2. ESSENTIAL NUTRIENTS - PROTEINS AND AMINO ACIDS

2.1 Proteins

The proteins are among the most important constituents of all living cells and represent the largest chemical group in the animal body, with the exception of water; the whole fish carcass contains on average 75% water, 16% protein, 6% lipid, and 3% ash. Proteins are essential components of both the cell nucleus and cell protoplasm, and accordingly account for the bulk of the muscle tissues, internal organs, brain, nerves and skin.

2.1.1 Composition

Proteins are very complex organic compounds of high molecular weight. In common with carbohydrates and lipids, they contain carbon (C), hydrogen (H), and oxygen (O), but in addition also contain about 16 % nitrogen (N: range 12–19%), and sometimes phosphorus (P) and sulphur (S).

2.1.2 Structure

Proteins differ from other biologically important macromolecules such as carbohydrates and lipids in their basic structure. For example, in contrast to the basic structure of carbohydrates and lipids, which is often composed of identical or very similar repeating units (ie. the glucose repeating unit within starch, glycogen and cellulose), proteins may have up to 100 different basic units (amino acids). It follows therefore that greater compound variabilities and ranges are possible, not only to composition, but also to protein shape.

2.1.3 Chemical properties

Colloidal in nature, proteins vary in their solubility in water, from insoluble keratin to the highly soluble albumins. All proteins can be ‘denatured’ by heat, strong acid, alkali, alcohol, acetone, urea and by heavy metal salts. When proteins are denatured they loose their unique structure, and therefore possess different chemical, physical and biological properties (ie. inactivation of enzymes by heat).

2.1.4 Classification

Proteins maŷ be classified into three main groups according to their shape, solubility and chemical composition:

1. Fibrous proteins: insoluble animal proteins which are generally very resistant to digestive enzyme breakdown. Fibrous proteins exist as elongated filamentous chains. Examples of fibrous proteins include the collagens (main protein of connective tissue), elastin (present in elastic tissues such as arteries and tendons), and keratin (present in hair, nails, wool and hooves of mammals).

2. Globular proteins: include all enzymes, antigens and hormone proteins. Globular proteins can be further subdivided into albumins (water soluble, heat-coagulable proteins which occur in eggs, milk, blood and many plants); globulins (insoluble or sparingly soluble in water, and present in eggs, milk, and blood, and serve as the main protein reserve in plant seeds); and histones (basic proteins of low molecular weight, water soluble, occur in the cell nucleus associated with deoxyribonucleic acid - DNA).

3. Conjugated proteins: these are proteins which yield non-protein groups as well as amino acids on hydrolysis. Examples include the phosphoproteins (casein of milk, phosvitin of egg yolk), glycoproteins (mucous secretions), lipoproteins (cell membranes), chromoproteins (haemoglobin, haemocyanin, cytochrome, flavoproteins), and nucleoproteins (combination of proteins with nucleic acids present in the cell nucleus).

2.2 Protein function

The function of proteins may be summarised as follows:

• To repair worn or wasted tissue (tissue repair and maintenance) and to rebuild new tissue (as new protein and growth).

• Dietary protein may be catabolized as a source of energy, or may serve as a substrate for the formation of tissue carbohydrates or lipids.

• Dietary protein is required within the animal body for the formation of hormones, enzymes and a wide variety of other biologically important substances such as antibodies and haemoglobin.

2.3 Protein requirements

The study of dietary nutrient requirements in fishes and shrimp has been almost entirely based on studies comparable to those conducted with terrestrial farm animals. It follows therefore that almost all the available information on the dietary nutrient requirements of aquaculture species is derived from laboratory based feeding trials; the animals being kept in a controlled environment at high density and having no access to natural food organisms.

