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1
Dietary Requirements
Feeds and feedstuffs contain nutrients and energy sources essential for fish growth, reproduction, and health. Deficiencies of these substances can reduce growth rates or lead to diseases, and, in some cases, excesses can cause a reduction in growth rate. Dietary requirements can be established for energy, protein and amino acids, lipids, minerals, and vitamins.
ENERGY
Energy is not a nutrient—it is released during metabolic oxidation of carbohydrates, fats, and amino acids. Absolute energy requirements of the animal can be quantified by measuring either oxygen consumption or heat production. However, estimates of dietary allowances must be determined by equating animal performance with feed materials in which the amount of available energy is accountable.
This section familiarizes the user with those aspects of nutritional energetics that deal with feed energy use by the animal, energy value of feedstuffs, and dietary energy requirements. Readers who require more detailed information on physiological energetics of fish may refer to the review of Brett and Groves (1979). Those who wish to read further on nutritional energetics, with an emphasis on determining dietary energy allowances for captive fish, should refer to the reviews of Smith (1989) and Cho and Kaushik (1990).
Partitioning of Dietary Energy
The energy of ingested feed is divided into many components in the animal's body. An illustration of energy flow in the animal with accepted abbreviations of energy metabolism terms (National Research Council, 1981) is shown in Figure 1-1. There are many places where energy is lost between intake and recovered products. Losses occur in feces, in urine and gill excretions, and as heat. Ideally, the fish feeder needs to minimize these losses and thereby obtain maximum return as useful products. The magnitude of these losses depends primarily on characteristics of the diet and the level of feeding. The difference between intake energy (IE) and digestible energy (DE) is energy lost in the feces (FE). The inclusion of fibrous materials that are poorly digested by fish will increase the FE loss. Metabolizable energy (ME) represents DE corrected for energy lost by
FIGURE 1-1 Schematic presentation of the fate of dietary energy for fish, categorizing the losses that occur as feed is digested and metabolized, leaving a fraction of the energy to be retained as new tissue. Source: Adapted from National Research Council. 1981. Nutritional Energetics of Domestic Animals and Glossary of Energy Terms. Washington, D.C.: National Academy Press.
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excretion through the gills (ZE) and urine (UE). The difference between ME and energy recovered as growth and/or reproductive products (RE) is energy lost as heat (HE). Heat loss occurs primarily by two processes: the heat increment of feeding (HiE) and maintenance heat loss (HEm).
The HiE is the increase in heat production subsequent to ingestion of feed. The factors contributing to HiE are the digestion and absorption processes (HdE), the transformation and interconversion of the substrates and their retention in tissues (HrE), and the formation and excretion of metabolic wastes (HwE). The main biochemical basis for HiE in mammals and birds is the energy required for the ingested amino nitrogen (N) to be deaminated and excreted (Kleiber, 1975); however, this represents less of an energy loss in fish because they can eliminate and products of protein metabolism (ammonia, bicarbonate, and carbon dioxide) without the need to synthesize urea, uric acid, or other similar compounds. Energy expenditures associated with diet ingestion and digestion are small compared with that associated with metabolic work (Brody, 1945). This conclusion has been reinforced by the observation that intravenous infusion of amino acids increases heat production to the same extent as does the oral administration of the amino acids (Benedict and Emmes, 1912; Borsook, 1936). HiE depends to a large extent on the balance of dietary nutrients and the plane of nutrition (Brody, 1945) and, in fish, the water temperature (Cho and Slinger, 1979). Thus, measurement of HiE for balanced feeds is more meaningful than measurement of the HiE of individual feed ingredients, because the metabolic fate of absorbed nutrients depends on the mixture absorbed and, hence, the variety of metabolic processes that are possible.
The HiE in fish is greater for diets with a high protein content than for diets with a low protein content (Cho, 1982). In mammals and birds, however, the effect of high dietary protein on heat increment is even more marked, partly because of the energy expenditure during synthesis of urea or uric acid from the deaminated nitrogen. The energy cost of synthesis for urea and uric acid is 3.1 and 2.4 kcal/g N, respectively (Martin and Blaxter, 1965). In contrast, ammonia is the primary nitrogenous waste product of protein catabolism in fish (Goldstein and Forster, 1970). Because this form of nitrogen can be readily released into the water, energy expenditure on urea or uric acid synthesis is not needed (Cowey, 1975). Cho et al. (1982) found that HiE for rainbow trout at 15°C was 5 to 15 percent of the gross energy consumed (IE) and fell as the ratio of protein to energy decreased. The HiE for livestock can be as much as 20 to 30 percent of the IE (Farrell, 1974; National Research Council, 1984). Thus, because of the lower heat increment of fish, the net energy (NE), which is the energy that is useful to the animal for maintenance and growth, in production diets is higher for fish than for warm-blooded animals.
Maintenance energy (HEm) is that required to maintain those functions of the body immediately essential to life. A major portion of this maintenance energy is spent for basal metabolism (HeE), such as respiration, transport of ions and metabolites, body constituent turnover, and circulation. A smaller portion is spent for voluntary or resting activity (HjE) and, in the case of homeothermic animals, thermoregulation of body temperature. Since fish do not regulate body temperature and they expend less energy in maintaining position in the water than do terrestrial animals in maintaining their posture, the HEm requirement of fish is lower than for homeotherms. The fasting heat production (HEf) is an approximation of the HEm. Cho and Kaushik (1990) measured oxygen consumption of fasting rainbow trout weighing 96 to 145 g at 15°C and calculated their HEf, in kcal/fish/day to be 8.85 W0.82 where W is body weight in kilograms. Smith (1989) reported an HEf value of 4.41 W0.63 for rainbow trout weighing 4 to 50 g at 15°C where fasting heat production was measured directly by placing the fish in a calorimeter. Brett and Groves (1979) recommended the exponent 0.8 for metabolic body size for fish. When these HEf values for fish are compared with 70 W0.75 for mammals and 83 W0.75 for birds (Brody, 1945), it is apparent that the fasting heat production of fish is much lower. The maintenance energy requirements of fish are one-tenth to one-twentieth of those of homeothermic animals of similar size in a thermoneutral environment (Brett, 1973). The lower maintenance requirement for fish means that the percentage of net energy that is not dissipated as heat but retained within the body as new tissue or recovered energy is greater.
Energy Value of Feedstuffs for Fish
The energy content of a diet depends on its chemical composition, with the mean values of heat of combustion of protein, lipid, and carbohydrate being 5.64, 9.44, and 4.11 kcal/g, respectively. However, the chemical makeup of the diet influences only its heat of combustion, or gross energy, and yields no information on whether the energy and nutrients are available to fish through the digestive process. Prior to formulating diets, therefore, it is necessary to know the bioavailability of the energy in the feedstuffs for the animal being fed.
Available energy values for feedstuffs for fish have been determined on a DE and ME basis. ME, where applicable, is a more exact measure of the energy value for a complete diet that becomes available for metabolism by the animal. Practically, ME offers little advantage over DE in evaluating useful energy in feedstuffs for fish because FE accounts for most of the excretory losses. Energy losses through ZE and UE by fish are smaller than nonfecal energy losses by mammals and birds, and they do not vary among feedstuffs as much as do FE losses. Furthermore, determining ME values with fish is difficult because of the need to force feed
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and restrain the fish in metabolism chambers with the aid of a collar for simultaneous collection of fecal, gill, and urinary excretions (Smith, 1976). DE values are generally easier to determine and the fish feed voluntarily (Page and Andrews, 1973; Cruz, 1975; Cho and Slinger, 1979; Takeuchi et al., 1979). However, the use of proper techniques is necessary to give reliable DE values for fish. The collection of feces without the leaching of nutrients is important in determining DE with fish. Early studies (Smith and Lovell, 1973; Windell et al., 1978) showed that improper collection of feces, such as allowing feces to remain in the fish tank too long, caused serious overestimation of digestion coefficients. Methods for determining DE and ME in fish are discussed in Chapter 4.
Both proteins and lipids are highly available energy sources for fish (Cruz, 1975; Smith, 1976; Popma, 1982). The value of carbohydrate as an energy source is variable among species. Nile tilapia (Popma, 1982) and channel catfish (Wilson and Poe, 1985), which are warm-water omnivorous species, digest over 70 percent of the gross energy in noncooked starch while rainbow trout, a cold-water carnivore, may digest less than 50 percent (Cho and Slinger, 1979). Cooking, as in extrusion processing of feeds, increases digestibility of starch for fish. Extrusion processed corn had a 38 percent higher DE for channel catfish than compression pelleted corn (Wilson and Poe, 1985) and gelatinized starch had a 75 percent higher DE for rainbow trout than raw starch (Cho and Slinger, 1979).
Energy Requirements
Energy intake is a basic nutritional requirement because maintenance of life processes takes priority over growth and other functions. Thus, energy concentration should be the
TABLE 1-1 Optimum Protein: Energy Ratio for Different Fish
Species
Digestible Protein (DP) (%)
Digestible Energy (DE) (kcal/g)
Final DP/DE (mg/kcal)
Weight (g)
Response Criteria
References
Channel catfish
22.2
2.33
95
526
Weight gain
Page and Andrews (1973)
28.8a
3.07a
94
34
Weight gain
Garling and Wilson (1976)
27.0
2.78
97
10
Protein gain
Mangalik (1986)
27.0
3.14
86
266
Protein gain
Mangalik (1986)
24.4a
3.05a
81
600
Weight gain
Li and Lovell (1992)
Red drum
31.5a
3.20a
98
43
Weight gain
Daniels and Robinson, (1986)
Hybrid bass
31.5a
2.80
112
35
Weight gain
Nematipour et al. (1992)
Nile tilapia
30
2.90
103
50
Weight gain
El-Sayed (1987)
Common carp
31.5a
2.90a
108
20
Weight gain
Takeuchi et al. (1979)
Rainbow trout
33
3.6
92
90
Weight gain
Cho and Kaushik (1985)
42
4.10
105
94
Weight gain
Cho and Woodward (1989)
a Digestible protein and energy were estimated from ingredient composition of the diet.
first nutritional consideration in diet formulation for fish. In practice, however, protein is usually given first priority because it is more expensive than other energy yielding components. Protein and energy should be kept in balance. A dietary deficiency or an excess of DE can reduce growth rates of fish. A diet deficient in energy in relation to protein will mean that protein is used for energy to satisfy maintenance before growth. In contrast, a diet containing excess energy can reduce feed consumption and thus lower the intake of the necessary amount of protein and other essential nutrients for maximum growth. Excessively high ratios of energy to nutrients can also lead to deposition of large amounts of body fat, which can be undesirable in food fish.
Ratios of digestible protein to DE (mg/kcal) for maximum weight gain for several fish species have been measured in growth studies (Table 1-1). Values range from 81 mg/kcal to 117 mg/kcal and are substantially higher than protein-energy ratios for swine and poultry, which range from 40 to 60 mg/kcal (National Research Council, 1984, 1988). The reason the protein-energy ratio for fish is higher than that for farm animals is not because fish have a higher protein requirement (fish convert dietary protein into tissue protein about as efficiently as warm-blooded animals [Smith, 1989]) but because fish require less energy for maintenance and the synthesis of uric acid.
Since lipid is the primary nonprotein energy source in salmonid diets, the protein-energy allowance for these diets is sometimes reported as the ratio of protein to lipid. The optimum combination for weight gain for rainbow trout was 35 to 36 percent protein and 15 to 16 percent lipid (Watanabe et al., 1979; Cho, 1982).
