ANNEXES (Cont.)

ANNEX H
Protocol for Tissue Sample Preparations

5 % TCA Method

1. Stun fish by blow to head.
2. Severe caudal peduncle.
3. Bleed out fish 2 minutes (collect blood sample).
4. Eviscerate fish, leave gills with carcass.
5. Grind carcass to minced mass in meat grinder.
6. Weigh mass, add equal volume of 5% TCA.
7. Homogenize in blendor.
8. Remove 2.0 gm sample.
9. Add 8 mL 5% TCA.
10. Homogenize in Polytron one minute
11. Transfer to centrifuge tube.
12. Centrifuge until clear (10–20 min).
13. Remove 3–5 mL. supernatant with syringe.
14. Filter through 0.45 um disc into sample tube.
15. Inject 10–20 uL filtrate into HPLC.

Microwave Method :

Repeat steps 1, 2, 3, 4, 5

6. Weigh mass, add equal volume of HPLC water.
7. Repeat as above.
8. Remove 2 gm sample, add 8 gm. Water.
9. Place in 100 mL Beaker with cover glass, weigh.
10. Microwave medium heat 30 sec, check 30 sec, and microwave again 30 sec.
11. Cool, weigh, add water to original weight Repeat steps 12, 13, 14, 15

ANNEX J

EXTRUDED CATFISH FEED

Operating Conditions

Feeder rate 300 rpm
Barrel temperature >= 120 degree C
Die Bore = 2 mm

Dryer temperature = 145 degree C

ANNEX K
THA/89/003 - FISH NUTRITION AND AQUACULTURE DIETS

LABORATORY NEEDS : SUPPLIES

ANNEX L

Draft

THA/89/003 - FISH NUTRITION AND AQUACULTURE DIETS

We are pleased to provide the detailed justification you expected in your letter of 20 August 1991 for the needed extension to 30 June 1992 to complete all elements of the project reviewed and agreed upon in principle at the Tripartite Review on 14–15 March 1991.

1. Development and testing of diets made in the pilot plant feed mill (delivery was delayed and prevented this activity until completion and installation of equipment). Mill is currently in operation but various type diet and operation procedures are not finalized. The Feed Technologist consultant is on duty and will complete these preliminary tests and personnel training for various feed type operations by 31 December 1991. These type diets prepared from local ingredients can then be rapidly tested in early 1992 leading to adoption of appropriate formulae for shrimp and fish farmers of Thailand by 30 June 1992, thus completing this major objective of project THA/89/003 as amended.

2. Installation and testing of the equipment in the quality control laboratory has delayed completion of the gross nutrient assays of the ingredients of the national survey of potential feedstuffs for local diet formulations. The lipid extraction equipment needs an exhaust hood to minimize employee danger when using volatile solvents. This hood with exhaust fan could be installed and this allow rapid completion of the assays to generate data for diet formulations.

3. Digestion and utilization of feedstuffs and final diet formulations manufactured under various pressure, temperature and moisture conditions in the new extruder need to be conducted before final diet formulae can be field tested then adopted for national use. The Feed Technologist is currently scheduling these manufacturing condition tests for shrimp and finish diet formulae made up primarily from national available underutilized ingredients. These tests will be completed by 31 December 1991 and results then need to be field tested early in 1992 before release to the fish farmers who rely on NIFI advice for their production improvements.

4. The Fatty Acid Consultant and the Vitamin and Amino Acid Consultant are on duty and will complete the training and demonstration of techniques for quantitative resolution of these required nutrients in feed formulations by late 1991. Alteratious in quantitative levels of essential fatty acids, amino acids, and critical vitamins by various extruder diet manufacturing conditions need to be tested before the field tests are completed by June 1992, and growth/health production results rationalized by the trained scientists using these advanced scientific instruments.

5. The general and specific research programme in fish nutrition and aquaculture diet development will have been developed and documented by the research leaders involved during the tenure of the consultants. When approved by NIFI and DOF this master plan and the work units which can be completed by 30 June 1992 will constitute completion of the major objective of the project for research and development planning.

6. Training has been completed for :

   1. Vitamin Nutrition (BL 31-03) ; Mr. Pairat Kosutarak
   2. Fatty Acids Nutrition (BL 31-04) ; Ms. Pisamai Somsueb
   3. Energy and Metabolism in Fishes (BL 31–05) ; Mr. Wichien Sakares
   4. Fish Feed Technology and Nutrition (BL 31–06) ; Mr. Thavee Wiputhanumas.

   Mr. Prasert Munsiri and Ms. Sonkphand Lumlertdecha is currently t Auburn for their Fish Nutrition Study (BL 31–01, 31–02). Ms. Sonkphand umlertdecha has been accepted and urged to matriculate directly into the Ph.D programme. This would be most desirable because another well trained scientist ould greatly strength the research and development capability of NIFI and the OF. Salary and consultancy cost savings could be used to subsidize this study.