2.3.1 Optimum dietary protein level

Based on feeding techniques pioneered and developed for terrestrial animals the dietary protein requirements of fish were first investigated in the Chinnok salmon (Oncorhynchus tshawytscha) by Delong, et al, (1958). Fish were fed a balanced diet containing graded levels of a high quality protein (casein:gelatin mixture supplemented with crystalline amino acids to simulate the amino acid profile of whole hen's egg protein) over a 10-week period and the observed protein level giving optimum growth was taken as the requirement (Fig. 2). Since these early studies the approach used by workers today has changed very little if at all, with the possible exception of the use by some researchers of maximum tissue protein retention or nitrogen balance in preference to weight gain as the criterion of requirement (Ogino, 1980). Dietary protein requirements are normally expressed in terms of a fixed dietary percentage or as a ratio of protein to dietary energy.

Fig. 2. Typical dose response curve

To date over 30 fish and shrimp species have been examined in this manner and the results show a uniformly high dietary protein requirement in the range 24–57%, or equivalent to 30–70% of the gross energy content of the diet in the form of protein (Table 1). Whilst a high protein requirement might have been expected for carnivorous fish species, such as plaice (Pleuronectes platessa-50%; Cowey et al, 1972) or snakehead (Channa micropeltes-52%; Wee and Tacon, 1982), the fact that a relatively high protein requirement was also observed in the herbivorous grass carp (Ctenopharyngodon idella 41–43%; Dabrowski, 1977) suggests that the requirement may in part be a function of the methodology used for its determination. The use by different workers of different dietary protein sources, non-protein energy substitutes, feeding regimes, fish age classes and methods for the determination of dietary energy content and dietary requirement leaves little common ground for direct comparisons to be made within or between fish speceis. For example, the high protein requirement observed for grass carp fry (41–43%; Dabrowski, 1977) almost certainly arose from all experimental fish being fed a restricted ration (fish fed only twice daily, and fixed on the lowest recorded ad libitum feed take) and consequently fish fed the lower protein diets not being able to consume sufficient feed to meet their dietary protein and energy requirements. A critical review of the methods used for the estimation of dietary protein and amino acid requirements in fish and crustacea has been made by Tacon and Cowey (1985) and Cowey and Tacon (1983) respectively.

The high dietary protein requirement of fish and shrimp is generally attributed to their carnivorous/omnivorous feeding habit, and their preferential use of protein over carbohydrates as a dietary energy source (Cowey, 1975). In contrast to terrestrial farm animals fish and shrimp are able to derive more metabolizable energy from the catabolism of proteins than from carbohydrates.

2.3.3 Abiotic factors - temperature and salinity

The influence of water temperature on protein requirement and fish growth has been the subject of numerous investigations. The early study of DeLong and co-workers with fingerling Chinook salmon (O. tshawytscha) was said to show an increase in the dietary protein requirement from 40% to 55% with an increase in water temperature from 8.3°C to 14.4°C (DeLong, et al., 1958). More recently, a similar rise in dietary protein requirement was reported in fingerling Striped bass (Morone saxatilis) from 47% to 55% with an increase in water temperature from 20.5°C to 24.5°C (Millikin, 1983; Table 1). In contrast, fingerling rainbow trout (Salmo gairdneri) showed no difference in growth at dietary protein levels of 35%, 40% and 45% at temperatures of 9°C, 12°C, 15°C and 18°C in one study (Slinger et al., 1977) or in another study with temperatures of 9°C, 15°C and 18°C (Cho and Slinger, 1978). Although distinct temperature effects were observed in terms of growth, the greater absolute need for protein at the higher water temperatures was apparently satisfied through the increased consumption of the lower protein diets. These latter studies are in line with the hypothesis that an increase in water temperature (up to an optimum level) is accompanied by an increased feed intake (Brett, et al., 1969; Choubert, et al., 1982), increased growth rate and metabolic rate (Jobling, 1983) and a faster gastro-intestinal transit time (Fauconneau, et al., 1983; Ross and Jauncey, 1981) under conditions where food supply is not limiting. The weight of evidence is that increased water temperature does not lead to an increased protein requirement. In both cases where such a requirement was claimed, the effect of water temperature on dietary protein requirement was investigated by comparing the results obtained in successive experiments at different water temperatures. In addition, the sub-optimal growth and increased feed intake observed with fish fed the higher protein diets suggests that the ad libitum feeding regime employed in fact led to a restricted feed intake.