Empirical calculation of energy requirements of fish based on energy losses and expected energy recovery are possible with reliable information on energy balances in the
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animal under a given set of conditions. Energy balances for rainbow trout have been established under laboratory conditions, as discussed previously. Cho and Kaushik (1990) constructed a model for calculating the DE required to grow 1 kg of rainbow trout, from 1 g to 100 g size at 15°C, based on derived heat and excretory losses and estimated recovery of energy in the fish. The model indicated that 3.56 Mcal of DE would be required to produce 1.91 Mcal of recovered energy in 1 kg of fish biomass with an RE:DE efficiency ratio of 0.54, which is comparable to a value of 0.56 reported for channel catfish (Gatlin et al., 1986). However, several factors significantly affect energy balance in fish, such as diet composition, feeding rate, and composition of body gain. Therefore, this approach to calculating energy requirements for production diets must be used cautiously until sufficient information is available to establish reliable energy budgets for a variety of production conditions for a specific aquaculture species.
PROTIEN AND AMINO ACIDS
Proteins are composed of up to 20 α-amino acids linked into chains by peptide bonds. The chains are cross-linked by disulfide bridges, hydrogen bonds, and van der Waals forces. The amino acid content of proteins, particularly feed proteins, may differ markedly. Some, such as gelatin (a mixture of proteins derived from collagen) or zein (a protein from maize gluten), are largely, or even entirely, deficient in one or more amino acids. Others, such as fishmeal, have a balance of amino acids that more closely meets the requirements of fish. Consequently the capacity of different feed proteins to meet the amino acid needs of the fish will differ considerably. Ingested protein is hydrolyzed to free amino acids, dipeptides, and tripeptides by digestive enzymes secreted into the gastrointestinal tract. These products are absorbed by the mucosal cells where intracellular digestion of small peptides occurs; thus only amino acids appear to be released into the portal vein as products of protein digestion (Murai et al., 1987). Some evidence has shown that small amounts of certain whole proteins may be absorbed through the wall of the gastrointestinal tract, but the quantities involved have not been confirmed as being of any quantitative significance (Ash, 1985).
In the context of animal feeding, protein generally refers to crude protein (CP); that is, N × 6.25, a definition based on the assumption that proteins contain 16 percent N. The requirement for dietary protein has two components:
a need for indispensable amino acids that the fish cannot synthesize either at all or at a rate commensurate with its need for protein deposition or commensurate with the synthesis of a variety of other compounds with metabolic functions and
a supply of either dispensable amino acids or sufficient amino nitrogen to enable the fish to synthesize them.
Insofar as synthesis of dispensable amino acids requires expenditure of energy, feeding dietary proteins that most nearly meet the needs of fish for both indispensable and dispensable amino acids will result in the most efficient growth by the fish. Thus, the concept of balance or pattern of amino acids is basic to protein requirement.
Protein Requirements
The protein requirements, meaning the minimum amount needed to meet requirements for amino acids and to achieve maximum growth, have now been measured in juvenile fish of many species (see Tables 1-2 to 1-13). They have been obtained mainly from dose-response curves in which graded amounts of high-quality protein were fed in partially defined diets. The response measured was weight gain. The values are expressed as a percentage of dry diet. Although the expression of protein as a proportion of dietary energy would have focused attention on protein as a substantial source of dietary energy, this approach was not possible for many of the data because in formation on the DE content of the diets was unavailable and values used for the energy density of dietary components varied between authors.
The protein allowances in fish diets are appreciably higher than those in the diets of terrestrial warm-blooded animals. The methods used to determine protein requirements, however, may overestimate requirements, in that excess dietary protein or amino acids, which cannot be stored, are catabolized preferentially over carbohydrates and fats and used for energy by some fishes (Wilson, 1989). In addition, adequate consideration has not always been given to factors such as concentration of DE in the diet, amino acid composition of the dietary protein, and digestibility of the dietary protein (Wilson and Halver, 1986; Wilson, 1989). Understanding the nutritional constraints and limitations used in arriving at these reported protein requirements is important for their proper application.
Protein requirements, as a proportion of the diet, decrease as fish approach maturity. For example, 25 percent protein was adequate in the diet of channel catfish of 114 to 500 g, but 35 percent protein produced faster gains than did 25 percent protein in 14- to 100-g fish (Page and Andrews, 1973). Somewhat similar results have been obtained with salmonids, common carp, and tilapia (Wilson and Halver, 1986).
Little convincing evidence exists to show that protein requirement, expressed as a percentage of dry matter, is affected by water temperature. In general, all feeding and growth functions increase in parallel as water temperature rises, although growth rate may increase more rapidly because of an increased feed conversion efficiency coupled
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TABLE 1-2 Estimated Dietary Protein Requirement for Maximal Growth of Some Species of Juvenile Fish (As Fed Basis)
Species
Protein Source
Estimated Protein Requirement (%)
Reference
Atlantic salmon
Casein and gelatin
45
Lall and Bishop (1977)
Channel catfish
Whole egg protein
32–36
Garling and Wilson (1976)
Chinook salmon
Casein, gelatin, and amino acids
40
DeLong et al. (1958)
Coho salmon
Casein
40
Zeitoun et al. (1974)
Common carp
Casein
31–38
Ogino and Saito (1970); Takeuchi et al. (1979)
Estuary grouper
Tuna muscle meal
40–50
Teng et al. (1978)
Gilthead sea bream
Casein, fish protein concentrate, and amino acids
40
Sabaut and Luquet (1973)
Grass carp
Casein
41–43
Dabrowski (1977)
Japanese eel
Casein and amino acids
44.5
Nose and Arai (1972)
Largemouth bass
Casein and fish protein concentrate
40
Anderson et al. (1981)
Milkfish
Casein
40
Lim et al. (1979)
Plaice
Cod muscle
50
Cowey et al. (1972)
Puffer fish
Casein
50
Kanazawa et al. (1980)
Rainbow trout
Fishmeal, casein, gelatin, and amino acids
40
Satia (1974)
Red sea bream
Casein
55
Yone (1976)
Smallmouth bass
Casein and fish protein concentrate
45
Anderson et al. (1981)
Snakehead
Fishmeal
52
Wee and Tacon (1982)
Sockeye salmon
Casein, gelatin, and amino acids
45
Halver et al. (1964)
Striped bass
Fishmeal and soy proteinate
47
Millikin (1983)
Blue tilapia
Casein and egg albumin
34
Winfree and Stickney (1981)
Mossambique tilapia
White fishmeal
40
Jauncey (1982)
Nile tilapia
Casein
30
Wang et al. (1985)
Zillii's tilapia
Casein
35
Mazid et al. (1979)
Yellowtail
Sand eel and fishmeal
55
Takeda et al. (1975)
with a higher intake per meal (Brett, 1979). Protein requirement for rainbow trout was unchanged from 35 percent (in diets containing 3,580 kcal DE/kg) at water temperatures ranging from 9° to 18°C (see Figure 5 in National Research Council, 1981).
The high concentrations of dietary protein necessary
TABLE 1-3 Amino Acid Requirements of Juvenile Chinook Salmon
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
40
6.0
2.4
Chemically defined
Klein and Halver (1970)
Histidine
40
1.8
0.7
Chemically defined
Klein and Halver (1970)
Isoleucine
41
2.2
0.9
Chemically defined
Chance et al. (1964)
Leucine
41
3.9
1.6
Chemically defined
Chance et al. (1964)
Lysine
40
5.0
2.0
Purified
Halver et al. (1958)
Methioninea
40
4.0
1.6
Chemically defined
Halver et al. (1959)
Phenylalanineb
41
5.1
2.1
Chemically defined
Chance et al. (1964)
Threonine
40
2.2
0.9
Chemically defined
DeLong et al. (1962)
Tryptophan
40
0.5
0.2
Chemically defined
Halver (1965)
Valine
40
3.2
1.3
Chemically defined
Chance et al. (1964)
a Diet contained 1.0 percent cystine.
b Diet contained 0.4 percent tyrosine.
for maximal growth rates of fish do not mean that they use more protein as an energy source than is the case with homeothermic vertebrates. Values for net protein retention are in the range of 20 to 50 percent for both types of vertebrate; Bowen (1987) summarized a number of data that showed a median value for fish of 31 percent and for other
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TABLE 1-4 Amino Acid Requirements of Juvenile Common Carp
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
38.5
4.3
1.6
Chemically defined
Nose (1979)
Histidine
38.5
2.1
0.8
Chemically defined
Nose (1979)
Isoleucine
38.5
2.5
0.9
Chemically defined
Nose (1979)
Leucine
38.5
3.3
1.3
Chemically defined
Nose (1979)
Lysine
38.5
5.7
2.2
Chemically defined
Nose (1979)
Methioninea
38.5
3.1
1.2
Chemically defined
Nose (1979)
Phenylalanineb
38.5
6.5
2.5
Chemically defined
Nose (1979)
Threonine
38.5
3.9
1.5
Chemically defined
Nose (1979)
Tryptophan
38.5
0.8
0.3
Chemically defined
Nose (1979)
42
0.3
0.1
Purified
Dabrowski (1981)
Valine
38.5
3.6
1.4
Chemically defined
Nose (1979)
a In the absence of dietary cystine.
b In the absence of tyrosine, with 1 percent tyrosine in the diet phenylalanine requirement was 3.4 percent of protein or 1.3 percent of dry matter.
vertebrates of 29 percent. Broadly similar proportions of dietary protein are therefore used as an energy source in fish as in warm-blooded terrestrial vertebrates; this, notwithstanding the fact that fish have lower presumed energy requirements than do homeotherms. Attempts have been made to compare absolute protein intake rates (mg protein ingested/g body weight/day); this is a difficult undertaking both because accurate measurement of feed intake by fish is in itself difficult and because the use of data for fish of different physiological ages, held under different conditions of temperature and photoperiod, introduces considerable variation.
TABLE 1-5 Amino Acid Requirements of Juvenile Channel Catfish
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
24
4.3
1.0
Chemically defined
Robinson et al. (1981)
Histidine
24
1.5
0.4
Chemically defined
Wilson et al. (1980)
Isoleucine
24
2.6
0.6
Chemically defined
Wilson et al. (1980)
Leucine
24
3.5
0.8
Chemically defined
Wilson et al. (1980)
Lysine
24
5.1
1.2
Chemically defined
Wilson et al. (1977)
30
5.0
1.5
Chemically defined
Robinson et al. (1980b)
Methioninea
24
2.3
0.6
Chemically defined
Harding et al. (1977)
Phenylalanineb
24
5.0
1.2
Chemically defined
Robinson et al. (1980a)
Threonine
24
2.0
0.5
Chemically defined
Wilson et al. (1978)
Tryptophan
24
0.5
0.12
Chemically defined
Wilson et al. (1978)
Valine
24
3.0
0.71
Chemically defined
Wilson et al. (1980)
a In the absence of dietary cystine.
b Diet contained 0.3 percent tyrosine. With 0.6 percent tyrosine in the diet, phenylalanine requirement was 2.0 percent of protein or 0.5 percent of dry matter.
Amino Acid Requirements
An absolute requirement for 10 amino acids (arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine) has been demonstrated in all fish species examined so far. Quantification of essential amino acid requirements has relied largely on dose-response curves in which the response measured has been weight gain. Various types of chemically defined, purified, and natural ingredient diets have been used to provide graded increments of the amino acid under test. Most studies have used test diets in which the nitrogen component consisted of either amino acids or a mixture of amino acids,
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TABLE 1-6 Amino Acid Requirements of Juvenile Japanese Eel
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
38
4.5
1.7
Chemically defined
Nose (1979)
Histidine
38
2.1
0.8
Chemically defined
Nose (1979)
Isoleucine
38
4.0
1.5
Chemically defined
Nose (1979)
Leucine
38
5.3
2.0
Chemically defined
Nose (1979)
Lysine
38
5.3
2.0
Chemically defined
Nose (1979)
Methioninea
38
3.2
1.2
Chemically defined
Nose (1979)
Phenylalanineb
38
5.8
2.2
Chemically defined
Nose (1979)
Threonine
38
4.0
1.5
Chemically defined
Nose (1979)
Tryptophan
38
1.1
0.4
Chemically defined
Nose (1979)
Valine
38
4.0
1.5
Chemically defined
Nose (1979)
a In the absence of dietary cystine.
b In the absence of tyrosine, with 2.0 percent tyrosine in the diet, phenylalanine requirement was 3.2 percent of protein or 1.2 percent of dry matter.
casein, and gelatin formulated to provide an indispensable amino acid composition identical with some reference protein (such as whole hen's egg protein or fish body protein) minus the amino acid under test. For many fish species, growth rates produced by diets with large amounts of free amino acids are inferior to diets of similar amino acid composition in which the nitrogen component is protein (Wilson et al., 1978; Robinson et al., 1981; Walton et al., 1982, 1986). Thus amino acid requirements obtained in this way are based on growth rates below the optimum.