7. The Larval Feed Consultant may not need to be recruited resulting in a large element of funds which can be used for the extension of the administrative Assistant and the Research Assistants until 30 June 1992. Dr. ardy, Dr. Halver and Thomas Wilson have each prepared and instructed students in preparation and use of larval feeds. NIFI does not have advanced feed engineering equipment to prepare flake, micro encapsulated, micro particulate, microcoated larval feeds and has limited facilities to prepare fortified natural larval feeds like EPA fortified or vitamin fortified rotifers, yeasts, artemia or copepods. Thus this element of the project should be rescheduld for future operations since several large institutes and industries are now roviding specific larval feed preparations for aquaculture.

8. The Administrative Assistant position must be extended for the full tenure of the project in order to conduct administrative support business services for all personnel engaged in any element of the overall project.

ANNEX M
DIETARY MINERAL REQUIREMENTS
OF FISH AND SHRIMP

ABSTRACT

The available literature on the dietary essenetiality of minerals in aquatic animals is reviewed. Based on the current literature dietary requirements for approximately nine minerals have been identified for fish (calcium, phosphorus, magnesium, copper, iron, zinc, manganese, selenium and iodine) and seven (calcium, phosphorus, potassium, magnesium, copper, zinc and selenium) have been recommended for shrimp and lobster.

Due to lack of studies concerning dietary mineral requirements of marine fish and crustaceans, methodologies and requirment parameters from freshwater fish should be used as guideline for marine fish and crustacean research. When evaluating the dietary essentiality of a mineral, the nutritionist must not only evaluate growth parameters, but also assess tissue stores, biochemical indicators (e.g., enzyme reaction rates), and effects on the general development of the animal.

INTRODUCTION TO MINERAL NUTRITION

A dietary source of 23 minerals has been demonstrated as essential in one or more animal species. These elements are divided into two groups, the seven major minerals (calcium, chloride, magnesium, phosphorus, potassium, sodium, and sulfur) and 16 trace elements (aluminum, arsenic, cobalt, chromium, copper, fluorine, iodine, iron, manganese, molybdenum, nickel, selenium, silicon, tin, vanadium, and zinc). For the chicken thirteen minerals have been identifed as essential in the diet and six have been identified as producing beneficial effects (Scott et. al., 1982). Thirteen minerals are required in the diet of by most terrestrial animals and may be essential for aquatic animals. Concerning these mineral eight are cations: calcium (Ca⁺⁺), sodium (Na⁺), potassium (K⁺), magnesium (Mg⁺⁺), manganese (Mn⁺⁺), zinc (Zn⁺⁺), iron (Fe⁺⁺) and copper (Cu⁺⁺); and five are anions or are usually found in anionic groupings chloride (Cl⁺ ), iodide (I), phosphate (PO³₄), molybdate (MoO³₄) and selenite (SeO²₃) (Scott et al. 1982). Nine minerals have been identified as essential in the diet of fish (Table 1) and six have been recommended for includsion in diets of shrimp and lobster (Table 2). This large discrepancy in knowledge of mineral nutrition of terrestrial and aquatic species illustrates the relative infancy of aquatic animal nutrition and the related difficulty of research in mineral nutrition of these species.

Minerals serve as structural components of hard-tissue matrices (e.g., bone, fin rays, scales, teeth and exoskeleton) and components of soft tissues (e.g., sulfur in proteins, phosphorus in phospholipids and nucleic acids). They also are components of metalloproteins (e.g., iron in hemoglobin, copper in hemocyanin, zinc in carboxypeptidase), and serve as cofactors and/or activators of a variety of enzymes (e.g., zinc activation of alkaline phosphatase). The more soluble minerals (calcium, phosphorus, sodium, potassium and chloride) function in osmoregulation, acid-base balance, and in the production of membrane potentials.

With the exception of osmoregulation, the maintenance of osmotic balance between body fluids and the water in which the animal lives, the biochemical functions of minerals in aquatic species appear to be similar to those in terrestrial animals (Lovell 1989). Freshwater species lose ions to the hypotonic environment and therefore suffer from hydration; whereas the reverse is true for marine species. Unlike terrestrial animals which are primarily limited to a dietary source for minerals, aquatic animals may be able to utilize, to some extent, minerals dissolved in the water to meet physiological requirements. Independent of the mode of intake, a deficiency will occur if the animal's physiological requirements are not met.

DIETARY ESSENTIALITY/REQUIREMENT OF MINERALS BY FISH AND SHRIMP MACROMINERALS

Calcium and Phosphorus

Calcium and phosphorus are two of the major constituents of the inorganic portion of feeds. Quantitatively, calcium and phosphorus function primarily as structural components of hard tissues (e.g., bone, exoskeleton, scales and teeth). In addition to structural functions of calcium, it is essential for blood clotting (vertebrates), muscle function, proper nerve impulse transmission, osmoregulation, and as a cofactor for enzymatic processes (National Research Council 1983). Phosphorus is a component of a variety of organic phosphates such as adenosine triphosphate (ATP), phospholipids, coenzymes, deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Inorganic phosphates also serve as important buffers to maintain normal pH of intra- and extracellular fluids (Zubay 1983).