¹ Fish size range: fry 0–0.5g, fingerling 0.5–10g, juvenile 10–50g, grower 50g and above.
² Feeding regime: %bw/d - fixed feed intake expressed as a percentage of body weight per day, or Ad libitum feeding two to four times daily.
^a No difference in protein requirement at three stocking densities of 400, 600 and 800 fish/m³, using 5m³ cages.
^b 200m² earthen ponds, fish density of 2/m², ponds also fertilized with poultry litter at a rate of 5kg/pond/week.
^c Fish stocking density of 300/m³.
^d/^e Fish stocking density of 9880/hectare.
^f Plastic lined ponds, with fish stocking density of 3000–3700/hectare.
^g Increased dietary protein requirement reported for fingerling striped bass from 47 to 55% with an increase in water temperature from 20.5 to 24.5°C.
^h Feed intake fixed within all groups to the lowest recorded Ad libitum feed intake observed.
ⁱ Protein requirement said to increase from 40 to 45% with increasing salinity.
^j Outdoor concrete ponds, 5 animals/m², infrequent water exchange, all animals fed at same fixed rate based on highest recorded intake.
^k outddor fibreglass tanks, 17 animals/m², high water exchange.
^l Animals housed within pens in earthen pond, 10 animals/m².
^m All animals fed at fixed rate of 5mg feed/larvae/day (PL 1–10), 15mg feed/larvae/day (PL 11–50), and 20mg feed/larvae/day (PL 24–42).
ⁿ All animals fed to excess once or twice daily.
^o Diet formulated to 55% crude protein, but actual level after diet processing was 45%.

Very few studies have been undertaken concerning the effect of salinity on protein requirement. Experiments conducted with fingerling rainbow trout ( a euryhaline fish) are reported to show an increase in the absolute dietary requirement for protein from 40% to 45% with a salinity increase from 10 to 20 parts per thousand (Zeitoun, et al., 1973; Table 1). However, no increase in dietary protein requirement was observed in a similar experiment conducted with Coho salmon fingerlings (O. kisutch); Zeitoun et al., 1974). In view of the speculative method for arriving at dietary requirement from the dose response curve (Zeitoun et al., 1973), and the lack of information on the protein requirements of these fish species in full strength sea water (35 parts per thousand), there are no firm data demonstrating that the protein requirements of fish are elevated with increased salinity. There is no information on the effect of salinity on the protein requirement of shrimp.

2.4 Amino acids

Although over 100 different amino acids have been isolated from biological materials, only 25 of these are commonly found in proteins. Individual amino acids are characterised by having an acidic carboxy group (-COOH) and a basic nitrogenous group (generally an amino group: -NH₂). In view of the presence of both acidic and basic groups, amino acids are amphoteric (ie. have both acidic and basic properties) and consequently act as buffers by resisting changes in pH. The chemical structure of the more commonly occurring amino acids is shown below:

2.5 Amino acid function

Amino acids occupy a central position in cellular metabolism since almost all biochemical reactions are catalysed by enzymes composed of amino acid residues. Amino acids are essential for carbohydrate and lipid metabolism, for the synthesis of tissue proteins and many important compounds (ie. adrenalin, thyroxine, melanin, histamine, porphyrins - haemoglobin, pyrimidines and purines - nucleic acids, choline, folic acid and nicotinic acid - vitamins, taurine - bile salts etc), and as a metabolic source of energy or fuel.

2.6 Amino acid requirements

For nutritional purposes, amino acids may be divided into two groups; the essential amino acids (EAA), and the non-essential amino acids (NEAA). EAA are those amino acids that cannot be synthesized within the animal body or at a rate sufficient to meet the physiological needs of the growing animal, and must therefore be supplied in a ready made form in the diet. NEAA are those amino acids that can be synthesized in the body from a suitable carbon source and amino groups from other amino acids or simple compounds such as diammonium citrate, and consequently do not have to be supplied in a ready made form in the diet.