Other approaches to quantifying indispensable amino acid requirements have included using proteins with poor amino acid patterns that differ substantially from that required, such as zein (Dabrowski, 1981) or maize gluten (Halver et al., 1958; Ketola, 1983). Comparatively small amounts of crystalline amino acids are then added to balance
TABLE 1-7 Amino Acid Requirements of Juvenile Nile Tilapia
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
28
4.20
1.18
Chemically defined
Santiago and Lovell (1988)
Histidine
28
1.72
0.48
Chemically defined
Santiago and Lovell (1988)
Isoleucine
28
3.11
0.87
Chemically defined
Santiago and Lovell (1988)
Leucine
28
3.39
0.95
Chemically defined
Santiago and Lovell (1988)
Lysine
28
5.12
1.43
Chemically defined
Santiago and Lovell (1988)
Methioninea
28
2.68
0.75
Chemically defined
Santiago and Lovell (1988)
Phenylalanineb
28
3.75
1.05
Chemically defined
Santiago and Lovell (1988)
Threonine
28
3.75
1.05
Chemically defined
Santiago and Lovell (1988)
Tryptophan
28
1.00
0.28
Chemically defined
Santiago and Lovell (1988)
Valine
28
2.80
0.78
Chemically defined
Santiago and Lovell (1988)
a Cystine 0.54 percent of dietary protein, 0.15 percent of dry diet.
b Tyrosine 1.79 percent of dietary protein, 0.5 percent of dry diet.
the protein component, leaving it deficient only in one amino acid. Concerns about this approach center on protein digestibility, amino acid availability and rate of transit, and absorption of supplemented free amino acids compared with those from dietary protein. In addition, imbalanced proteins may have high percentages of certain amino acids, such as leucine, and these may depress the assimilation of other amino acids.
Ogino (1980) measured the retention of indispensable amino acids in the whole body protein of carp and rainbow trout and used the increase in indispensable amino acid content measured over periods of 14 to 28 days to estimate requirements. This method assumes that the maintenance requirements of young growing fish are low (although it is not easy to reconcile this view with the fact that only 30 to 40 percent of dietary nitrogen is retained by growing fish), so
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TABLE 1-8 Amino Acid Requirements of Juvenile Rainbow Trout
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
36
3.3
1.2
Purified
Kaushik (1979)
45
3.6
1.6
Purified
Walton et al. (1986)
35
4.0
1.4
Chemically defined
Kim et al. (1983)
33
4.7
1.6
Purified
Cho et al. (1989)
47
5.9
2.8
Purified
Ketola (1983)
Lysine
35
3.7
1.3
Chemically defined
Kim and Kayes (1982)
45
4.2
1.9
Purified
Walton et al. (1984a)
47
6.1
2.9
Purified
Ketola (1983)
Methionine
46.4
2.2a
1.0a
Chemically defined
Walton et al. (1982)
35
3.0b
1.1b
Chemically defined
Rumsey et al. (1983)
35
2.9c
1.0c
Chemically defined
Kim et al. (1984)
35
1.4
0.5
Chemically defined
Kim et al. (1992)
41
1.5
0.6d
Purified
Cowey et al. (1992)
Tryptophan
55
0.5
0.3
Purified
Walton et al. (1984b)
35
0.6
0.2
Chemically defined
Kim et al. (1987)
42
1.4
0.6
Chemically defined
Poston and Rumsey (1983)
a Diet lacked cystine.
b Diet contained 0.3 percent cystine.
c Diet contained 0.5 percent cystine.
d Diet contained 0.16 percent cystine.
that the pattern of amino acids deposited in body weight gain is the main determinant of patterns of amino acids required.
Relationship of Amino Acid Requirements to Protein Intake
In warm-blooded animals, a constant relationship was shown between indispensable amino acid requirements and protein intake up to the level of protein required for maximum growth (Almquist, 1972). For several indispensable amino acids, intake and weight gain were apparently linearly related and this relationship was presumed to hold for all indispensable amino acids. On this basis amino acid requirements of fish were expressed as a percentage of dietary protein as well as on a dry matter basis (National Research Council, 1981, 1983).
Later studies bear on the finding of Almquist (1972) in
TABLE 1-9 Amino Acid Requirements of Juvenile Coho Salmon
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
40
5.8
2.3
Chemically defined
Klein and Halver (1970)
Histidine
40
1.8
0.7
Chemically defined
Klein and Halver (1970)
Tryptophan
40
0.5
0.2
Chemically defined
Halver (1965)
that they show the relationship to be not a linear but an exponential function (Finke et al., 1987). The response of an animal to dietary increments of a limiting nutrient does not break at one particular point. An accurate representation of the so-called ''diminishing returns" area of the response curve is claimed (Finke et al., 1989) to be critical in assessing the efficiency of incremental increases of dietary amino acid concentration as the response approaches the maximum The use of a logistic model supports a more accurate assessment, than that provided by broken-line analysis, of the diminishing returns area of the response curve and of the maximum response (Finke et al., 1989).
The implication of these later studies is that indispensable amino acid requirements are not best expressed as a percentage of dietary protein. Nevertheless, because the dose-response relationship is, for all practical purposes, linear for much of its length (Gahl et al., 1991), amino acid
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TABLE 1-10 Amino Acid Requirements of Juvenile Chum Salmon
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
40
6.0
2.6
Chemically defined
Akiyama (1987)
Histidine
40
1.6
0.7
Chemically defined
Akiyama et al. (1985)
Isoleucine
40
2.4
1.0
Chemically defined
Akiyama (1987)
Leucine
40
3.8
1.5
Chemically defined
Akiyama (1987)
Lysine
40
4.8
1.9
Chemically defined
Akiyama et al.(1985)
Methionine + cystine
40
3.0
1.2
Chemically defined
Akiyama (1987)
Phenylalanine + tyrosine
40
6.3
2.5
Chemically defined
Akiyama (1987)
Threonine
40
3.0
1.2
Chemically defined
Akiyama et al.(1985)
Tryptophan
40
0.7
0.3
Chemically defined
Akiyama (1987)
Valine
40
3.0
1.2
Chemically defined
Akiyama (1987)
requirements in Tables 1-3 to 1-13 have again been expressed both as a percentage of dietary protein and on a dry matter basis.
Diets in which the nitrogen component is made up of casein, gelatin, and crystalline amino acids have been referred to in the tables as chemically defined diets. Purified diets are those in which proteins, with an amino acid pattern (g amino acid/16 g nitrogen) that differs substantially from that required, supply the bulk of the nitrogen together with some supplementary amino acids. Natural ingredient diets use normal feed ingredients such as fishmeal, soya meal, blood meal, and wheat middlings.
The values in Tables 1-3 to 1-13 suggest that large differences exist among fish species in their requirements for certain amino acids. Where several estimates are available for one amino acid in a single species, as in the case of rainbow trout (Table 1-8), marked discrepancies occur. Some of these may be due to differences in growth rate, amino acid sources, feed intake, and other aspects of methodology.
Pathologies Resulting from Deficiencies
For most indispensable amino acids, deficiency is manifest as a reduction in weight gain. In certain species of fish, however, a deficiency of methionine or tryptophan leads to
TABLE 1-11 Amino Acid Requirements of Juvenile Mossambique Tilapia
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
40
4
1.6
Natural ingredient
Jackson and Capper (1982)
Lysine
40
4.1
1.6
Natural ingredient
Jackson and Capper (1982)
Methioninea
40
40
1.3
Natural ingredient
Jackson and Capper (1982)
a Diet contained 0.7 percent cystine.
pathologies, because these amino acids are not only incorporated into proteins but also used for the synthesis of other essential compounds.
Salmonids, including rainbow trout, Atlantic salmon (Salmo salar), and lake trout (Salvelinus namaycush), suffer from cataracts when given a diet deficient in methionine (Poston et al., 1977). The lens begins to become opaque after 2 to 3 months, depending on the extent to which the fish are deficient in sulfur amino acids. As the deficiency increases, lens opacity gradually progresses, causing a large reduction in light transmission. Cataracts also occur as a consequence of tryptophan deficiency in rainbow trout (Poston and Rumsey, 1983; Walton et al., 1984b); the developmental pattern of the cataracts is similar to that occurring in methionine deficiency (Poston and Rumsey, 1983).
Tryptophan deficiency leads to scoliosis (lateral curvature of the vertebral column) and to a derangement of mineral metabolism in certain salmonids, including rainbow trout (Walton et al., 1984b), sockeye salmon (Oncorhynchus nerka) (Halver and Shanks, 1960), and chum salmon (Oncorhynchus keta) (Akiyama et al., 1986). Scoliosis in chum salmon may be reversed by restoring tryptophan to normal concentrations in the diet. The condition may be related to a decline in levels of the brain neurotransmitter serotonin, which is formed from tryptophan. Thus, inclusion of serotonin
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TABLE 1-12 Amino Acid Requirements of Juvenile Gilthead Sea Bream
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Arginine
34
5.0
1.7
Purified
Luquet and Sabaut (1974)
Lysine
34
5.0
1.7
Purified
Luquet and Sabaut (1974)
Methioninea
34
4.0
1.4
Purified
Luquet and Sabaut (1974)
Tryptophan
34
0.6
0.2
Purified
Luquet and Sabaut (1974)
a Cystine content of diet not stated.
in tryptophan-deficient diets greatly reduces the incidence of scoliosis (Akiyama et al., 1986).
Changes in mineral metabolism were observed in tryptophan-deficient rainbow trout (Walton et al., 1984b). Significantly greater concentrations of calcium (Ca) (a fourfold increase over control trout), sodium (Na), and potassium (K) were found in the kidneys of tryptophan-deficient trout. Concentrations of Ca, magnesium (Mg), Na, and K in the livers of tryptophan-deficient trout were also significantly greater than in normal trout. The metabolic lesion(s) responsible for these changes have not been resolved.
Relationships Among Amino Acids
Cystine can be formed metabolically from dietary methionine at a rate sufficient to meet the requirements of fish. The reverse sequence of reactions does not occur, however, and fish have an absolute requirement for methionine. Methionine can thus meet the total sulfur amino acid requirement of fish, although some of this requirement may be met by cystine.
Rainbow trout can use D-methionine to replace L-methionine on an equimolar basis (Kim et al., 1992). D-methionine is deaminated by D-amino acid oxidase and subsequently reaminated to L-methionine. This metabolic capacity is probably also characteristic of other fish.
A similar relationship exists between aromatic amino acids. Fish readily convert phenylalanine to tyrosine so that phenylalanine alone can meet requirements for aromatic
TABLE 1-13 Amino Acid Requirements of Juvenile Lake Trout
Amino Acid
Protein in Diet (%)
Requirement as Percentage of Dietary Protein
Requirement as Percentage of Dry Diet
Type of Diet
Reference
Isoleucine
27
2.0-2.6
0.5-0.7
Purified
Hughes et al. (1983)
Leucine
27
3.5-4.6
1.0-1.3
Purified
Hughes et al. (1983)
Valine
27
2.6-3.3
0.6-0.8
Purified
Hughes et al. (1983)
amino acids. However, the presence of tyrosine in the diet will reduce some of the requirement for phenylalanine.