There are many factors influencing the absorption, distribution and excretion of calcium and phosphorus. Dietary calcium is primarily absorbed from the intestine by active transport. In vertebrates, the vitamin 1,25 dihydroxycholecalciferol functions as a hormone in the maintenance of serum calcium and phosphorus levels by altering the rate of intestinal absorption (via synthesis of Ca²⁺ -binding protein), renal resorption and bone mobilization (Lovell 1989). Regardless of the form in which calcium and phosphorus are ingested, their absorption is dependent upon solubility at the point of contact with the absorbing membranes; hence, mineral sources that are soluble at luminal pH are potentially more available. In the presence of adequate phosphorus, Nakamuara and Yamada (1980) determined the availability of calcium for common carp (Cyprinus carpio) to be 58, 37 and 27% from calcium lactate, tribasic calcium phosphate and calcium carbonate, respectively.

The National Research Council (1983) has summarized the relative availability or apparent absorption of various sources of phosphorus for four species of fishes. In general bioavailability of phosphorus (and minerals in general) has been found to be positively correlated with the solubility of the mineral in water. Monobasic phosphates of sodium and potassium are highly available (90–95%) sources of phosphorus for channel catfish (Ictalurus punctatus) (Loveil 1978), common carp (Ogino et al. 1979), red sea bream (Crysophrys major) (Sakamoto and Yone 1979b), and rainbow trout (Oncorhynchus mykiss) (Ogino et al. 1979). Di- and tri-basic calcium phosphates are highly available to red sea bream (Sakamoto and Yone 1979b). For the channel catfish, di-basic calcium phosphate has an availability of 65% (Lovell 1989), and di- and tri-basic calcium phosphates have availabilities of 71 and 64% respectively, for the rainbow trout (Ogino et al. 1979). For the carp, which lacks an acidic stomach, the bioavailability of calcium and phosphorus from different sources is more variable; tribasic calcium phosphate has an availability of only 13%, dibasic calcium phosphate has an availability of 46% and monobasic calcium phosphate has an availability of 94% (Ogino et al. 1979).

In addition to the chemical form (solubility) affecting mineral availability, other nutrients may also affect the availability of certain minerals. For exampel lactose may interact with absorptive cells to increase their permeability to calcium ions; large intakes of iron, aluminum and magnesium interfere with the absorption of phosphorus by forming insoluble phosphates, and fats interact with calcium to form insoluble soaps (Maynard et al. 1979). Hence, availability of calcium and phosphorus is dependent upon the mineral source (solubility at intestinal pH), calcium to phosphorus ratio (Ca:P ratio), lactose intake, and dietary levels of calcium, phosphorus, vitamin D, iron, aluminum, manganese, potassium, magnesium and fat.

Phosphorus in fish meal exists mainly in the form of insoluble hydroxyapatite originating from hard tissues such as bones and scales. It has been shown that the availability of phosphorus contained in fish meal is fairly low for the carp (10–33%) as compared to rainbow trout (60–81%) (Watanabe et al. 1988). Akiyama and Dominy (1989) reported the apparent absorption of phosphorus from fish meal to be 46.5% for Penaeus vannamei.

Although calcium requirements of crustacea have been reported (Gallagher et al. 1978, Kitabayashi et al. 1971, Kanazawa et al. 1984), recent work indicates that P. vannamei raised in seawater does not require supplemental calcium (Davis 1990). Similarly, calcium deficiencies in fish have only been produced in low-Ca water (Robinson et al. 1984, 1986, 1987). It appears that both shrimp and fish can absorb some minerals from the water via drinking, and by direct absorption via the gills, skin, or both (National Research Council 1983, Deshimaru et al. 1978). Hence, the calcium requirement may be totally or partially met through absorption of calcium from the water. Due to the low concentration of phosphorus in natural waters (Boyd 1981), the absorption of significant amounts of phosphorus from freshwater and saltwater is unlikely, making a dietary source of phosphorus potentially more critical.

Dietary phosphorus requirements of 1% (Kitabayashi et al. 1971), 1–2% (Kanazawa et al. 1984) and 2% (Deshimaru and Yone 1978) have been recommended for shrimp. Davis (1990), demonstrated that the dietary phosphorus requirement of P. vannamei was dependent upon the calcium content of the diet and that in the absence of calcium supplements the basal diet (0.03% Ca, 0.34% P%) contained adequate phosphorus for normal growth. The dietary phosphorus requirements of fish range from 0.3 to 0.8% of the diet (National Research Council 1983). The large disparity between dietary requirements of fish and shrimp may indicate that dietary phosphorus requirements of shrimp have been overestimated and/or the bioavailability of phosphorus sources utilized are very low.