The dietary EAA for fish and shrimp are as follows:

Although the NEAA are not dietary essential nutrients, they perform many essential functions at the cellular or metabolic level. They are termed dietary non-essential nutrients only because the body tissues can synthesize them on demand. In fact it is often quoted that the NEAA are physiologically so essential that the body ensures an adequate supply by synthesis. From a feed formulation viewpoint, it is important to know that the NEAA's cystine and tyrosine can be synthesized within the body from the EAA's methionine and phenylalanine respectively, and consequently the dietary requirement for these EAA is dependent on the concentration of the corresponding NEAA within the diet.

2.6.1 Optimum dietary essential amino acid levels

(a) Dose response and carcass deposition method: The quantitative EAA requirements of fish have traditionally been determined by feeding graded levels of each amino acid within an amino acid test diet so as to elicit a dose response curve (for review see Ketola, 1982; Cowey and Luquet, 1983; Wilson, 1985). Dietary requirement is then usually taken at ‘break point’ on the basis of the observed growth response. In addition to growth, several workers have also used free amino acid levels within specific tissue pools (whole blood, blood plasma or muscle; Kaushik, 1979), or the oxidation of radioactively labelled amino acids (administered orally or by injection; Walton, Cowey and Adron, 1982) as the criterion for estimating dietary requirement. Within the amino acid test diets used the protein component is supplied almost entirely in the form of crystalline amino acids or in combination with selected ‘whole’ protein sources (commonly either casein, gelatin, zein, gluten or fish meal); the amino acid profile of the total protein component of the diet being controlled so as to simulate the amino acid profile of a specific reference protein.

In contrast to the above standard method where fish are fed graded levels of crystalline amino acids, Ogino (1980a) determined the quantitative EAA requirement of fish simultaneously on the basis of the daily deposition of individual amino acids within the fish carcass. In the Ogino method fish are fed a diet containing a ‘whole’ protein source of high biological value, and the dietary EAA requirement computed on the basis of the observed daily EAA tissue deposition value.

Table 2 summarises the known quantitative EAA requirements of fish studied to date using the above mentioned techniques. Quantitative dietary requirements for all 10 EAAs have been established for only five fish species (common carp C. carpio, rainbow trout S. gairdneri, channel catfish I. punĎtatus, Japanese eel A. anguilla, and the Chinook salmon O. tshawytscha). At present there is no quantitative information on the dietary EAA requirements of shrimp; in the main this has been due to the poor growth observed with shrimp fed synthetic amino acid test diets and the inherent problems of nutrient leaching due to the extended feeding habits of shrimp.

Although numerous independent studies have recently been performed on the amino acid requirements of rainbow trout, significant differences in requirement (g amino acid/100g protein) exist within and between individual fish species (Table 2). For example, differences of the order of 65%, 72% and 114% were observed between independent laboratories for the lysine, arginine and methionine requirement of fingerling/juvenile rainbow trout. Similarly, inter-species variations ranged from 22% for valine to as high as 122% for tryptophan. Whilst one would have expected the quantitative EAA requirements of fish to decrease with age and decreasing protein synthesis (growth), one may well question whether or not the observed variations in requirement are real or merely an artifact of the method employed. In contrast to the variations in requirement observed for the same fish species fed conventional amino acid test diets, there was no significant difference in the EAA requirement of carp and trout on the basis of the carcass deposition method of Ogino (1980a). However, the dietary requirements observed are within the range reported for fish fed amino acid test diets (Table 2).

Compared with the conventional method of feeding graded levels of individual amino acids, the carcass deposition method of Ogino (1980a) offers numerous advantages:

• Fish are fed rations in which the protein component is supplied in the form of a ‘whole’ protein of high biological value. Amino acid requirements can therefore be ascertained in fish displaying optimal growth.

• The dietary requirement for all ten EAAs can be determined simultaneously in one single experiment. Using conventional amino acid test diets up to 10 separate experiments have to be performed, each experiment involving the use of up to six dietary regimes employing varying dietary concentrations of the single EAA under test.