Some adverse interactions may occur between amino acids that are structurally related when their concentrations in the diet are imbalanced. Well-known examples in homeotherms are antagonisms arising from dietary imbalances of lysine-arginine and of leucine-valine. No convincing evidence exists, however, for lysine-arginine antagonism in fish. Robinson et al. (1981) could not demonstrate any effects when diets with excess lysine in the presence of adequate or marginal arginine were fed to channel catfish; diets containing excess arginine in the presence of adequate or marginal lysine similarly failed to show any antagonistic effect. Nor did excess lysine affect the growth rates of rainbow trout fed low concentrations of arginine (Kim et al., 1983).
Antagonism between branched-chain amino acids generally arises in mammals from an excess of leucine over isoleucine and valine; the first two steps of the catabolic breakdown of all three branched-chain amino acids are catalyzed by the same enzymes. Data on antagonisms among branched-chain amino acids in fish are not clear-cut and are inconsistent between species. Thus the isoleucine requirement of chinook salmon (Oncorhynchus tshawytscha) increased slightly with increasing concentrations of dietary leucine (Chance et al., 1964). Hughes et al. (1983) observed changes in concentrations of branched-chain amino acids in lake trout given diets containing increasing amounts of valine. Plasma isoleucine and leucine were both elevated in valine-deficient fish, and their concentrations decreased as
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dietary valine was increased. No changes in plasma valine concentration occurred until valine in the diet reached the required concentration, after which the plasma valine increased about 2.5-fold. In contrast, rainbow trout showed a high tolerance for dietary leucine; no growth depression occurred with concentrations as high as 9.2 percent. Even with excessive dietary leucine concentrations (13.4 percent), which were overtly toxic, the concentrations of free valine and isoleucine in plasma, liver, and muscle were not depressed (Choo, 1990).
Another interaction characteristic of some homeotherms, and referred to as an imbalance, occurs when diets are supplemented with the second most limiting amino acid, or with all indispensable amino acids other than the first limiting amino acid. This leads to a fall in the concentration of the first limiting amino acid in the blood and eventually to reduced feed intake even though retention of the first limiting amino acid is not affected. No data are available on such interactions in fish. Nevertheless, oversupplementation with the second most limiting amino acid should be avoided as it may exacerbate a primary deficiency. There is considerable information available on amino acid interrelationships in mammals. Further information on these relationships can be found in Czarnecki et al. (1985), Baker (1987), and May et al. (1991).
LIPIDS
Dietary lipids are important sources of energy and of essential fatty acids (EFA) that are needed for normal growth and development. They also assist in the absorption of fat-soluble vitamins. Dietary lipids, mainly in the form of triacylglycerols, are hydrolyzed by digestive enzymes to a mixture of free fatty acids and 2-monoglycerides. These compounds are then absorbed and either used for the synthesis of various cellular components or catabolized for energy.
Dietary lipids contain both saturated and unsaturated fatty acids. Fatty acids may be designated by numbering either from the methyl or carboxyl terminal. The notation from the methyl terminal is most convenient for many nutritional purposes and is used here. It involves three numbers given in sequence, the first denoting the number of carbon atoms; the second, following a colon, the number of double bonds; and the third, designated as (n-) indicates the number of carbon atoms between the methyl terminal and the first double bond. The term polyunsaturated fatty acid (PUFA) normally refers to fatty acids with 18 or more carbon atoms and two or more double bonds.
Essential Fatty Acids
In common with other vertebrates, fish cannot synthesize either 18:2(n-6) or 18:3(n-3) de novo. Hence one or both of these fatty acids must be supplied preformed in the diet, depending on the EFA requirements. In addition, fish vary considerably in their ability to convert 18-carbon unsaturated fatty acids to longer-chain, more highly unsaturated fatty acids of the same series (Owen et al., 1975). The EFA requirement of the fish is thus related, to some extent, to their ability to modify these fatty acids metabolically.
The quantitative EFA requirements of several fish species are summarized in Table 1-14. A major difference appears to exist between freshwater and stenohaline marine fish (those unable to withstand a wide variation in water salinity). In general, freshwater fish require either dietary linoleic acid, 18:2(n-6), or linolenic acid, 18:3(n-3), or both, whereas stenohaline marine fish require dietary eicosapentaenoic acid (EPA), 20:5(n-3), and/or docosahexaenoic acid (DHA), 22:6(n-3).
Among the freshwater species, the ayu, channel catfish, coho salmon, and rainbow trout require 18:3(n-3) or EPA and/or DHA. Chum salmon, common carp, and Japanese eel require an equal mixture of 18:2(n-6) and 18:3(n-3); whereas, Nile tilapia and Zillii's tilapia require only 18:2(n-6) for maximum growth and feed efficiency. Striped bass, however, require n-3 PUFA and cannot chain elongate 18:3(n-3) (Webster, 1989; Webster and Lovell, 1990).
The principal gross signs of EFA deficiency reported for various fishes are dermal signs (fin rot), a shock syndrome, myocarditis, reduced growth rate, reduced feed efficiency, and increased mortality (Castell et al., 1972; Takeuchi and Watanabe, 1977a,b; Takeuchi et al., 1980; Satoh et al., 1989). Essential fatty acid deficiency has also been shown to reduce the reproductive performance of common carp (Shimma et al., 1977), rainbow trout (Watanabe, 1982; Watanabe et al., 1984c; Leray et al., 1985) and red sea bream (Watanabe et al., 1984a,b).
In fish species that can further desaturate and chain elongate 18:2(n-6) or 18:3(n-3), an absence of either of these fatty acids in the diet leads to the desaturation and chain elongation of oleic acid, 18:1(n-9), to 20:3(n-9), which is characteristic of an EFA deficiency in many terrestrial animals. Thus when EFAs are deficient, increased concentrations of 20:3(n-9) are incorporated into tissue polar lipids in place of 20:4(n-6), 20:5(n-3), or 22:6(n-3). Castell et al. (1972) suggested that the ratio of 20:3(n-9)/20:5(n-3) in polar lipids from the liver of rainbow trout might be a useful index of EFA status. By analogy with mammals, the diet is considered satisfactory with respect to EFA if this ratio is not greater than 0.4.
Watanabe et al. (1983) have reported that n-3 PUFAs such as EPA and DHA, are required for normal growth and development of ayu and red sea bream larvae. High mortalities and abnormalities, such as underdeveloped swim bladder and scoliosis, have been observed in red sea bream larvae reared on rotifers and Artemia spp., either devoid of n-3 PUFAs or containing only low concentrations of n-3 PUFAs
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have been identified in several other species. The requirements are affected by size, age, and growth rates as well as by various environmental factors and nutrient interrelationships. Thus, different researchers have reported fairly wide ranges in requirement values for growth in the same species (see Table 1-15). Recent studies with spring chinook salmon indicate that the dietary requirements for certain vitamins may be lower than previously reported for this species (Leith et al., 1990). In addition, the requirement values listed in Table 1-15, as determined by maximum liver storage or based on certain enzyme data, are often much higher than the requirement values based on weight gain and absence of deficiency signs; therefore, professional judgment must be used in selecting which requirement value best fits the user's needs. Thus, more studies are needed to refine the requirements for various species for normal growth, health, and enhancement of defense mechanisms, as suggested by Ikeda (1985). A summary of vitamin deficiency signs reported in several cultured fishes are presented in Appendix Table A-3. Further information on vitamin nutrition research in fishes is discussed by Halver (1989).
Fat-Soluble Vitamins
The fat-soluble vitamins, A, D, E, and K, are absorbed in the intestine along with dietary fats; therefore, conditions favorable for fat absorption also enhance the absorption of fat-soluble vitamins. Fat-soluble vitamins are stored by animals if dietary intake exceeds metabolic needs. Thus, animals can accumulate enough fat-soluble vitamins in their tissues to produce a toxic condition (hypervitaminosis). This has been demonstrated in the laboratory with trout for vitamins A, D, and E, but it is unlikely to occur under practical conditions (Poston et al., 1966; Poston, 1969a; Poston and Livingston, 1969).
Since fat-soluble vitamins can be stored in the body, the nutritional history of experimental fish prior to their use in requirement studies becomes critical. The time required to deplete fish of their stored fat-soluble vitamins is highly variable. Differences in vitamin intake prior to an experiment may be responsible for some of the conflicting findings on the induction and severity of deficiency signs.
VITAMIN A
Vitamin A is required in vertebrates for the regeneration of the light-sensitive compound rhodopsin in the retina of the eye. Vitamin A has also been shown to be essential for proper growth, reproduction, resistance to infection, and the maintenance of differentiated epithelia and mucus secretions. Blomhoff et al. (1992) have presented a recent review of metabolic functions of vitamin A in vertebrates.
Vitamin A occurs in three forms: as an alcohol (retinol), an aldehyde (retinal), and an acid (retinoic acid). Vitamin A1 (retinol) is found in mammals and marine fishes, whereas both vitamin A1 and vitamin A2 (3-dehydroretinol) are found in freshwater fishes (Braekkan et al., 1969; Lee, 1987). In freshwater fish, the oxidative conversion of retinol to 3-dehydroretinol occurs (Goswami, 1984) as well as the reversible oxidation and reduction reactions of retinol to retinal and of 3-dehydroretinol to 3-dehydroretinal (Wald, 1945-1946). For example, tilapia has been shown to convert dietary retinol into 3-dehydroretinol and retinal into 3-dehydroretinal (Katsuyama and Matsuno, 1988). Channel catfish were found to convert β-carotene to vitamin A1 and A2 in about a 1:1 ratio (Lee, 1987).
Cold-water fish can use β-carotene as a vitamin A precursor (Poston et al., 1977). Dupree (1970) found that channel catfish could use β-carotene as a vitamin A source only if the dietary concentration exceeded 2,000 international units per kilogram (IU/kg). It has recently been shown that β-carotene and canthaxanthin can be biotransformed in the liver of tilapia into vitamin A1 and that dihydroxycarotenoids such as astaxanthin, zeaxanthin, lutein, and tunaxanthin were directly bioconverted into vitamin A2 (Katsuyama and Matsuno, 1988). In mammals, carotenoids have been found to fulfill various biological functions independent of vitamin A (Olson, 1989). Thus, more studies are needed on the metabolic role of carotenoids in fish and on the possibility that carotenoids serve as a provitamin A.
Vitamin A deficiency in rainbow trout causes anemia, twisted gill opercula, and hemorrhages in the eyes and base of fins (Kitamura et al., 1967a). Brook trout exhibited poor growth, high mortality, and eye lesions, such as edematous eyes, displaced lens, and degeneration of the retina, when fed a vitamin A-deficient, purified diet from first feeding (Poston et al., 1977). Channel catfish fed 0.4 mg of β-carotene/kg of diet for 3 years developed exophthalmia, edema, and hemorrhagic kidney (Dupree, 1966). Anorexia, pale body color, hemorrhagic skin and fins, exophthalmia, and twisted gill opercula occurred in common carp fed a vitamin A-deficient diet after 8 to 11 weeks (Aoe et al., 1968). Rapidly growing yellowtail fingerlings fed a vitamin A-deficient diet developed deficiency signs in 20 days including arrested growth of gill opercula, dark pigmentation, anemia, and hemorrhage in the eyes and liver, accompanied by high mortality (Hosokawa, 1989).
High dietary intake (2.2 million IU/kg diet) of retinyl palmitate caused slow growth, anemia, and severe necrosis of the caudal fin of brook trout at 8.3°C (Poston et al., 1966). Feeding up to 2.5 million IU retinyl palmitate to trout at 12.4°C also reduced body fat and liver size (Poston, 1971a). A high intake of dietary protein (Poston and Livingston, 1971) or methionine (Eckhert and Kemmerer, 1974) by young trout reduced the toxicity of excess dietary vitamin A observed in fish fed a low-protein diet.