Gallagher et al. (1978) examined the effects of varying dietary Ca:P ratios on juvenile lobsters. Based on growth and histological studies of the endocuticle, a Ca:P ratio of 0.51 (0.56:1.10) was found to be best for the juvenile lobster, while ratios of 1.55 and greater resulted in abnormalities of the endocuticle. In P. japonicus, a dietary Ca:P ratio of 1:1 has been recommended (Kanazawa et al. 1984; Kitabayashi et al. 1971). Davis (1990) reported that the supplementation of calcium to the basal diet appeared to inhibit phosphorus bioavailability and that the calcium to phosphorus ratio was inadequate to explain the inhibitory effects of calcium. Based on these studies it appears that calcium may affect phosphorus availability and that calcium levels in excess of 2.5% should be avoided.

In addition to calcium affecting availability, phytate phosphorus, which constitutes approximately 67% of the phosphorus in grains, is poorly available to fish (Lovell 1989) and may not be utilized by shrimp. Civera et al. (1990) found that the presence of phytate inhibited the availability of dietary calcium and phosphorus, presumably due to the formation of insoluble complexes in the digestive system of P. vannamei and P. japonicus. The apparent availability of phytate phosphorus was determined to be 47.3% for P. japonicus and 8.4% for P. vannamei. Similarly, phytate phosphorus was found to be unavailable to P. vannamei and the presence of 1.5% phytate inhibited the availability of dietary phosphorus and zinc (Davis 1990).

Care should be taken to minimize the oversupplementation of dietary phosphorus. Considering that there is a great deal of interest in the deleterious effects of phosphorus pollution from effluent waters of aquaculture facilities, the minimization of phosphorus supplements will not only economically benefit the feed producer but it will also reduce the effects of phosphorus pollution.

Sodium, Potassium and Chloride

Sodium, potassium, and chloride are recognized as being essential for a number of physiological processes. Dietary deficiencies of sodium and chloride have not been demonstrated in fish (National Research Council 1983). Recent work with the red drum, Sciaenops ocellatus, has demonstrated that at low salinities the supplementation of sodium chloride to the diet resulted in increased growth (Holsapple 1990). One possible explanation for this positive response is an increase in amino acid absorption. Boge et al. (1983) demonstrated that, in addition to being energized by a Na⁺-gradient, amino acid transport by brush-border membrane vesicles prepared from enterocytes of the sea bass (Dicentrachus labrax) is dependent upon the presence of Clions. The addition of dietary sodium chloride at low salinities may increase the absorption of amino acids and/or satisfy other metabolic requirements of this species which are normally obtained from the seawater, thus resulting in a physiological advantage.

A dietary potassium requirement has been identified for chinook salmon (Oncorhynchus tshawytscha) in fresh water (Shearer 1988), but not for red sea bream in salt water (Sakamoto and Yone 1978b). Kanazawa et al. (1984) reported that diets containing 0.9% potassium improved growth of P. japonicus as compared to diets containing 1.8% potassium. Deshimaru and Yone (1978) recommended dietary supplementation of 1% potassium, based on the comparative growth of shrimp fed a diet without magnesium and potassium supplementation; however, potential interactions between potassium and magnesium were not evaluated. Davis (1990), utilizing semipurified diets found that the individual deletion of potassium did not result in a significant depression in tissue potassium or growth; however, tissue levels of magnesium were affected, indicating a potential interaction.

Most freshwater and all seawater probably contains sufficient amounts of sodium, potassium and chloride ions to satisfy physiological needs of fish (Lovell 1989). These ions are also found in substantial amounts in most feedstuffs, making the necessity of dietary supplementation unlikely.

Magnesium

Magnesium is one of the major minerals recognized as essential for animals. In vertebrates, approximately 60% of total body magnesium is located in bone where about one-third of it is combined with phosphate and the remainder is adsorbed loosely on the surface of the mineral structure (Pike and Brown, 1975). In soft tissues magnesium occurs intra-and extracellularly.

Magnesium is essential for maintenance of intra-and extra-cellular homeostasis in fish (Moyle and Cech 1982) and crustacea (Mantel and Farmer 1983). Magnesium is essential for cell respiration and phosphate transfer reactions being complexed with adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate. It is an activator for all thiamine pyrrophosphate reactions and is involved in the metabolism of fats, carbohydrates and proteins.

Dietary magnesium deficiencies have been documented in a variety of freshwater fish (National Research Council 1983). Deficiency symptoms include: poor growth, anorexia, lethargy, flaccid muscles, convulsions, vertebral curvature, high mortality and depressed magnesium levels in the whole-body, blood serum and bone (Lovell 1989). Fish in freshwater, which only contains 1 to 3 mg Mg/L, require 0.025% to 0.07% magnesium in the diet (Lovell 1989). Seawater typically contains high levels of magnesium (1,350 mg/L), and magnesium is excreted by marine crustacea and fish, resulting in blood levels lower than that of the external medium. Thus, saltwater species may not require a dietary source of magnesium (Dall and Moriarty, 1983).