• Quantitative EAA requirements can equally be established for first feeding fry and brood-stock fish with no loss of precision.

¹ Feeding regime: indicates feeding level and number of feedings per day.
² In the presence of dietary cystine
a 2%;
b 0.24%;
c 1%;
d 1%;
e 0.3%;
f 2%;
g 0.74%;
h 1%
³ In the absence of dietary cystine.
⁴ In the presence of dietary tyrosine
h 1%;
i 1%;
j 0.4%;
k 2%
⁵ In the absence of dietary tyrosine.

(b) Carcass analysis method: Interestingly, recalculation of the data obtained by Ogino (1980a) shows that there is no difference between the relative proportions of individual EAAs required in the diet and the relative proportions of the same 10 EAAs present within the fish carcass (Tacon and Cowey, 1985). A similar relationship has also been seen in the growing pig and chick (Boorman, 1980), and to a lesser extent within the four fish species for which EAA requirements have been determined using amino acid test diets (Fig. 3). Similarly, Wilson and Poe (1985) obtained a regression coefficient of 0.96 when the EAA requirement pattern for the channel catfish was regressed against the whole body EAA pattern found in a 30g channel catfish. Since the amino acid composition of fish body tissue does not differ greatly (if at all) between individual fish species (Njaa and Utne, 1982; Wilson and Cowey, 1985), it follows, therefore, that the pattern of requirement for different species will also be similar. Although not proven, it is not unreasonable to suppose that a similar relationship also exists for shrimps and freshwater prawns. For comparative purposes Table 3 presents the dietary EAA requirement pattern for fish, as determined by Ogino (1980a), together with the carcass EAA pattern of whole fish body tissue, Penaeus japonicus

Figure 3 Relationship between pattern of EAA requirements found by feeding experiments using amino acid test diets with carp (•), Japanese eel (■), channel catfish (□) and chinook salmon (o) and the pattern of the same amino acids in fish carcass. The level of each amino acid is represented as a percentage of the sum of all 10 EAA's in each pattern. The line represents coincidence of requirement and tissue patterns. larvae and juveniles, Penaeus paulensis juveniles, short-necked clam tissue (Venerupis philippinarum; regarded as an excellent and ideal natural food for marine shrimp), and the tail muscle of Macrobrachium rosenbergii. On the basis of the amino acid profiles presented it would appear that shrimp have a higher dietary requirement for arginine, tryptophan and tyrosine, and a lower dietary requirement for valine, threonine and lysine than fish.

NA - data not available (not analysed).
* - Non-essential amino acids.All values are expressed as a percentage of total EAA plus cystine and tyrosine.

In the absence of firm quantitative information on the dietary EAA requirements of shrimp and the majority of farmed fish species, dietary requirement can initially be computed on the basis of the carcass EAA pattern present within 35% of the known dietary protein requirements of the said species; on a general basis EAAs (including the NEAAs cystine and tyrosine) constitute about 35% of the total dietary protein required by fish (Table 2). Thus if a shrimp or fish is known to have a dietary protein requirement of 45%, then dietary EAA requirement would be computed on a carcass EAA pattern of 35% of the dietary protein level. For example, if the carcass EAA pattern for lysine is 16.9% of the total EAA plus cystine and tyrosine present, then dietary requirement level for lysine would be or 2.66% of the dry diet (ie. 45% protein fish ration).

As a guide line Table 4 presents the calculated dietary EAA requirements of fish and shrimp at varying dietary protein levels based on the mean carcass EAA pattern of whole fish tissue and short-necked clam tissue respectively (short-necked clam tissue is used here in the absence of a mean carcass EAA pattern for shrimp).

¹ Carcass EAA pattern of whole fish tissue (Wilson & Cowey, 1985)
² Carcass EAA pattern of short-necked clam (Deshimaru et al., 1985)
* Non-essential amino acids

2.6.2 Utilization of free amino acids

Fish or juvenile shrimp fed rations in which a significant proportion of the dietary protein is supplied in the form of ‘free’ or crystalline amino acids generally display sub-optimal growth and feed conversion efficiency compared with animals fed protein-bound amino acids or ‘whole’ proteins (Wilson et al., 1978; Robinson et al., 1981; Yamada et al., 1981; Walton et al., 1982; Deshimaru, 1981; Deshimaru & Kuroki, 1974a, 1975).