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TABLE 1-15 Vitamin Requirements for Growing Fish Determined with Chemically Defined Diets in a Controlled Environment
Vitamin and Fish
Requirement (units/kg diet)
Response Criteria
Reference
Vitamin A
Pacific salmon
R
Halver (1972)
Rainbow trout
2,500 IU
WG, ADS
Kitamura et al. (1967a)
Channel catfish
1,000-2,000 IU
WG
Dupree (1970)
Common carp
4,000-20,000 IU
WG, MLS
Aoe et al. (1968)
Yellowtail
5.68 mg
WG, MLS
Shimeno (1991)
Vitamin D
Pacific salmon
NR
Halver (1972)
Rainbow trout
1,600-2,400 IU
WG, FE
Barnett et al. (1982a)
Channel catfish
500 IU
WG
Lovell and Li (1978)
1,000 IU
WG
Andrews et al. (1980)
250 IU
WG
Brown (1988)
Yellowtail
NR
Shimeno (1991)
Vitamin E
Atlantic salmon
35 mg
WG, ADS
Lall et al. (1988)
Pacific salmon
30 IU
WG, ADS
Woodall et al. (1964)
40-50 mg
WG, MLS
Halver (1972)
Rainbow trout
30 IU
WG, ADS
Woodall et al. (1964)
25 mg
WG, ADS
Hung et al. (1980)
100 mg
MLS
Watanabe et al. (1981b)
50 mg
AASLP
Cowey et al. (1983)
Channel catfish
25 mg
WG, ADS
Murai and Andrews (1974)
50 mg
AASLP
Wilson et al. (1984)
Common carp
100 mg
WG, ADS
Watanabe et al. (1970b)
Yellowtail
119 mg
MLS
Shimeno (1991)
Blue tilapia
25 mg
WG
Roem et al. (1990)
Nile tilapia
50-100 mg
WG, ADS
Satoh et al. (1987)
Vitamin K
Pacific salmon
R
Halver (1972)
Lake trout
0.5-1 mg
NHV
Poston (1976a)
Channel catfish
R
Dupree (1966)
NR
Murai and Andrews (1977)
Yellowtail
NR
Shimeno (1991)
Thiamin
Pacific salmon
10-15 mg
MLS
Halver (1972)
Rainbow trout
1-10 mg
WG, ADS
McLaren et al. (1947)
1 mg
WG, ED
Morito et al. (1986)
Channel catfish
1 mg
WG, ADS
Murai and Andrews (1978b)
Common carp
0.5 mg
WG, ADS
Aoe et al. (1969)
Yellowtail
11.2 mg
MLS
Shimeno (1991)
Riboflavin
Pacific salmon
20-25 mg
MLS
Halver (1972)
7 mg
WG, ADS
Leith et al. (1990)
Rainbow trout
5-15 mg
WG, ADS
McLaren et al. (1947)
6 mg
MLS
Takeuchi et al. (1980)
3 mg
ED
Hughes et al. (1981a)
2.7 mg
MLS, ED
Amezaga and Knox (1990)
Channel catfish
9 mg
WG, ADS
Murai and Andrews (1978a)
Common carp
4 mg
WG, ADS
Aoe et al. (1967c)
6.2 mg
MLS
Aoe et al. (1967c)
7 mg
MLS
Takeuchi et al. (1980)
Yellowtail
11 mg
MLS
Shimeno (1991)
Blue tilapia
6 mg
WG, ADS
Soliman and Wilson (1992b)
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Vitamin and Fish
Requirement (units/kg diet)
Response Criteria
Reference
Vitamin B6
Atlantic salmon
5 mg
WG, ADS
Lall and Weerakoon (1990)
Pacific salmon
10-20 mg
MLS
Halver (1972)
6 mg
WG, ADS
Leith et al. (1990)
Rainbow trout
1-10 mg
WG, ADS
McLaren et al. (1947)
2 mg
WG, ADS
Woodward (1990)
3-6 mg
ED
Woodward (1990)
Channel catfish
3 mg
WG, ADS
Andrews and Murai (1979)
Common carp
5-6 mg
WG, ADS
Ogino (1965)
Yellowtail
11.7 mg
MLS
Shimeno (1991)
Pantothenic acid
Pacific salmon
40-50 mg
MLS
Halver (1972)
17 mg
WG, ADS
Leith et al. (1990)
Rainbow trout
10-20 mg
WG, ADS
McLaren et al. (1947)
20 mg
WG, ADS
Cho and Woodward (1990)
Channel catfish
10 mg
WG, ADS
Murai and Andrews (1979)
15 mg
WG, ADS
Wilson et al. (1983)
Common carp
30-50 mg
WG, ADS
Ogino (1967)
Yellowtail
35.9 mg
MLS
Shimeno (1991)
Blue tilapia
10 mg
WG, ADS
Soliman and Wilson (1992a)
Niacin
Pacific salmon
150-200 mg
MLS
Halver (1972)
Rainbow trout
1-5 mg
WG, ADS
McLaren et al. (1947)
10 mg
WG, ADS
Poston and Wolfe (1985)
Channel catfish
14 mg
WG, ADS
Andrews and Murai (1978)
Common carp
28 mg
WG, ADS
Aoe et al. (1967b)
Yellowtail
12 mg
MLS
Shimeno (1991)
Biotin
Pacific salmon
1-1.5 mg
MLS
Halver (1972)
Rainbow trout
0.05-0.25 mg
WG, ADS
McLaren et al. (1947)
0.08 mg
WG, ADS
Woodward and Frigg (1989)
0.14 mg
ED
Woodward and Frigg (1989)
Lake trout
0.1 mg
WG, ADS
Poston (1976b)
0.5-1 mg
OSS
Poston (1976b)
Channel catfish
R
Robinson and Lovell (1978)
Common carp
1 mg
WG, ADS
Ogino et al. (1970b)
Yellowtail
0.67 mg
MLS
Shimeno (1991)
Vitamin B12
Pacific salmon
0.015-0.02 mg
MLS
Halver (1972)
Rainbow trout
R
Phillips et al. (1964)
Channel catfish
R
Limsuwan and Lovell (1981)
Common carp
NR
Kashiwada et al. (1970)
Yellowtail
0.053 mg
MLS
Shimeno (1991)
Nile tilapia
NR
Lovell and Limsuwan (1982)
Folate
Pacific salmon
6-10 mg
MLS
Halver (1972)
2 mg
WG, ADS
Leith et al. (1990)
Rainbow trout
1.0 mg
WG, ADS
Cowey and Woodward (1993)
Channel catfish
1.5 mg
WG, NHV
Duncan and Lovell (1991)
Common carp
NR
Aoe et al. (1967a)
Yellowtail
1.2 mg
MLS
Shimeno (1991)
Choline
Pacific salmon
600-800 mg
MLS
Halver (1972)
Rainbow trout
50-100 mg
WG, ADS
McLaren et al. (1947)
714-813 mg
WG, LLC
Rumsey (1991)
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Vitamin and Fish
Requirement (units/kg diet)
Response Criteria
Reference
Choline
Lake trout
1,000 mg
WG
Ketola (1976)
Channel catfish
400 mg
WG, LLC
Wilson and Poe (1988)
Common carp
1,500 mg
WG, LLC
Ogino et al. (1970a)
Yellowtail
2,920 mg
MLS
Shimeno (1991)
Myoinositol
Pacific salmon
300-400 mg
MLS
Halver (1972)
Rainbow trout
250-500 mg
WG, ADS
McLaren et al. (1947)
Channel catfish
NR
Burtle and Lovell (1989)
Common carp
440 mg
WG, ADS
Aoe and Masuda (1967)
Yellowtail
423 mg
MLS
Shimeno (1991)
Vitamin C
Atlantic salmon
50 mg
WG, ADS
Lall et al. (1990)
Pacific salmon
50 mg
MKS
Halver et al. (1969)
Rainbow trout
250-500 mg
WG, ADS
McLaren et al. (1947)
100 mg
MKS
Halver et al. (1969)
40 mg
WG, ADS
Hilton et al. (1978)
Channel catfish
60 mg
WG, ADS, VC
Lim and Lovell (1978)
45 mg
WG, ADS
Robinson (1990)
11 mg
WG, ADS, VC
El Naggar and Lovell (1991)
Common carp
R
Dabrowski et al. (1988)
Yellowtail
122 mg
WG, ADS
Shimeno (1991)
Blue tilapia
50 mg
WG, ADS
Stickney et al. (1984)
NOTE: Abbreviations: AASLP, ascorbic acid stimulated lipid peroxidation; ADS, absence of deficiency signs; ED, enzyme data; FE, feed efficiency; LLC, liver lipid content; MLS, maximum liver storage; MKS, maximum kidney storage; NHV, normal hematocrit values; NR, no requirement determined; OSS, optimum swimming stamina; R, required but no value determined; VC, vertebral collagen content; and WG, weight gain.
Vitamin A is added to fish feeds as the acetate, palmitate, or propionate ester in the form of free-flowing beadlets in a multivitamin premix.
Vitamin D
The two major natural sources of vitamin D are ergocalciferol (vitamin D2, which occurs predominantly in plants) and cholecalciferol (vitamin D3, which occurs in animals). Both forms of vitamin D are hydroxylated in the liver to the 25-hydroxy forms. The 25-hydroxy-D3 is further hydroxylated in the kidney to 1,25-dihydroxyvitamin D3, which is the biologically active form of vitamin D responsible for facilitating mobilization, transport, absorption, and use of calcium and phosphorus in concert with the actions of parathyroid hormone and calcitonin.
Cholecalciferol has been shown to be at least three times more effective than ergocalciferol in meeting the vitamin D requirement of rainbow trout (Barnett et al., 1982a). Andrews et al. (1980) found that vitamin D3 was used more effectively by catfish than vitamin D2 at dietary concentrations of 2,000 IU/kg of diet and that high concentrations of vitamin D3 (20,000 to 50,000 IU/kg of diet) reduced weight gain. Brown (1988), however, found that vitamin D2 was utilized as well as vitamin D3 up to 1,500 IU/kg of diet, but higher concentrations of vitamin D2 depressed weight gain and feed efficiency in channel catfish reared in calcium-free water.
Rainbow trout fed a vitamin D-deficient diet exhibited poor growth, elevated liver lipid content, impaired calcium homeostasis manifested by tetany of white skeletal muscles, and ultrastructural changes in the white muscle fibers of the epaxial musculature (George et al., 1981). However, in a similar study also with rainbow trout, no hypocalcemia or changes in bone ash were observed (Barnett et al., 1982a). A lordosis-like droopy tail syndrome observed in vitamin D-deficient trout (Barnett et al., 1982b) was suggested to be related to an epaxial muscle weakness. Channel catfish fed a vitamin D-deficient diet for 16 weeks showed poor growth, lowered body calcium and phosphorus levels, and lowered total body ash (Lovell and Li, 1978). Andrews et al. (1980) reported that vertebral ash level in channel catfish was not significantly affected by vitamin D deficiency.
Fingerling brook trout fed 3.75 × 106 IU vitamin D3/kg diet for 40 weeks had hypercalcemia and increased hematocrit levels but no difference in rates of growth and survival (Poston, 1969a). However, Hilton and Ferguson (1982) did not detect any incidence of renal calcinosis in rainbow trout
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fed a diet containing up to 1 × 106 IU vitamin D3/kg diet. Supplementation of 50,000 IU vitamin D3/kg diet significantly depressed the growth rate of channel catfish (Andrews et al., 1980). By contrast, a diet of 1 × 106 IU vitamin D3/kg has been reported to show no toxic effects in channel catfish reared in calcium-free water for 14 weeks (Brown, 1988).
Vitamin D3 is added to fish feeds either in a beadlet with vitamin A or as a spray or drum-dried powder in a multivitamin premix.
