A primary objective in diet formulation for fish is to provide a nutritionally balanced mixture of ingredients to support the maintenance, growth, reproduction, and health of the animal at an acceptable cost. The mixture should also facilitate the manufacturing process to produce a diet with the desired physical properties. The diet should be palatable to the fish and not contain antinutritional components at concentrations that would impede the performance of the fish. The diet should be compatible with desirable flesh qualities of the fed fish and have minimum effect on water quality in the culture system. Readers needing to have more details on formulation and processing of fish diets may refer to recent publications of Cho et al. (1985), Halver (1989), Lovell (1989), Robinson (1991), and Wilson (1991) as well as the bulletin of the Food and Agriculture Organization (FAO) and the United Nations Development Program (UNDP) (1978).

The energy and nutrient requirements presented in this report were determined primarily with small fish under optimum growth conditions and represent levels affecting maximum growth rate. Fish size, metabolic function, management, and environmental factors have slight to profound effects on dietary nutrient levels for optimum performance. Thus, these data represent approximations and should be used with discretion. Also, these requirement data were determined with diets containing chemically defined and purified, highly digestible ingredients; therefore, the data represent near 100 percent digestibility to the fish. Allowances should be made when formulating natural ingredient diets for bioavailability of nutrients, processing and storage losses, and cost.

If the dietary energy and nutrient requirements are not known for a species, the requirements established for a related species can be discretely substituted. Generally, variation among fishes should be expected between warm-water and cold-water species and between freshwater and saltwater ones. As more information becomes available on nutrient requirements of various species, the recommended nutrient allowances for specific needs will become refined and commercial feeds will be more cost-effective.

Protein is usually the first nutrient considered, with the level of energy in the diet being adjusted to provide the optimum ratio. The protein has to be balanced for essential amino acids. The amount of carbohydrate in the diet varies with fish species, depending on their ability to use it as an energy source, and processing requirements. The type and concentration of lipids used in the diet are selected to satisfy essential fatty acid (EFA) and energy requirements. The vitamin requirements are mostly supplied from a supplemental premix because of uncertainty over content and bioavailability of vitamins in the feedstuffs. Mineral contents of feedstuffs are more consistent, so mineral supplementation usually is made on the basis of the composition of the major ingredients. Overfortification of labile nutrients in processed fish feeds is necessary as a safety factor. Amino acids, several vitamins, and inorganic nutrients are relatively stable to heat, moisture, and oxidation that occur under normal processing and storage conditions. Some of the vitamins are subject to some loss, however, and should be used in excess of the requirement.

Least-cost formulation using linear programming methods is commonly used to derive minimum-cost diets for fish and other food animals. The following information must be available: nutrient requirements of the animal; bioavailable nutrient and energy content of the ingredients; minimum and maximum restrictions on concentrations of various ingredients;

and cost of ingredients. The bioavailability of nutrients to fish from various feedstuffs must be known in order to make computerized substitutions among ingredients. These values are often quite variable among fish and among feedstuffs. For example, it is well known that cold-water fishes to not utilize carbohydrates as energy sources as well as do warm-water species; digestibility of phosphorus is less for fish than for livestock, especially for fish without gastric secretions in the digestive tract (Nose and Arai, 1976); and the lysine in cottonseed meal is less digestible than the lysine in soybean meal (Wilson et al., 1981).

Restrictions can be placed on minimum or maximum concentration levels of certain ingredients because of their effects on manufacturing process and palatability or their potential adverse effects on fish performance, flesh quality, or water quality. For example, fishmeal and other animal protein sources have been found to be beneficial in catfish diets for reasons not explained on the basis of meeting amino acid requirements (Mohsen and Lovell, 1990), therefore, minimum levels are usually assigned. The content of cottonseed meal in fish diets is sometimes restricted because of free gossypol toxicity (Herman, 1970). Carotenoid concentrations should be controlled because xanthophylls impart undesirable yellow pigmentation to light-fleshed fish (Lee, 1987), whereas red pigmentation sources are necessary in the diets of salmonids.

Ingredients used in commercial fish diets can be classed as sources of protein (amino acids), energy, EFA, vitamins, and minerals. Special ingredients may be used to enhance growth, pigmentation, or sexual development and to prepare diets having the required physical, palatability, and preservation properties. The nutrient composition of some ingredients commonly used in fish diets is presented in Chapter 8.

Fishmeal prepared from good-quality, whole fish is one of the highest-quality protein sources commonly available. It is also a rich source of energy, EFA, and minerals and is highly digestible and palatable to most fishes. fishmeal made from fish parts, such as waste from fish processing and canning plants, has a lower percentage of high-quality protein than that of meal from whole fish. It is also high in ash and should be used prudently in fish diets as it can produce mineral imbalances.