Red sea bream reared in saltwater showed no signs of dietary deficiencies when fed diets containing as low as 0.012% magnesium (Sakamoto and Yone 1979a). Deshimaru and Yone (1978) found that supplementation of 0.3% magnesium did not improve the nutritive value of a semi-purified diet for P. japonicus. Kanazawa et al. (1984) reevaluated the magnesium requirement of P. japonicus and reported that dietary supplementation of 0.1–0.5% magnesium improved the nutritive value of the diet. Davis (1990) reported a depression in hepatopancreas magnesium levels of P. vannamei in response to the deletion of magnesium from in a semipurified diet, however, growth depression was not observed. Based on the high levels of magnesium in seawater and the magnesium requirements of fish, a dietary requirement for marine species would not be expected; however, further investigation into the dietary requirements of marine fish and shrimp is needed to evaluate the essentiality. Since most feed ingredients, especially those of plant oragin, are high in magnesium, the supplementation of magnesium to practical diets is generally not necessary.

MICROMINERALS

Copper

Copper functions in hematopoiesis and in numerous copper-dependent enzymes including lysyl oxidase, cytochrome c oxidase (CCO), ferroxidase, tyrosinase and superoxide dismutase (SOD) (O'Dell 1976). Lysyl oxidase functions in the formation of crosslinks during the synthesis of collagen and elastin. The failure of collagen maturation (crosslinking) in the organic matrix of bone accounts for increased fragility of bones and the associated abnormalities of copper deficiencies (O'Dell 1976). Failure of collagen and elastin crosslinking and a undefined muscular defect result in enlargement of the heart and cardiac failure in copper-deficient animals.

Copper is also involved in the absorption and metabolism of iron and functions in the formation of hemoglobin in vertebrates. Some marine animals such as mollusks and crustaceans have hemocyanin or cyanodin as oxygen-carrying pigments in blood. These copper containing pigments have an analogous role to hemoglobin in red-blooded animals (Lovell 1989). Depledge (1989) estimated that on a fresh-weight basis 40% of the whole-body copper load in shrimp is found in hemocyanin. This would indicate suggest a considerable increase in the physiological demand for copper above that required by vertebrates.

In addition to the physiological functions of copper, high levels of environmental copper have been found to be toxic to a variety of marine species (Bryan 1976). Copper contamination in the marine environment is generally due to an increase in anthropogenic input and occurs primarily in coastal and estuarine areas, where copper concentrations (up to 0.6 mg Cu/L) far exceed the background copper level in seawater (0.5 ug Cu/L) (Bjerregaard and Vislie 1986). Due to toxic effects of dissolved copper, shrimp hatcheries routinely use water which has been treated with ethylenediaminetetraacetic acid (EDTA) to chelate free copper (Lawrence et al. 1981). Although a great deal of research has been conducted on the toxicity of dissolved copper, the dietary essentiality of this nutrient in marine species has received little attention.

Dietary deficiencies of copper have been documented in several freshwater fish (Murai et al. 1981; Gatlin and Wilson 1986b; Ogino and Yang 1980, Julshamn et al. 1988) but the dietary essentiality of copper has not been evaluated in marine fish. Dietary requirements for copper range from 1.5 to 5 mg Cu/kg diet. In addition to dietary deficiencies resulting in reduced growth and feed efficiency, copper-dependent enzymes such as ceruloplasmin, copper and zinc dependent SOD and CCO have been shown to be excellent indicators of copper nutriture (Gatlin and Wilson, 1986b).

Kanazawa et al. (1984) found that the dual deletion of iron and copper had no significant effect on the growth and survival of P. japonicus. It should be noted that in this series of experiments, percent weight gain was very low (40%) and survival was very poor (57%); hence, the nutritional stress or the quality of the diet may not have been adequate to induce a dietary deficiency. Davis (1990) demonstrated a dietary copper deficiency in P. vannamei fed semi-purified diets containing less than 34 mg Cu/kg diet. Deficiency symptoms included poor growth, reduced copper levels in the carapace, hepatopancreas and hemolymph, and enlargement of the heart. These results indicate that P. vannamei can not meet there physiological needs for copper from seawater and that a dietary source is required for maximum growth and tissue mineralization. Thus indicating that species utilizing copper as a component of there respiratory pigment may have an increased copper requirement over species utilizing iron-based respiratory pigments.

Iron

Iron is a trace element that is essential for the production and normal functioning of hemoglobin, myoglobin, cytochromes, and many other enzyme systems. In vertebrates, the principal role of iron is as a component of hemoglobin. Red blood cells are regenerated periodically and most of the iron is recycled. That which is not recycled is excreted via the bile into the intestine. Like other elements of low solubility such as zinc and copper, iron is absorbed and transported in the body in a protein-bound form (Lovell 1989). In vertebrates, mucosal transferrin binds Fe²⁺ in the intestinal lumen and transports it across the mucosal brush border. Within the cell Fe¹⁺ is bound to apoferritin forming ferritin. The amount of apoferritin in the mucosa is regulated by physiological needs for iron. Iron released into the blood must be reduced to Fe²⁺ by reducing agents (e.g., vitamin C) prior to reacting with transferrin. The iron-bound transferrin is then transported in the blood where the iron is again released at target tissues (liver and hemopoietic tissue). Iron and other minerals of low solubility are not easily excreted; thus, mineral excesses are deposited in cells of the digestive system which are sloughed into the digestive tract for excretion with the feces.