VITAMIN E
Vitamin E is a generic descriptor for all the molecules that possess the biological activity of α-tocopherol. Natural forms of vitamin E are all d-stereoisomers and consist of a substituted aromatic ring and a long isoprenoid side chain. There are eight naturally occurring compounds with vitamin E activity: d-α-; d-β-; d-γ-; d-δ-tocopherols, which differ in the number and position of the methyl groups in the aromatic ring; and their corresponding tocotrienols. The compound with the highest biopotency is d-α-tocopherol. The other tocopherol isomers have some, but very low, biological activity. No interconversion between a-tocopherol and the other tocopherol forms has been detected in liver or muscle tissue of rainbow trout (Watanabe et al., 1981c). The free tocopherol form of vitamin E is unstable to oxidizing conditions; whereas the acetate and succinate esters are quite stable. These ester forms possess no antioxidant activity, but they are readily hydrolyzed in the digestive tract to the biologically active free tocopherol. One IU of vitamin E is defined as the biological activity of 1 mg of DL-α-tocopheryl.
Vitamin E functions in vitro as a very good antioxidant in a manner similar to several synthetic antioxidants. In vivo, vitamin E and selenium (via glutathione peroxidase) function as parts of a multicomponent antioxidant defense system. This system protects the cell against the adverse effects of reactive oxygen and other free radical initiators of the oxidation of polyunsaturated membrane phospholipids, critical proteins, or both.
Vitamin E deficiency signs have been described for chinook salmon (Woodall et al., 1964), Atlantic salmon (Poston et al., 1976), channel catfish (Dupree, 1968; Murai and Andrews, 1974; Lovell et al., 1984; Wilson et al., 1984), common carp (Watanabe et al., 1970a,b, 1981a), rainbow trout (Cowey et al., 1981, 1983; Hung et al., 1981; Watanabe et al., 1981b; Moccia et al., 1984) and yellowtail (Toyoda, 1985). The deficiency signs of vitamin E in various fishes are similar and include muscular dystrophy involving atrophy and necrosis of white muscle fibers; edema of heart, muscle, and other tissues due to increased capillary permeability allowing exudates to escape and accumulate, which are often green in color as a result of hemoglobin breakdown; anemia and impaired erythropoiesis; depigmentation; and ceroid pigment in the liver. The incidence and severity of these deficiency signs have been shown to be enhanced when diets deficient in both vitamin E and selenium were fed to Atlantic salmon (Poston et al., 1976), rainbow trout (Bell et al., 1985), and channel catfish (Gatlin et al., 1986). These latter observations demonstrated a significant interaction between selenium and vitamin E in the nutrition of fish.
Erythrocyte fragility has been used as an indicator of vitamin E status in some animals (Draper and Csallany, 1969). Peroxide hemolysis of red blood cells has been used to determine vitamin E deficiency in rainbow trout (Hung et al., 1981); however, this procedure was not sensitive enough to aid in determining the vitamin E requirement in rainbow trout (Cowey et al., 1981) and channel catfish (Wilson et al., 1984). Cowey et al. (1981) found that in vitro ascorbic acidstimulated lipid peroxidation in liver microsomes of rainbow trout accurately reflected -tocopherol status. This latter procedure has also been used to assess vitamin E status in channel catfish (Wilson et al., 1984; Gatlin et al., 1986).
When high concentrations of dietary polyunsaturated fatty acids are involved in the diets of common carp (Watanabe et al., 1981a) and rainbow trout (Watanabe et al., 1981b; Cowey et al., 1983), the requirement for vitamin E is increased. Vitamin E-deficient rainbow trout have been reported to have significantly reduced immune and nonspecific responses to infection (Blazer and Wolke, 1984a); however, Salte et al. (1988) could show no beneficial effect of dietary vitamin E supplementation alone or in combination with selenium as a prophylaxis for Hitra disease in Atlantic salmon.
High dietary concentrations of vitamin E (5,000 mg of DL-α-tocopherol/kg of diet) have been shown to cause reduced concentrations of erythrocytes in trout blood (Poston and Livingston, 1969).
Vitamin E is added to fish feeds as a dry powder form of DL-α-tocopheryl acetate.
VITAMIN K
Vitamin K is required for stimulation of prothrombin activity in plasma and synthesis of blood clotting factors VII, IX, and X. The metabolic role of vitamin K involves the vitamin K-dependent carboxylase, which carries out the posttranslational conversion of specific glutamyl residues in the vitamin K-dependent plasma proteins to γ-carboxy-glutamyl residues. These residues are essential for the normal, Ca2+-dependent, interaction of the vitamin K-dependent clotting factors with phospholipid surfaces (Suttie, 1985).
The term vitamin K is used as a generic descriptor for both 2-methyl-1,4-naphthoquinone and all 3-substituted derivatives of this compound, which exhibit an antihemorrhagic activity in animals fed a vitamin K-deficient diet. The three major forms of vitamin K include: vitamin K1 or
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phylloquinone, which can be isolated from plants; vitamin K2 or the menaquinones, which are synthesized by bacteria; and vitamin K3 or menadione which is a synthetic product.
Many animals do not require vitamin K in the diet because of bacterial synthesis in the intestinal tract, but intestinal vitamin K-synthesizing microflora have not been described in fish (Margolis, 1953). Supplementation of sulfaguanidine to a vitamin K-deficient diet and low water temperature caused prolonged blood coagulation time and low hematocrit values without affecting growth performance of trout (Poston, 1964). Dupree (1966) reported hemorrhages in channel catfish fed a vitamin K-deficient diet. However, Murai and Andrews (1977) failed to detect any deficiency signs in channel catfish fed a diet devoid of vitamin K and supplemented with sulfaguanidine. The addition of dicumarol, a vitamin K antagonist, did not increase prothrombin time in catfish. The addition of pivalyl, a stronger (20 times) vitamin K antagonist than dicumarol, completely blocked the blood coagulation of channel catfish (Murai and Andrews, 1977). High-dietary concentrations of menadione sodium bisulfite (2,400 mg/kg of diet) had no adverse affect on growth, survival, blood coagulation, or the number of erythrocytes of young trout (Poston, 1971b).
Vitamin K is added to fish feeds as a menadione salt—menadione sodium bisulfite (50 percent K3), menadione sodium bisulfite complex (33 percent K3), or menadione dimethylpyrimidinol bisulfite (45.5 percent K3).
Water-Soluble Vitamins
The water-soluble vitamins, with the exception of two water-soluble growth factors (choline and myoinositol) and ascorbic acid, have unique coenzyme functions in cellular metabolism. Yet, it is not always possible to correlate a sign of deficiency with a diminished function of an enzyme system for which that vitamin is essential. For some warm-water fishes, intestinal synthesis by microorganisms supplies the requirement for certain vitamins. Thus, deficiency signs result only in those cases when antibiotics are fed along with a deficient diet. A constant supply of essential water-soluble vitamins is required to prevent deficiency signs in fish, since these vitamins are not stored in body tissues.
THIMAIN
The coenzyme form of thiamin is thiamin pyrophosphate. Thiamin pyrophosphate functions in the oxidative decarboxylation of α-keto acids, such as pyruvate and α-ketoglutarate, and in the transketolase reaction in the pentose shunt.
Dietary thiamin deficiency has been shown to result in neurological disorders such as hyperirritability in salmonids (Halver, 1957; Coates and Halver, 1958; Kitamura et al., 1967b; Lehmitz and Spannhof, 1977), channel catfish (Dupree, 1966; Comacho, 1978), Japanese eel (Hashimoto et al., 1970), and Japanese parrotfish (Ikeda et al., 1988). However, Murai and Andrews (1978a) did not observe neurological disorders in thiamin-deficient channel catfish. Arai et al. (1972) found only subcutaneous hemorrhages and congested fins in subadult Japanese eels, and Hashimoto et al. (1970) observed neurological disorders in small Japanese eels. Similar deficiency signs with varying degrees of mortality have been reported in common carp (Aoe et al., 1969), red sea bream (Yone and Fujii, 1974), turbot (Cowey et al., 1975), and yellowtail (Hosokawa, 1989).
Erythrocyte transketolase activity has been used as a specific indicator of thiamin status in the turbot (Cowey et al., 1975). Kidney or liver transketolase activity in rainbow trout (Lehmitz and Spannhof, 1977; Masumoto et al., 1987) and thiamin content in the blood of yellowtail (Hosokawa, 1989) also have been shown to decrease much earlier than the appearance of external deficiency signs.
Thiamin is added to fish feeds as thiamin mononitrate, which is 91.9 percent thiamin. Thiamin mononitrate is stable in vitamin premixes that do not contain trace minerals and choline chloride.
RIBOFLAVIN
Riboflavin functions in the intermediary transfer of electrons in metabolic oxidation-reduction reactions as a component of two coenzymes, flavin monouncleotide (FMN) and flavin adenine dinucleotide (FAD). These coenzymes serve as prosthetic groups of oxidation-reduction enzymes involved in the metabolism of keto-acids, fatty acids, and amino acids in the mitochondrial electron transport system.
Species-specific deficiency signs are found in fish. The only common signs are anorexia and poor growth. The first sign of riboflavin deficiency observed in salmonids (McLaren et al., 1947; Halver, 1957; Steffens, 1970; Takeuchi et al., 1980; Hughes et al., 1981a,b) appeared in the eyes and included photophobia, cataracts, corneal vascularization, and hemorrhages. Lack of coordinated swimming and dark skin coloration have also been reported for riboflavin-deficient chinook salmon (Halver, 1957) and rainbow trout (Kitamura et al., 1967b; Steffens, 1970). In contrast, Woodward (1984) did not observe cataracts or corneal occlusion in riboflavin-deficient rainbow trout fry and fingerlings; however, severe fin erosion and light skin coloration accompanied by high mortality were observed. The eye lesions and dark skin coloration followed by high mortality have also been observed in riboflavin-deficient yellowtail fingerlings (Hosokawa, 1989). Riboflavin-deficient common carp (Aoe et al., 1967c; Ogino, 1967; Takeuchi et al., 1980) and Japanese eel (Arai et al., 1972) exhibited hemorrhages in various parts of the body, nervousness, and photophobia but no evidence of cataract development. Monolateral or bilateral
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cataracts have been reported in riboflavin-deficient channel catfish (Dupree, 1966), but Murai and Andrews (1978b) found only poor growth and short-body dwarfism in two independent feeding trials with channel catfish. Lethargy and high mortality have been reported in Japanese parrotfish fed riboflavin-deficient diets (Ikeda et al., 1988).
Hughes et al. (1981a) used the activation coefficient (ratio of activity following preincubation with FAD:basal activity) of erythrocyte glutathione reductase to measure the riboflavin status of rainbow trout. However, Woodward (1983) found the activity of D-amino acid oxidase to be a more sensitive indicator of the riboflavin status in rainbow trout, since the low activity of erythrocyte glutathione reductase made its quantification difficult. Amezaga and Knox (1990) also found that hepatic D-amino acid oxidase was a reliable indicator of riboflavin status in rainbow trout. They pointed out, however, that an assay for glutathione reductase activity in erythrocytes would be advantageous since it could be used on live fish. Woodward (1985) reported that the riboflavin requirement was not affected by temperature or by genetic differences in growth rate. This might be one reason why the riboflavin requirement values shown in Table 1-15 agree fairly well even among different species.
Hughes (1984) found that feeding high concentrations of riboflavin (up to 600 mg/kg diet) had no adverse effects on growth of rainbow trout. These results were expected since riboflavin has not been shown to cause hypervitaminosis in other animals. However, two previous studies (McLaren et al., 1947; Woodward, 1982) had reported depressed growth in rainbow trout fed moderate concentrations of riboflavin. It was concluded that the growth depression observed in the earlier studies must have resulted from some factor other than riboflavin.
Riboflavin is added to fish feeds as a dry powder in a multivitamin premix.
VITAMIN B6 (PYRIDOXINE)
The term vitamin B6 is the generic descriptor for the 2-methylpyridine derivatives that have the biological activity of pyridoxine. Pyridoxine is the main form found in plant products, whereas pyridoxal and pyridoxamine are the principal forms found in animal tissue. All three forms are readily converted in animal tissue to the coenzyme forms, pyridoxal phosphate and pyridoxamine phosphate. Pyridoxal phosphate is required for many enzymatic reactions involving amino acids such as transamination, decarboxylation, and dehydration. Pyridoxal phosphate also functions in the biosynthesis of porphyrins and in the catabolism of glycogen.