Other animal protein sources are by-products, such as meat and bone meal and poultry by-product meal, that contain about 45 to 55 percent crude protein. The quality of the protein in these by-products is less than that of whole fishmeal, and the ash content is usually high because a significant amount of the material comes from bone and other nonmuscle tissue. Flash or spray-dried blood meal is rich in protein (80 to 86 percent) but low in methionine and unbalanced in branched-chain amino acids. Feather meal is high in crude protein (80 percent) but, unless the feathers are thoroughly hydrolyzed during processing, digestibility is low (Cho and Slinger, 1979).

Soybean meal is universally available and has one of the best amino acid profiles of all protein-rich plant feedstuffs for meeting most of the essential amino acid requirements of fish (Mohsen, 1989). Some fish, such as young salmon, find soybean meal unpalatable (Hardy, 1989) while others, such as channel catfish, readily consume diets containing up to 50 percent soybean meal (Robinson, 1991). Soybeans contain several antinutritional factors (discussed in Chapter 3), but heating during commercial oil extraction destroys much of the activity. Meals from cottonseed and peanut (groundnut) are concentrated sources of protein and have been used in fish feeds in the United States. Compared with soybean meal, however, these meals are seriously limiting in lysine and methionine. Also, most cottonseed meals contain free gossypol, which is moderately toxic to monogastric animals and limits its use in fish feeds. Lupin flour effectively replaces full-fat soybean flour as a protein source in feeds for rainbow trout (Hughes, 1988). Meal from canola seed (low-glucosinolate rapeseed) has been used in experimental feeds for salmonids with success (Higgs et al., 1983). It has an amino acid profile comparable to soybean meal, but it is lower in protein and higher in fiber and tanins. When oilseed meals replace fishmeal or other animal by-product proteins in the diet, the losses in energy, minerals, and lipids should be considered. Dehulled soybean meal, for example, contains 25 percent less metabolizable energy (for rainbow trout), 86 percent less available phosphorus (for channel catfish), and 90 percent less (n-3) fatty acids than anchovy fishmeal on an equal dry matter basis (Lovell, 1984).

Carbohydrates are the primary nutritional contribution of grains. Whole grains contain 62 to 72 percent starch, which is 60 to 70 percent digestible by warm-water fish (Popma, 1982; Wilson and Poe, 1985) but markedly less digestible by salmonids (Smith, 1976; Cho and Slinger, 1979). Starch in grains is an important binding agent in steam-pelleted and extruded fish feeds. Fats and oils are used as energy sources, to provide EFA, and to coat the outside of pellets to reduce abrasiveness and dustiness. Marine fish oils are rich sources of essential (n-3) fatty acids, containing 10 to 25 percent of the highly unsaturated (n-3) fatty acids. Fatty acid composition of fats and oils from various sources is presented in Table 8-5.

Major ingredients used in fish diets should be analyzed regularly for proximate composition and for selected nutrients, such as limiting amino acids (usually lysine and sulfur amino acids) or EFA. Animal by-products that may contain

protein from bone, feathers, or connective tissues should be subjected to in vitro enzyme assays for an estimate of protein digestibility. All feed ingredients should be tested for mycotoxins before purchase. Periodic screening for pesticides and other contaminants (discussed in Chapter 3) are recommended. Standards may be established for some ingredients that vary considerably in quality and composition. Table 5-1 presents an example of quality standards for fishmeal and oil for use in salmonid diets.

Fish feeds should be processed into water-stable, particulate forms (granules, pellets) for efficient consumption by the fish and to minimize fouling of the water. Most manufactured fish feed is processed by compression pelleting or extrusion; other manufactured forms include moist (or semimoist), microencapsulated, and micropulverized feeds. These processes will be introduced here; for more details on fish feed processing, refer to FAO/UNDP (1978), Hardy (1989), and Lovell (1989).

Steam pelleting, through compression, produces a dense pellet that sinks rapidly in water. This process involves the use of moisture, heat, and pressure to agglomerate ingredients into compact and larger particles. Steam added to the ground feed mixture (mash) during pelleting assists in partially gelatinizing starch, which aids in the binding of the ingredients. Generally, an amount of steam is added to the feed mixture to increase moisture content by approximately 5 to 6 percent and to assist in the elevation of temperature between 70° and 90°C. The pellets are cooled and dried by forcing air over the surface of the hot pellets immediately after they leave the pelleter. Steam-pelleted feeds must be firmly bonded to prevent rapid disintegration in water, which will reduce feed efficiency and water quality. Processing conditions and ingredient composition are both important. All ingredients should be finely ground to a particle size of 0.5 mm, or smaller, prior to pelleting. Starch and gluten are important for good pellet binding while fiber and fat are antagonistic to firm bonding. Thus, supplemental fats should not be added to the feed until after pelleting, and highly fibrous feedstuffs should not be used in large quantity. Special binding agents (discussed in Chapter 2) are sometimes used in quantities of 0.5 percent to 3 percent of the ingredient formula.

