart of Rotifer Culture

Fig. 23. Flow Diagram of Rotifer Culture

Note: Kitchen blender may be used to dissolve Baker's yeast in water.

Table 9. Hatching Characteristics of Commercially-Available Artemia

* After 24-hr incubation (Instar I nauplii with yolk)

Note: Samples are taken from newly opened cans

In order to have a good hatching rate, incubate 3.6 grams of cysts per liter of seawater. Hatching container may be made of glass, plastic, fiberglass or wood. The bottom may be flat or conical as long as the seawater is vigorously aerated. Thus, the eggs are in constant suspension.

6.1.3.2 Separation of unhatched eggs from nauplii

After incubation, the newly hatched Artemia nauplii can be separated from the unhatched eggs. The early stages of Artemianauplii are highly phototactic and can be attracted to a corner by means of light. The following procedure is followed when separating the nauplii from the eggs:

1. Shut off the aeration in the incubating container and wait for 5–10 minutes. This allows the unhatched cysts or egg shells to float or sink. While most of the cysts would sink, the newly hatched nauplii would tend to swim towards the surface and light.

2. Siphon the nauplii with a polyvinyl tubing and concentrate them with a filter net. Wash thoroughly the harvested nauplii with a clear fresh seawater and place them in a clear glass or plastic container about 20 liters in capacity. Separation process should be done more than once or until a minimal quantity of egg shells or unhatched cysts are left with the nauplii. The Artemia nauplii are then ready for feeding to the larvae (Fig. 24).

6.1.4 Preparation of processed fresh tissue of acetes, clams or mussels

1. For bivalve tissue preparation, the bivalve is shucked and the byssus removed prior to processing.

2. The fresh meat must be thoroughly washed with water. For clam and mussel meat, the body must be squeezed out during washing. Washed meat are drip-dried then blended.

3. Blend the dried tissue with water (1:1 by volume) for about three minutes. Blending time is dependent on the desired particle size of the tissue to be used as food for a particular larval stage.

4. The blended tissue is passed through a series of nylon sieves. Sieve mesh size range from 500u, 350u and 100u. Tissue particles that pass through 100u sieve are used as feed for protozoea. Intermediate sized particle tissues of 105–250u are used as feed for mysis (M¹-M³). Tissue particles that are retained in 250 μ sieve but can pass through the 500μ sieve are utilized as feed for postlarvae. Particles that are retained in 500usieve are discarded as these usually consist the shell or hard tissues which cannot be blended.

5. Processed tissue are then drip-dried and stored in the freezer until ready for use.

A. HARVEST ARTEMIA FROM HATCHING TANK	B. PLACE IN A NEW CONTAINER	C. COVER THE CONTAINER
D. ARTEMIA SWIM TOWARD LIGHT SOURCE	E. HARVESTED

Fig. 24. Harvesting of Artemia

6.1.5 Preparation of dry acetes diet

1. Freshly caught acetes are sun dried to remove about 80% moisture.

2. Prior to grinding the dry acetes, these are further dried in an oven for 2 hours at 80°C.

3. The over-dried acetes are then grounded and allowed to pass through a series of metal sieves with the following mesh sizes: 50u, 100u, 250u, 350u and 500u. The different particle sizes of grounded dried acetes are collected and used as feed according to the larval stage of the shrimp:

6.1.6 Preparation of egg custard

Contrary to the use of boild egg yolk, preparation and use of egg custard is preferred for use as larval feed. Chicken egg is broken open and the egg white and yolk placed in a bowl. Add a small volume of water while manually stirring the solution or mixing can be done with the use of an electric blender. Then steam the homogenized solution until it hardens.

The egg custard is then passed through a siever with a desired mesh size. Weigh the particulate eggcustard according to the desired quantity, i.e. mg/no. of larvae.