In general, dietary free amino acids are more rapidly assimilated in fish than protein-bound amino acids. Experiments with rainbow trout (Yamada et al., 1981), common carp (Plakas et al., 1980) and tilapia (Oreochromis niloticus; Yamada et al., 1982) fed free amino acid test diets showed that peak plasma amino acid concentrations occurred sooner (12–24h, 2–4h, 2h, respectively) than with an equivalent casein-based diet (24–36h, 4h, 4h, respectively). Furthermore, in carp. individual free amino acids appear to be absorbed at varying rates from the gastro-intestinal tract, and consequently peak plasma concentrations of individual amino acids do not occur simultaneously (Plakas et al., 1980). In juvenile shrimp the situation appears to be the reverse. For example, Deshimaru (1981) showed that the assimilation rate of dietary free arginine into muscle protein by Penaeus japonicus juveniles was extremely low (assimilation less than 0.6%) compared to that of protein-bound arginine (assimilation above 90%). However, although Deshimaru (1981) reported no beneficial effect on growth of free amino acid supplemented diets with P. japonicus juveniles, recent studies have demonstrated that the larvae of the same species is capable of utilizing amino acid supplemented diets for growth (Teshima, Kanazawa & Yamashita, 1986).

For optimal protein synthesis to occur, it is essential that all amino acids (whether they be derived from whole proteins or amino acid supplements) are presented simultaneously to the tissue. If such an equilibrium is not achieved, then amino acid catabolism (breakdown) ensues with consequent loss of growth and and feed efficiency. For those warm water fish species which display a rapid uptake and assimilation of free amino acids, it is therefore essential that either; (1) the release or absorption of free amino acids from the diet is reduced so as to minimise the variations in absorption rate observed between free and protein-bound amino acids (achieved by coating individual amino acids with casein, zein or nylon-protein membranes; Murai et al., 1982; Teshima, Kanazawa & Yamashita, 1986); or (2) that the frequency of feed presentation is increased from two or three feeds per day to up to 18 feeds per day so as to minimise the variations observed in plasma amino acid concentration (Yamada, Tanaka & Katayama, 1981).

2.6.3 Amino acid composition and protein quality

On the basis of the above discussions it is evident that the protein quality of a feed ingredient is dependent upon the amino acid composition of the protein and the biological availability of the amino acids present. In general, the closer the EAA pattern of the protein approximates to the dietary EAA requirement of the species, the higher its nutritional value and utilization. For example, Table 5 presents the ‘chemical score’ or potential protein value of some commonly used feed proteins. Chemical scores of 100 indicate that the level of a particular EAA within the feed protein is identical to the dietary EAA requirement level for fish (when expressed as a percentage of the total EAAs plus cystine and tyrosine) as determined by Ogino (1980a). The chemical score of the protein is taken to be the percentage of the EAA in greatest deficit relative to the dietary requirement pattern. This method of assessing protein quality is based on the concept that the nutritive value of a protein depends primarily on the amount of the EAA in greatest deficit in that protein, compared to a reference protein (in this case the reference protein is the dietary EAA requirements of fish as determined by Ogino. 1980a). It can be seen from Table 5 that compared to fish meal or fish muscle, which has a well balanced EAA profile and high chemical score (c. 80), the majority of protein sources presented have amino acid imbalances which render them unsuitable as a sole source of dietary protein for fish within complete diets intended for intensive farming systems. The aim of feed formulation is to mix proteins of various qualities to obtain the desired EAA pattern of the fish or shrimp species in question (complete diet feeding).