Extrusion is a process by which the feed mixture, in the form of a dough, is forced through a small orifice at high pressure and temperature. This process allows entrapment of water vapor by the feed particles, which on drying will float on water. Extrusion requires more elaborate equipment and higher inputs of moisture, heat, and pressure than pelleting. Usually the mixture of finely ground ingredients is conditioned with steam into a ''mash" that may or may not be precooked before entering the extruder. The mash, which contains around 25 percent moisture, is compacted and heated to 104° to 148°C under pressure in the barrel of the extruder. As the material is squeezed through die holes at the end of the barrel, and external pressure decreases, part of the water in the superheated dough immediately vaporizes and causes expansion of the feed particles. The extruded particles contain more water than steam-pelleted particles and require external heat for drying. Thus, after extrusion, the particles must pass through a drying tunnel to reduce moisture to a safe storage level. Heat-sensitive vitamins, especially L-ascorbic acid, are added in excess prior to processing or applied to the surface after processing. Extruded feeds are firmly bound due to gelatinization of the starch and denaturation of the protein; this results in few fines and long water stability. Extruded feeds are preferred by many farmers, especially those feeding in large ponds, because they allow observation of the feeding process.

Granule diets for small fish are usually prepared by pelleting the ingredient mixture and subsequently reducing the size of the pellets by crumbling. The particles from the crumbled pellets are separated into various sizes by screening. Fat is usually sprayed onto the surface of the particles after processing. Considerable loss of water-soluble nutrients due to leaching may occur with small-particle diets because of the large amount of surface area, therefore,

overfortification of water-soluble vitamins is recommended to compensate this loss.

Microencapsulation involves coating a small particle of diet with a thin layer of a compound that will reduce disintegration, leaching, or, in some cases, bacterial degradation until the material is consumed by the fish or removed from the rearing container. The materials should be water insoluble but digestible by enzymes in the digestive tract of the fish. Several published and patented processes for microen-capsulation and microbinding vary with the encapsulation material and the substrate being coated. Nylon (N—N bonds) cross-linked proteins, calcium alginate, and oils have been used as encapsulation materials.

Moist or semimoist feeds are prepared by adding moisture and a hydrocolloidal binding agent, such as carboxymethylcellulose, gelatinized starch, or ground, wet animal tissue with the dry ingredients and forming the mixture into soft, moist pellets. The advantages of moist feeds are that some fish species find moist diets more palatable than dry diets, a steampelleting machine is not needed (a feed grinder will suffice), and heating and drying are avoided. Disadvantages of moist feeds are their susceptibility to microorganism or oxidation spoilage unless fed immediately or frozen. Fish parts going into moist feeds should be pasteurized to destroy possible pathogens and thiaminase. Some moist diets do not require frozen storage. They contain humectants, like propylene glycol, which lower water activity below that which will allow bacterial growth; they also contain fungistats, like propionic or sorbic acid, which retard mold growth (Lovell, 1989). These diets must be packaged in hermetically sealed containers, preferably under nitrogen, and stored at low temperatures for best keeping quality.

Research laboratories and fish farming operations often have a requirement for a nutritionally complete, production-type reference diet to serve as a basis for developing and testing new feeds. Examples of production diets for salmonids and channel catfish are presented in Table 5-2. These diets contain commonly available ingredients, and they have been shown to produce good results under experimental and production conditions. Diets similar to the channel catfish diet have been used satisfactorily with other warm-water species such as tilapia and carp. It is important that these diets be produced with proper processing procedures and with high-quality ingredients, particularly the fishmeal and fish oil.

All diets in an experiment should be alike in all respects except the variable being tested; this includes nutrient composition, energy level, palatability, water stability, and particle size. Experimental diets for evaluating nutrient requirements should be prepared from highly chemically defined ingredients. Casein and gelatin are a good protein combination for purified diets. Low-vitamin casein is available for use in vitamin experiments, however, regular casein is satisfactory for other experiments. Blood fibrin is a desirable protein for mineral studies, and chicken egg protein can be used in protein or amino acid experiments. These protein sources and others are available in highly purified forms. Dextrin is traditionally used as a carbohydrate source. Cooked starch is satisfactory for warm-water fish. Some of the lipid should be from fish oil to provide essential highly unsaturated (n-3) fatty acids. Purified cellulose can be used as a nonnutritive filler. The diets should contain a binding agent that will hold the particles together for a reasonable time in water. Gelatin, agar, alginic acid, wheat gluten, or carboxymethylcellulose can be used.

Examples of purified reference diets for salmonids and channel catfish are presented in Table 5-3. These diets can be prepared by compression-pelleting in a manner similar to that used in making low-moisture commercial diets, or as a moist diet using a laboratory feed grinder. Moist diets may be prepared by mixing the dry ingredients, then adding the oil with further mixing, followed by the addition of water (30 to 40 percent) to give the mixture a plastic consistency. If gelatin is to be the primary binding agent, it is mixed with hot water along with the oil; this mixture is added to the dry mixture. The dough is then extruded through a feed grinder and the extruded strands are broken into smaller pieces and dried or frozen.

Purified diets are sometimes unpalatable to some species. Palatability can sometimes be improved by substituting a natural feedstuff, such as fish flour, for a chemically defined ingredient in the diet formula. The nutritional contribution of the commercial to the purified diet should be known and should not confound the experimental diet.