In crustaceans, the hepatopancreas has been found to be the organ richest in iron. Storage cells containing iron have been reported in the crayfish (Procambarus clarkii) (Ogura 1959; Miyawaki et al. 1961) and the crab (Cancer irroratus) (Martin 1973). Iron-transporting proteins have been found in the hemolymph of two species of crabs (Ghidalia et al. 1972 and Depledge et al. 1986). These observations indicate a regulatory mechanism similar to that of vertebrates. In addition to the digestive system, gills appear to play a active role in iron metabolism. In the crab (Cancer irroratus), iron accumulates by forming a coating around the branchial lamellae during the intermolt cycle, which is then rejected at ecdysis along with the integument (Martin 1973). Absorption of iron from the water through the gills would potentially provide an additional source of iron.

Iron deficiencies have been documented for several species of fish (National Research Council 1983); however, dietary deficiencies for shrimp have not been observed (Deshimaru and Yone 1978; Kanazawa et al. 1984, Davis 1990). Iron deficiency causing hypochromic microcytic anemia has been reported for freshwater fish such as the brook trout (Salvelinus fontinalis) (Kawatsu 1972) and carp (Cyprinus carpio) (Sakamoto and Yone 1978d) and in marine fish such as the red sea bream (Sakamoto and Yone 1976b) and the yellowtail (Seriola quinquerodiata) (Ikeda et al. 1973). However, a growth depression was not observed in these iron-deficient fish. Gatlin and Wilson (1986a) characterized iron deficiency signs of the channel catfish, and found that fish fed the basal diet (9.6 mg Fe/kg) exhibited suppressed growth and feed efficiency, as well as reduced hemoglobin, hematocrit, plasma iron, transferrin saturation and erythrocyte count values. They concluded that a minimum of 20 mg supplemental Fe/kg diet (30 mg total Fe/kg) was required by channel catfish for best growth and hematological values.

In addition to deficiencies causing physiological problems, excessive levels can be toxic. Excessive dietary supplementation of iron appears to have potentially adverse effects on growth of P. japonicus (Deshimaru and Yone 1978; Kanazawa et al. 1984). Additionally, iron-catalyzed lipid oxidation increases with iron supplementation which in turn adversely affects feed stability (Desjardins et al. 1987). Iron is one of the primary metals involved in lipid oxidation and ferrous iron is a more potent catalyst of lipid peroxidation than ferric iron (Chvapil et al. 1974; Lee et al. 1981). Ferrous iron catalyzes the formation of hydroperoxides and free radical peroxides by providing a free radical initiator in the presence of unsaturated fatty acids and oxygen. Crustacean diets primarily contain polyunsaturated fats; therefore, the supplementation of ferrous iron to the diet could be expected to affect the stability of the diet through increased lipid oxidation (rancidity) and reduced stability of ascorbic acid (Hilton 1989).

Iodine

Iodine is an essential element for a variety of animals. Nearly every cell in the body contains iodine; however, in vertebrates the thyroid gland is the main location of iodine reserves. The thyroid hormones, which contain iodine, are known to have a role in thermoregulation, intermediary metabolism, reproduction, growth and development, hematopoiesis and circulation, as well as neuromuscular functioning (National Research Council 1980). The minimum dietary requirement of fish has not been defined; however, 1 to 5 mg/kg feed has been found to be an adequate level (National Research Council 1983). The physiological essentiality of iodine has not been evaluated in shrimp.

Manganese

Manganese functions as a cofactor in several enzyme systems, including those involved in synthesis of urea from ammonia, amino acid metabolism, fatty acid metabolism, and glucose oxidation (National Research Council 1980).

Principal signs of manganese deficiency in terrestrial species include reduced growth rate, skeletal abnormalities, convulsions, reduced righting ability, abnormal reproductive function in males and females, and ataxia in the newborn (National Research Council 1980). Dietary deficiencies in fish have resulted in poor growth, skeletal abnormalities, high embryo mortalities and poor hatch rates (National Research Council 1983). A total dietary manganese content of 12 to 13 mg/kg has been recommended for the common carp and rainbow trout (Ogino and Yang 1980); however, Gatlin and Wilson (1984b) found that 2.4 mg Mn/kg diet was sufficient for normal growth and health of channel catfish. Kanazawa et al. (1984) found that the supplementation of 10 and 100 mg Mn/kg diet did not improve the growth of P. japonicus; however, it should be noted that percent weight gain was less than 70%. The nutritional stress placed on these shrimp would not be considered adequate to reduce body stores low enough to induce a deficiency of a trace element. Since the manganese content of seawater is very low (0.01 mg/L), significant absorption from the water is unlikely making a dietary source potentially necessary for marine shrimp and fish.