Pyridoxal phosphate is required for the synthesis of the neurotransmitters—5-hydroxytryptamine and serotonin—from tryptophan. Consequently, signs of pyridoxine deficiency include nervous disorders—erratic swimming, hyperirritability, and convulsions—that have been observed in salmonids (Halver, 1957; Coates and Halver, 1958), gilthead sea bream (Kissil et al., 1981), channel catfish (Andrews and Murai, 1979), common carp (Ogino, 1965), yellowtail (Sakaguchi et al., 1969), and Japanese eel (Arai et al., 1972).
Other deficiency signs such as anorexia and poor growth usually appear in the fish within 3 to 6 weeks after being fed a pyridoxine-deficient diet. Pyridoxine deficiency has been reported to cause various histopathological changes in rainbow trout liver (Jurss and Jonas, 1981) and kidney (Smith et al., 1974) and in the intestinal tissue of both rainbow trout (Smith et al., 1974) and gilthead sea bream (Kissil et al., 1981).
The activity of certain aminotransferase enzymes that require pyridoxal phosphate as a coenzyme has been used as an index of pyridoxine status in fish. Serum or tissue alanine and/or aspartate aminotransferase activities have been used to evaluate pyridoxine status in common carp (Ogino, 1965), rainbow trout (Smith et al., 1974; Jurss, 1978), chinook salmon (Hardy et al., 1979), turbot (Adron et al., 1978), and gilthead sea bream (Kissil et al., 1981).
Vitamin B6 is added to fish feeds as pyridoxine hydrochloride in a dry form as part of a multivitamin premix.
PANTOTHENIC ACID
Pantothenic acid is a component of coenzyme A (CoA), acyl CoA synthetase, and acyl carrier protein. The coenzyme form of the vitamin is therefore responsible for acyl group transfer reactions. Coenzyme A is required in reactions in which the carbon skeletons of glucose, fatty acids, and amino acids enter into the energy-yielding tricarboxylic acid cycle. Acyl carrier protein is required for fatty acid synthesis.
A deficiency of this vitamin impairs the metabolism of mitochondria-rich cells that undergo rapid mitosis and high-energy expenditure. Thus, deficiency signs have been found to appear within 10 to 14 days in rapidly growing fish such as fingerling yellowtail (Hosokawa, 1989). Gill lamellar hyperplasia or clubbed gills is a characteristic sign of pantothenic acid deficiency in most fish. In addition to clubbed gills, anemia and high mortality have been observed in pantothenic acid-deficient salmonids (Phillips et al., 1945; McLaren et al., 1947; Coates and Halver, 1958; Kitamura et al., 1967b; Poston and Page, 1982; Karges and Woodward, 1984), channel catfish (Dupree, 1966; Murai and Andrews, 1979; Brunson et al., 1983; Wilson et al., 1983), and yellowtail (Hosokawa, 1989). Pantothenic acid-deficient Japanese parrotfish exhibited anorexia, convulsions, and cessation of growth followed by high mortality (Ikeda et al., 1988). Similar deficiency signs were observed in red sea bream (Yone and Fujii, 1974). Slow growth, anorexia, lethargy, and anemia were observed in common carp (Ogino, 1967). Poor growth, hemorrhage, skin lesions, and abnormal swimming were found in Japanese eel (Arai et al., 1972) fed pantothenic acid-deficient diets.
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Pantothenic acid is added to fish feeds as either calcium d-pantothenate (92 percent activity) or calcium DL-pantothenate (46 percent activity) as a dry powder in a multivitamin premix.
Niacin
Niacin is used as the generic descriptor of pyridine 3-carboxylic acids and their derivatives that exhibit the biological activity of nicotinamide (the amide of nicotinic acid). Of the compounds with niacin activity, nicotinic acid and nicotinamide have the greatest biological activity. Niacin is widely distributed in both plant and animal tissue. Much of the niacin in plant material, however, is present in bound forms that have limited availability to fish.
Niacin is a component of the two coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). These coenzymes are essential for several oxidation-reduction reactions involving the transfer of hydrogen and electrons in carbohydrate, lipid, and amino acid metabolism. They are also involved in various energy yielding and biosynthetic pathways including the mitochondrial electron transport system. Tryptophan can be metabolically converted to niacin in many animals, but not in certain salmonid fish (Poston and DiLorenzo, 1973; Poston and Combs, 1980). The fact that niacin deficiency can readily be induced in various fish indicates that most if not all fish lack the capacity for niacin synthesis.
Trout and salmon fed niacin-deficient diets exhibited anorexia, poor growth, poor feed conversion, photosensitivity or sunburn, intestinal lesions, abdominal edema, muscular weakness, spasms, and increased mortality (McLaren et al., 1947; Phillips and Brockway, 1947; Halver, 1957). Channel catfish (Andrews and Murai, 1978) and common carp (Aoe et al., 1967b) showed skin and fin lesions, high mortality, skin hemorrhages, anemia, and deformed jaws when fed niacin-deficient diets for 2 to 6 weeks. Skin hemorrhages, dermatitis, anemia, abnormal swimming, and ataxia were observed in Japanese eels fed a niacin-deficient diet for 14 weeks (Arai et al., 1972).
Poston and Wolfe (1985) have experimentally demonstrated the interaction between the occurrence of dermal lesions and niacin deficiency. Two weeks after exposure of niacin-deficient rainbow trout to ultraviolet radiation, a total loss of mucus-producing cells was observed in histopathological sections of the epidermis.
High dietary intake of niacin (10,000 mg/kg) increased liver fat, decreased body fat, and tended to reduce growth rate in fingerling brook trout (Poston, 1969b).
Niacin is added to fish feeds as either nicotinic acid or niacinamide; both have similar biological activity. Nicotinic acid or niacinamide is added to the multivitamin premix in a dry form.
BIOTIN
Biotin acts in certain metabolic reactions as an intermediate carrier of carbon dioxide during carboxylation and decarboxylation reactions. Specific enzymes that require biotin include acetyl-CoA carboxylase, pyruvate carboxylase, and propionyl-CoA carboxylase. Metabolic pathways requiring biotin include the biosynthesis of long-chain fatty acids and the synthesis of purines.
In many animals, a biotin deficiency can only be induced by feeding avidin, a glycoprotein found in raw chicken egg white that binds biotin and prevents absorption of the vitamin from the intestine. Robinson and Lovell (1978) fed avidin in a biotin-free chemically defined diet to channel catfish and noted a growth suppression that led them to suggest some biotin synthesis by intestinal microflora in this species. However, in a later study by Lovell and Buston (1984) no synthesis of biotin by the intestinal microflora in channel catfish could be detected.
Common carp required 8 to 12 weeks (Ogino et al., 1970a) and channel catfish took 11 weeks (Lovell and Buston, 1984) to show growth depression when fed biotindeficient diets. A similar effect in rainbow trout took only 4 to 8 weeks in water temperatures of 15°C (Woodward and Frigg, 1989). Anorexia, reduced weight gain, and higher feed conversion were more noticeable in smaller than in larger rainbow trout fed biotin-deficient diets (Walton et al., 1984). Biotin-deficient channel catfish exhibited skin depigmentation (Robinson and Lovell, 1978), whereas biotin-deficient Japanese eels had darker skin coloration (Arai et al., 1972). Histological signs of biotin deficiency were not detected after 12 weeks in rainbow trout having an initial weight of 25 g (Walton et al., 1984). However, severe deficiency signs were produced in rainbow trout and lake trout having initial weights of 1.3 and 6.7 g, respectively (Poston and Page, 1982; Woodward and Frigg, 1989). Rainbow trout and lake trout developed biotin-related histopathological signs in the gills (Castledine et al., 1978; Poston and Page, 1982), liver (Poston, 1976b; Poston and Page, 1982), and kidney (Poston and Page, 1982).
Hepatic pyruvate carboxylase activity in rainbow trout fed a lipid-free and biotin-deficient diet decreased to 3.3 percent of that in fish fed a diet sufficient in lipid and biotin, although the enzyme activity was restored to about 50 percent of normal following the addition of lipid to the diet (Walton et al., 1984). In contrast, lipid supplementation of biotin-deficient diets did not increase hepatic pyruvate carboxylase activity in channel catfish (Robinson and Lovell, 1978).
Signs of biotin deficiency were not detected in rainbow trout (Castledine et al., 1978) or channel catfish (Lovell and Buston, 1984) fed natural ingredient diets without supplemented biotin for 24 and 17 weeks, respectively. These studies concluded that adequate biotin was available in the
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various feed ingredients in the natural ingredient diets used to meet the requirements of the fish.
Biotin is added to fish feeds when necessary as D-biotin in a dry form in the multivitamin premix.
FOLATE
The term folate is used as the generic descriptror for folic acid and related compounds exhibiting qualitatively the biological activity of folic acid. Folic acid is composed of a pteridine ring linked through a methylene bridge to p-aminobenzoic acid to form pteroic acid, which is in turn linked as an amide to glutamic acid. Folic acid undergoes enzymatic reduction in the tissues to its active coenzyme form, tetrahydrofolic acid. It functions as an intermediate carrier of one-carbon groups in a number of complex enzymatic reactions. In these reactions, methyl, methylene, and other one-carbon groups are transferred from one molecule to another. These reactions are found in the metabolism of certain amino acids and the biosynthesis of purines and pyrimidines along with the nucleotides found in DNA and RNA.
Trout and salmon fed folate-deficient diets exhibited anorexia; reduced growth; poor feed conversion; and macrocytic normochromic, megaloblastic anemia (Smith, 1968; Smith and Halver, 1969) characterized by pale gills, anisocytosis, and poikilocytosis. The erythrocytes were large with abnormally segmented and constricted nuclei, and a large number of megaloblastic proerythrocytes were present in the erythropoietic tissue of the anterior kidney. Production of erythrocytes decreased with time in fish fed the folate-deficient diet. Some of these signs have also been observed in the rohu (John and Mahajan, 1979).
Poor growth and dark skin coloration were noted in Japanese eels fed a folate-deficient diet for 10 weeks (Arai et al., 1972). Folate-deficient yellowtail fingerlings also showed congestion in fins and bronchial mantle, dark skin coloration, and anemia (Hosokawa, 1989). Folate deficiency signs in channel catfish included reduced growth, anemia, and increased sensitivity to bacterial infection (Duncan and Lovell, 1991). Deficiency signs were not observed in common carp (Aoe et al., 1967a) fed a folate-free diet, presumably due to bacterial synthesis of folate in the intestine (Kashiwada et al., 1971).
Folate is added to fish feeds as folic acid as a dry powder in a multivitamin premix.
VITAMIN B12
The term vitamin B12 should be used as the generic descriptor for all corrinoids exhibiting qualitatively the biological activity of cyanocobalamin. This vitamin was previously known as vitamin B12 or cyanocobalamin. Vitamin B12 is a large molecule (molecular weight 1355) that contains a cobalt atom. Neither higher plants nor animals can synthesize vitamin B12, but both depend on certain microorganisms for the trace amounts required. Vitamin B12 is required for normal maturation and development of erythrocytes, for the metabolism of fatty acids, in the methylation of homocysteine to methionine, and for the normal recycling of tetrahydrofolic acid. Thus, a deficiency of vitamin B12 can result in signs similar to folate deficiency.
Salmon (Halver, 1957) and trout (Phillips et al., 1964) fed low amounts of vitamin B12 showed a high variability in numbers of fragmented erythrocytes and in hemoglobin values, with a tendency for a microcytic, hypochromic anemia. Channel catfish fed a vitamin B12-deficient diet for 36 weeks exhibited reduced growth but no other clinical deficiency signs (Dupree, 1966). John and Mahajan (1979) observed reduced growth and lower hematocrit in rohu fed a vitamin B12-deficient diet. Japanese eel were found to require vitamin B12 for normal appetite and growth (Arai et al., 1972).