However, the above relationship between protein quality and EAA pattern will only hold true if the individual amino acids are equally biologically available to the animal. For example, under certain conditions some of the amino acids may be unavailable because the proteins in the diet are incompletely digested. Thus, for carnivorous fish and shrimp species, the cellulose cell wall within plant protein sources may render the proteins present within the cell inaccesible to the digestive enzymes. In other cases, digestion may be hindered by the presence of enzyme inhibitors within the food protein; trypsin inhibitor within raw soybeans. Although it is possible to inactivate these inhibitors by moderate heat processing, under conditions of excessive heat treatment proteins become more resistant to digestion due to peptide bond formation occurring between the side chains of lysine and dicarboxylic acid. The free epsilon amino groups of lysine are particularly susceptible to heat damage, forming addition compounds with non-protein compounds (ie. reducing sugars such as glucose) present in the food stuff (Cockerell, Francis & Halliday, 1972). This reaction is known as the Maillard reaction, and renders the lysine biologically unavailable. Substances other than reducing sugars which are known to react with the free epsilon amino group of lysine include gossypol; phenol based compound present in cottonseed meal. An estimate of the biological availability of amino acids within feed proteins, and hence an indicator of protein quality, can be made by chemically measuring the free or available lysine content of the feed protein (Cowey, 1979).

¹ Scores based on comparison with the mean essential amino acid requirements of rainbow trout andcarp (Ogino, 1980). Mean EAA requirement (expressed as % of total EAA) being: threonine 10.6;valine 9.5; methionine 5.4; cystine 2.7; isoleucine 7.5; leucine 13.5; phenylalanine 9.5;tyrosine 6.5; lysine 16.8; histidine 4.8; arginine 11.6; and tryptophan 1.7

² Source: 1-Kay (1979); 2-Gohl (1980); 3-Bolton and Blair (1977); 4-National Research Council(1983); 5-Tunnel AVEBE Starches Ltd., UK; 6-Cowey et al. (1971); 7-Unpublished data; 8-Coweyand Sargent (1972); 9-Connell and Howgate (1959); 10-Jackson, Kerr and Cowey (1984); 11-Tacon,Stafford and Edwards (1983); 12-Spinelli (1980)

* Limiting essential amino acids (present below 30% mean fish requirement)

2.7 Evaluation of protein quality

Apart from chemically measuring amino acids and their availability within feed proteins, there are many biological methods of evaluating protein quality:

Specific growth rate (SGR) The rate of growth of an animal is a fairly sensitive index of protein quality; under controlled conditions weight gain being proportional to the supply of essential amino acids. Daily SGR can be calculated by using the formula:

Food conversion ratio (FCR) Defined as the grams of feed consumed per gram of body weight gain.

	* As fed basis ie. dry weight
	** Wet or fresh weight gain

Food efficiency (FE) Defined as the grams of weight gained per gram of feed consumed. Units of expression as above.

Protein efficiency ratio (PER) Defined as the grams of weight gained per gram of protein consumed.

* With this method no allowance is made for maintenance: ie. method assumes that all protein is used for growth.

Apparent net protein utilization (Apparent NPU) Defined as the percentage of ingested protein which is deposited as tissue protein.

where Pb is the total body protein at the end of the feeding trial, Pa is the total body protein at the beginning of the feeding trial, and Pi is the amount of protein consumed over the feeding trial. In this calculation no allowance is made for endogenous protein losses. In contrast to the previous methods of evaluating protein quality, this method requires that a representative sample of animals be sacrificed at the beginning and end of the feeding trial for carcass protein analysis.

The main drawback of these methods of predicting diet or protein quality is that they have to be performed under controlled experimental conditions in the absence of natural food organisms. Consequently, these methods can only be used within intensive or clear water culture systems.

2.8 Nonprotein nitrogenous constituents

Amino acids are important not only as building blocks of protein but as the primary constituents or nitrogen precursors for many nonprotein nitrogencontaining compounds. Table 6 lists some of the more biologically important nonprotein nitrogenous compounds that originate from amino acids.

¹ Lloyd, McDonald & Crampton (1978)

² Pyrimidine and purine have been suggested to be essential dietary nutrients fornewly hatched fish larvae (Dabrowski & Kaushik, 1982) and Artemia salina (Hernandorena, 1983) respectively.