Selenium

Selenium is a trace element which functions as a component of the enzyme glutathione peroxidase. Glutathione peroxidase converts hydrogen peroxide and lipid hydroperoxides to water and lipid alcohols respectively; thus, protecting the cell from the deleterious effects of peroxides (Little et al. 1970). This enzyme acts along with vitamin E to function as a biological antioxidant which protects polyunsaturated phospholipids in cellular and subcellular membranes from peroxidative damage (Lovell 1989). In addition to participation in enzymatic functions, selenium helps protect against mercury toxicosis by forming a mercuric-selenium complex. This protein-bound complex is diverted from the kidney (where inorganic mercury devoid of selenium is deposited) to the liver and spleen where its toxicity is considerably reduced (National Research Council 1980).

Complementary functions of selenium and vitamin E may allow these nutrients to interact physiologically (Gatlin et al. 1986). Selenium and vitamin E interrelationships have been investigated in several animal species, and a variety of common and unique deficiency signs have been described (National Research Council 1983). Differing responses, especially with respect to gross deficiency signs, were observed when the Atlantic salmon (Salmo salar) (Poston et al. 1976), rainbow trout (Bell et al. 1985), and the channel catfish (Gatlin et al. 1986) were fed diets without supplemental selenium and vitamin E or both of these nutrients.

Selenium deficiencies have been reported for the Atlantic salmon (Poston et al. 1976), rainbow trout (Hilton et al. 1980) and the channel catfish (Gatlin and Wilson 1984a; Gatlin et al. 1986). A level of between 0.15 and 0.38 mg Se/kg diet (Hilton et al. 1980), and 0.25 mg Se/kg diet (Gatlin and Wilson 1984a) was required to provide maximum growth and glutathione peroxidase activity in rainbow trout and channel catfish, respectively. In zooplanktonic daphnids, a selenium deficiency in the medium resulted in a cuticle deformation and a depression in reproduction. In the presence of replete zinc, 1 ppb selenium was adequate (Keating and Dagbusan 1984); however, in the absence of detectable zinc, 5 ppb selenium was required to eliminate deficiencies characteristic of selenium deprivation (Keating and Caffrey 1989). Davis (1990) found that juvenile P. vannaemi grew best when fed semi-purified diets supplemented with 0.2–0.4 mg Se/kg diet. Although this response was not duplicated, it appears that shrimp have a dietary requirement for selenium.

Practical diets containing more than 15% fish meal should contain adequate selenium and not require supplementation. Diets formulated with predominantly plant ingredients may require a selenium supplement. Due to potentally toxic effects, selenium supplementation of greater than 0.3 mg/kg should be avoided.

Zinc

Zinc is required for normal growth, development, and function in all animal species that have been studied (National Research Council 1980). Zinc functions as a cofactor in several enzyme systems and is a component of a large number of metalloenzymes which include carbonic anhydrase, carboxypeptidases A and B, alcohol dehydrogenase, glutamic dehydrogenase, D-glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, malic dehydrogenase, alkaline phosphatase, aldolase, superoxide dismutase, ribonuclease and DNA polymerase (National Research Council 1980).

A dietary requirement for zinc has been demonstrated in a variety of freshwater fishes fed semi-purified diets: 20 mg Zn/kg diet for channel catfish (Gatlin and Wilson 1983) and blue tilapia (Oreochromis aurea) (McClain and Gatlin 1988): 15–30 mg Zn/kg diet for carp (Ogino and Yang 1979); 15–30 mg Zn/kg diet for rainbow trout (Ogino and Yang 1978). For daphnids, reared under controlled trace-element exposure utilizing a controlled media system (Keating 1985), the absence of detectable zinc resulted in a shortening of life span and increased demand on the animal's pool of available selenium (Keating and Caffrey 1989). The marine shimp P. vannamei has been found to require 33 mg Zn/kg diet to maintain normal tissue mineralization (Davis 1990). Although minimal dietary levels have not been well established for shrimp the zinc requirement of red drum has been determined to be 20 mg/kg diet (?).

Practical diets contain feedstuffs that are relatively rich sources of zinc (e.g., fish meal); however, for fish the bioavailability of zinc in these feedstuffs is generally very low, making supplementation essential (Watanabe et al. 1988). The bioavailability of zinc in various fish meals has been found to be inversely related to the tricalcium phosphate content of the meal and is thus generally lowest in white fish meal, which contains the highest level of tricalcium phosphate, and slightly better in brown fish meals (Watanabe et al. 1988).