Intestinal microfloral synthesis appeared to satisfy the B12 requirement of Nile tilapia (Lovell and Limsuwan, 1982), but channel catfish required dietary supplementation of B12 to prevent anemia (Limsuwan and Lovell, 1981). Intestinal microfloral synthesis of vitamin B12 has been demonstrated in common carp (Kashiwada et al., 1970; Sugita et al., 1991a), channel catfish (Limsuwan and Lovell, 1981; Sugita et al., 1990, 1991a), Nile tilapia (Lovell and Limsuwan, 1982; Sugita et al., 1990, 1991a), rainbow trout (Sugita et al., 1991b), and ayu and goldfish (Sugita et al., 1991a). Sugita et al. (1991a) found a close relationship between the amount of vitamin B12 and the viable counts of Bacteroides type A in the intestinal contents of the various fish studied. They found that this bacterium was present in the intestinal contents of fish that do not require vitamin B12 and absent in those fish that do require vitamin B12.
Vitamin B12 is added to fish feeds when necessary in a dry form as part of a multivitamin premix.
CHOLINE
Unlike the other water-soluble vitamins, choline has no known coenzyme function. Choline has three major metabolic functions: as a component of phosphatidylcholine, which has structural functions in biological membranes and in tissue lipid utilization; as a precursor of the neurotransmitter acetylcholine; and as a precursor of betaine, which serves as a source of labile methyl groups for methylation reactions such as the formation of methionine from homocysteine and creatine from guanidoacetic acid.
Rainbow trout fed a choline-deficient diet developed light yellow-colored livers, protruded eyes, anemia, and extended abdomens (kitamura et al., 1967a). Lake trout fed a choline-deficient diet for 12 weeks had depressed growth rate and increased liver fat content (Ketola, 1976). Depressed
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growth, loss of appetite, and white-gray colored intestines were observed in Japanese eels fed a choline-deficient diet (Arai et al., 1972). Increased liver lipid content has been observed in common carp and channel catfish fed choline-deficient diets (Ogino et al., 1970b; Wilson and Poe, 1988). In addition, common carp developed vacuolization of hepatic cells after being on such a diet for 10 weeks (Ogino et al., 1970b). A thinning of the intestinal wall muscle and focal degeneration of the exocrine pancreas were observed in choline-deficient sturgeon (Hung, 1989).
Channel catfish fed casein-gelatin diets containing excess methionine did not develop signs of choline deficiency; however, catfish fed diets adequate but not excessive in methionine did develop deficiency signs (Wilson and Poe, 1988). Rumsey (1991) has suggested that 50 percent of the choline requirement of rainbow trout can be met from betaine. These observations indicate that certain fish can meet a part of their choline needs through the synthesis of choline by the methylation of ethanolamine, which uses methyl groups from S-adenosyl methionine.
Choline is added to fish feeds as a 70 percent choline chloride solution or a 25 to 60 percent dry powder. Choline chloride can decrease the stability of other vitamins in a multivitamin premix during prolonged storage.
MYOINOSITOL
Inositol may exist in one of seven optically inactive forms and as one pair of optically active isomers. Only one of these forms, myoinositol, possesses biological activity. Inositol is a biologically active cyclohexitol and occurs as a structural component in biological membranes as phosphatidylinositol. Recently, phosphatidylinositol was shown to be involved in signal transduction of several metabolic processes (Mathews and van Holde, 1990). Although similar in many respects to the adenylate cyclase transduction system, the phosphoinositide system is distinctive in that the hormonal stimulus activates a reaction that generates two second messengers. Membrane bound phosphatidylinositol 4,5-bisphosphate is cleaved to release sn-1,2-diacylglycerol and inositol 1,4,5-triphosphate, following the interaction of a hormone or agonist with the receptor on the cell membrane. Inositol 1,4,5-triphosphate stimulates the release of calcium from its intracellular stores in the endoplasmic reticulum, and sn-1,2-diacylglycerol activates protein kinase C to phosphorylate specific target proteins. Examples of cellular processes controlled by the phosphoinositide second messenger system include amylase secretion, insulin release, smooth muscle contraction, liver glycogenolysis, platelet aggregation, histamine secretion, and DNA synthesis in fibroblasts and lymphoblasts.
Signs of inositol deficiency have been reported to include poor appetite, anemia, poor growth, fin erosion, dark skin coloration, slow gastric emptying, and decreased cholinesterase and certain aminotransferase activities in trout (McLaren et al., 1947; Kitamura et al., 1967b), red sea bream (Yone et al., 1971), Japanese eel (Arai et al., 1972), Japanese parrotfish (Ikeda et al., 1988), and yellowtail (Hosokawa, 1989). Rainbow trout fed a diet devoid of inositol had large accumulations of neutral lipids in the liver, increased levels of cholesterol and triglycerides, but decreased amounts of total phospholipid, phosphotidylcholine, phosphotidylethanolamine, and phosphotidylinositol (Holub et al., 1982).
Inositol appears to be synthesized in common carp intestine (Aoe and Masuda, 1967), but not in amounts sufficient to sustain normal growth of young fish without an exogenous source of this vitamin, because younger carp require a higher level of inositol than older fish. Burtle and Lovell (1989) demonstrated de novo synthesis of inositol in the liver of channel catfish, as well as intestinal synthesis. High concentrations of dietary glucose may increase the need for inositol in some fish (Yone et al., 1971).
Myoinositol is added to fish feeds when necessary as a dry powder in a multivitamin premix.
VITAMIN C
Most animals can synthesize vitamin C, or L-ascorbic acid, from D-glucose, but many fish cannot (Kitamura et al., 1965; Poston, 1967; Halver et al., 1969; Wilson, 1973; Dabrowski, 1990). Ascorbic acid is a strong reducing agent and is readily oxidized to dehydroascorbic acid. Dehydroascorbic acid can be enzymatically reduced back to ascorbic acid in animal tissue with glutathione or reduced NADP. Ascorbic acid is a cofactor in the hydroxylation of proline and lysine to hydroxyproline and hydroxylysine in procollagen, which is the precursor of collagen and thus is necessary for the formation of connective tissues, scar tissue in wound repair, and bone matrix (Sandel and Daniel, 1988). Ascorbic acid also facilitates the absorption of iron, thus preventing the anemia often observed in ascorbic acid-deficient fish. In addition, ascorbic acid functions with vitamin E to minimize peroxidation of lipids in fish tissues (Heikkila and Manzino, 1987).
Vitamin C-deficient salmon and trout exhibited structural deformities (scoliosis, lordosis, and abnormal support cartilage of the eye, gill, and fins) and internal hemorrhaging usually preceded by nonspecific signs such as anorexia and lethargy (Halver et al., 1969; Hilton et al., 1978; Tsujimura et al., 1978; Sato et al., 1983), ascites and hemorrhagic exophthalmia (Poston, 1967), and high level of plasma triglycerides and cholesterol (John et al., 1979). Similar structural deformities such as scoliosis and lordosis due to vitamin C deficiency have been observed in channel catfish (Wilson and Poe, 1973; Andrews and Murai, 1974; Lim and Lovell, 1978; Wilson et al., 1989), Indian major carp (Agrawal and Mahajan, 1980), common carp and roach (Dabrowski et al., 1988, 1989), blue tilapia (Stickney et al., 1984),
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Nile tilapia (Soliman et al., 1986a,b), and yellowtail (Sakaguchi et al., 1969). Japanese eels fed a vitamin C-deficient diet showed reduced growth after 10 weeks and hemorrhage in the head and fins after 14 weeks (Arai et al., 1972). Opacity of the cornea and kidney granulomatosis associated with hypertyrosinemia have been described as signs of vitamin C deficiency in turbot (Messager, 1986; Messager et al., 1986).
Phagocytic activity of cells of the immune system in fish produce reactive oxygen radicals that are potent microbicidal factors, but also autotoxic to fish macrophages (Secombes et al., 1988). Vitamin C appears to protect phagocytic cells and surrounding tissues from oxidative damage. An increased immune response due to high concentrations of vitamin C supplementation has been demonstrated in channel catfish (Durve and Lovell, 1982; Li and Lovell, 1985) and rainbow trout (Blazer and Wolke 1984b; Wahli et al., 1986; Navarre and Halver, 1989). However, Lall et al. (1990) observed no differences in humoral response and the complement system in Atlantic salmon fed diets containing 0 to 2,000 mg of vitamin C/kg after vaccination and subsequent live challenge with Aeromonas salmonicida and Vibrio anguillarum. Dietary and environmental contaminants, such as heavy metals (Yamamoto and Inoue, 1985) and chlorinated hydrocarbon pesticides (Mayer et al., 1978), increase the vitamin C requirements of fish.
Reproduction appears to increase maternal demands for vitamin C. Female tilapia fed vitamin C-free diets for 21 weeks produced eggs and fry containing no detectable ascorbic acid (Soliman et al., 1986b). Reduced reproductive performance has also been reported in rainbow trout fed vitamin C-deficient diets (Sandnes et al., 1984). Ascorbic acid reserves are rapidly depleted during embryonic (Sato et al., 1987) and larval development of certain fish (Dabrowski et al., 1988, 1989; Dabrowski, 1990), suggesting that requirements during early life stages may be higher than for fingerlings or adults.
Liver (Hilton et al., 1977; Sato et al., 1983) and kidney (Halver et al., 1969) ascorbic acid concentrations of less than 20 µg/g have been suggested as an indicator of vitamin C deficiency in salmonid fish. A similar value of less than 26 µg/g of liver has been suggested to indicate vitamin C deficiency in channel catfish (Lim and Lovell, 1978). A much higher value of 100 µg/g of kidney coincided with signs of vitamin C deficiency in snakehead (Mahajan and Agrawal, 1979).
Vertebral collagen levels have been shown to be a sensitive index of vitamin C status in channel catfish (Wilson and Poe, 1973; Lim and Lovell, 1978; El Naggar and Lovell, 1991) and rainbow trout (Sato et al., 1978).
Various derivatives of ascorbic acid, which are more stable than the parent compound, have been shown to provide antiscorbutic activity in fish. These include L-ascorbate-2-sulfate in rainbow trout (Halver et al., 1975; Grant et al., 1989), channel catfish (Murai et al., 1978; Brandt et al., 1985; Wilson et al., 1989), and tilapia (Soliman et al., 1986a); L-ascorbyl-2-monophosphate in channel catfish (Brandt et al., 1985; Lovell and El Naggar, 1990); and L-ascorbyl-2-polyphosphate in rainbow trout (Grant et al., 1989) and channel catfish (Wilson et al., 1989). Ascorbate-2-sulfate does not appear to be used as well as other more stable forms of ascorbic acid by certain fish (Murai et al., 1978; Soliman et al., 1986a; Dabrowski and Kock, 1989; Dabrowski et al., 1990), and in channel catfish it accounted for only 7 percent as much vitamin C activity as L-ascorbic acid or L-ascorbyl-2-monophosphate (Lovell and El Naggar, 1990).
Ascorbic acid is very labile and thus readily destroyed in the manufacturing process, especially in extruded feeds. Therefore it is not usually added to multivitamin premixes for fish feeds. Various coated forms of ascorbic acid, such as ethylcellulose or fat-coated products, have been used to increase retention of the vitamin in fish feeds. Nevertheless, approximately 50 percent of the supplemental ascorbic acid is destroyed during the manufacture of extruded catfish feeds (Lovell and Lim, 1978), and excess ascorbic acid is added to commercial formulations to ensure that an adequate concentration of the vitamin is retained during processing. Phosphorylated ascorbic acid, which is stable during extrusion processing (El Naggar and Lovell, 1991), is available for use in fish feeds but is presently relatively expensive. The form of the vitamin selected depends on how the fish feed is to be manufactured and how long it is to be stored before being fed to the fish. At present, it is still more economical to overfortify channel catfish feeds with the ethylcellulose coated product than to use the phosphate derivatives of ascorbic acid.
Representative terms from entire chapter:
channel catfish
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