2.9 Protein and amino acid pathology

2.9.1 Dietary Essential Amino Acid Deficiency

Although all fish examined to date display reduced growth when fed EAA deficient diets, the following additional gross anatomical deficiency signs have been observed under experimental conditions with juvenile fish fed synthetic rations deficient in one or more EAAs:

¹ 1-Walton, Cowey and Adron (1984); 2-Ketola (1983); 3-Mazid et al. (1978);4-Walton, Cowey and Adron (1982); 5-Poston et al. (1977); 6-Barash, Poston andRumsey (1982); 7-Walton et al. (1984); 8-Kloppel and Post (1975); 9-Poston andRumsey (1983); 10-Shanks, Gahimer and Halver (1962); 11-Halver and Shanks (1960);Akiyama et al. (1985a).

² Curvature of the vertebral column

Under intensive farming conditions dietary EAA deficiencies may arise from one of four possible routes:

• Poor feed formulation arising from the use of disproportionate amounts of feed proteins with natural specific EAA deficiencies (Table 5).

  Dietary imbalances may also arise from the presence of disproportionate levels of specific amino acids; including leucine/isoleucine antagonisms, and to a lesser extent arginine/lysine and cystine/methionine antagonisms. For example, blood meal is a rich source of valine, leucine and histidine, but is a very poor source of methionine and isoleucine. However, in view of the antagonistic effect of excess leucine on isoleucine, animals fed high dietary levels of blood meal suffer from an isoleucine deficiency caused by an excess of dietary leucine (Taylor, Cole and Lewis, 1977). Although similar antagonisms have also been reported for cystine/ methionine (use of hydrolysed feather meal; Ichhponani and Lodhi, 1976) and arginine/ lysine (Harper, Benevenga and Wohlhueter, 1970) in terrestrial farm animals, they have not been found to occur in fish fed synthetic amino acid diet combinations (Robinson, Wilson and Poe, 1981).

• Dietary EAA deficiencies may arise from excessive heat treatment of feed proteins during feed manufacture.

• Dietary EAA deficiencies may arise from the chemical treatment of feed proteins with acids (silage production) or alkalies, due to the loss of free tryptophan and lysine/cystine respectively (Kies, 1981).

• Dietary EAA deficiencies may arise from the leaching of free and protein bound amino acids into the water. For example, Grabner, Wieser and Lackner (1981) reported the loss, through leaching, of almost all the free and about one-third of the free plus protein bound amino acids from frozen or freezedried zooplankton (Artemia salina and Moina spp.) after a 10 minute water immersion period at 9°C. Considerable losses of water-soluble amino acids have also been observed in carp during mastication (Yamada and Yone, 1986). However, the problem of nutrient leaching of water soluble materials is probably greatest for crustaceans due to their very slow demersal feeding habit and necessity to masticate their food externally prior to ingestion (Farmanfarmaian, Lauterio and Ibe, 1982). For example, Bages and Sloane (1981) reported a 28% loss of dietary protein during the preparation and rehydration of a dry alginate-bound shrimp diet prior to feeding, and a total protein loss of 39–47% after a six hour immersion period in seawater. In general nutrient losses are greater in freshwater than in seawater (Balazs, Ross and Brooks, 1973). However, problems of nutrient leaching can be minimised by using an appropriate feeding regime (ie. regular rather than infrequent feeding; Sedgwick, 1979) and a suitable diet binding or micro-encapsulation technique (Goldblatt, Conklin and Duane Brown, 1980; Jones et al., 1976).

2.9.1 Toxic non-essential amino acids

Nutritional pathologies may also arise from the ingestion of feed proteins containing toxic amino acids. Commonly used feed proteins which are known to contain toxic amino acids include alkali-treated soybean (toxic amino acid - lysinoalanine), the legume Leucaena leucocephala or ‘ipil ipil’ (toxic amino acid - mimosine), and the faba bean Vicia faba (toxic amino acid - dihydroxyphenylalanine).