In addition to interactions with other minerals affecting the dietary requirement for zinc, practical diets often contain feedstuffs that are relatively high in phytate which may affect the bioavailability of zinc. This effect has been well documented in a variety of terrestrial animals (Oberleas et al. 1962; O'Dell et al. 1964; Savage et al. 1964; Lo et al. 1981) and fish (Gatlin and Wilson 1984c; Richardson et al. 1985; McClain and Gatlin 1988; Gatlin and Phillips 1989) and shrimp (Davis 1990).

FACTORS AFFECTING BIOAVAILABILITY OF MINERALS.

In order to met an animals physiological requirments for various minerals, dietary sources of these minerals must be available to the animal. Numerous factors affect the availability of minerals. The most soluble and consequently the most readily absorbed form is the simple ionic state of the atom or ionic group of atoms (e.g., Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, PO³). However, in nature compounds differing in electric charge bind with these minerals forming a stable compound that is less soluble in water. Although these compounds have low solubility in water, the acidic condition of the gastric stomach allows the dissociation into salts which can be easily absorbed by the intestine. Consequently, in animals without acidic digestive systems the bioavailability of minerals is generally inhibited.

Although the gastric stomach generally increases the availability of minerals, after solubilization in the stomach some minerals may interact after being released into the basic intestine and for insoluble precipitates. Excessive levels of calcium and phosphorus react with magnesium and zinc to form insoluble precipitates. Additionally, colloids such as particles of clay, insoluble salts of aluminum, magnesium, iron, and other element strongly absorb cations, and this absorption occurs both through chemical union with highly electronegative areas of the colloidal surface and through attraction of the cation by physical forces (Scott et al. 1982). Consequently, the bioavailability of a given mineral will be dependent upon the dissociation of the mineral as well as interactions with other dietary components.

Of the feed ingredients used in practical animal diets, fish meal is the richest source of endogenous minerals. Research on the bioavailability of minerals contained in fish meals has demonstrated that there is considerable variation in the bioavailability of these minerals to various species (probably due to luminal pH) and that the bioavailability of the minerals is also affected by meal type. Although fish meals are relatively rich in minerals and on a chemical basis should satisfy physiological requirements of fish, the low availability and inhibitory interactions require the supplementation of available sources of phosphorus, magnesium, zinc, manganese, and copper to prevent dietary deficiencies and maximize growth (Watanabe, et al. 1988). As the aquatic animal feed industry increases its use of less expensive plant protein sources, which are generlly poor sources of minerals and/or may contain factors that reduce the bioavilability of minerals, the need for mineral supplements should increase.

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Table 1. Dietary mineral requirements of fish.

¹ Fresh Water (F.W.). Salt Water (S.W.), Brackish Water (B.W.)

² Concentration in rearing water

³ Apparent available phosphorus

⁴ 1 & 99 mg Fe/kg diet tested

Table 2. Mineral requirements of Penaeid shrimp and lobstor.

¹ Level in the basal diet.

ANNEX N

School of Fisheries HF-15
University of Washington
Seattle, WA. 98195 USA
telex: 4740096 telephone: (206) 543–9619 FAX: (206) 685–3275

FINAL REPORT

ASTOS in Rainbow Trout in Seawater
July 1990--June 1991

ABSTRACT:

Rainbow Trout fingerlings were reared in freshwater at the Marrowstone Field Station, US Fish & Wildlife Service, Nordland, Washington until 30 to 50 grams in weight. Fish were then converted to a sea water environment over a 10 day gradual transition period. After transition into sea water the fish were converted to standard test diet H440 over a one week transition period. Duplicate lots of 200 trout were housed in 5 foot circular fiberglass tanks equipped with self cleaning central standpipes and cover, and were provided with 2 gallons of sea water per minute per tank. Six diet treatments were used: H440 base containing ASTOS (C₂) at 100 mg, 200 mg, or 300 mg per kg dry diet; STAY-C (C₃) at 200 mg/kg dry diet; L-ascorbic acid (C₁) at 0,100 mg/kg dry diet. Lots were randomly distributed throughout the wet laboratory. Tanks and diet containers were color coded. Research aids were not cognizant of diet treatments assigned to each tank. Fish were fed daily 0700–0900 hrs and 1600–1800 hrs for 274 days. Daily records of diet consumed, mortality, anomalies were recorded. Diets were formulated and manufactured at monthly intervals, then packed, sealed, frozen, and transported to the MFS site for use as needed. Fish were weighed at monthly intervals and populations reduced below 10 kg biomass per tank at each weigh period. Diet stability was measured indicating approximately 40% loss of C₁ after 45 days frozen storage, and little or no loss of C₂ or C₃ under similar storage conditions. No statistically significant differences in growth, FGR, PER, or ANPU were observed between diet treatments over 10X weight gain. Signs of early scurvy appeared in those lots fed diets devoid of Vitamin C. Tissue assays reflected normal growth and tissue storage levels for C₁ and C₂ in all lots receiving a dietary C supplement. ASTOS at levels used was a satisfactory Vitamin C source to rear Rainbow Trout from 70 to 700 grams under these experimental conditions.

ANNEX O