Proteins are complex, organic compounds composed of many amino acids
linked together through peptide bonds and cross-linked between chains by
sulfhydryl bonds, hydrogen bonds and van der Waals forces. There is a
greater diversity of chemical composition in proteins than in any other
group of biologically active compounds. The proteins in the various animal
and plant cells confer on these tissues their biological specificity.
(a) Simple proteins. On hydrolysis they yield only the amino acids
and occasional small carbohydrate compounds. Examples are: albumins,
globulins, glutelins, albuminoids, histones and protamines.
(b) Conjugated proteins. These are simple proteins combined with some
non-protein material in the body. Examples are: nucleoproteins,
glycoproteins, phosphoproteins, haemoglobins and lecithoproteins.
(c) Derived proteins. These are proteins derived from simple or
conjugated proteins by physical or chemical means. Examples are:
denatured proteins and peptides.
Structure
The potential configuration of protein molecules is so complex that
many types of protein molecules can be constructed and are found in
biological materials with different physical characteristics. Globular
proteins are found in blood and tissue fluids in amorphous globular form
with very thin or non-existent membranes. Collagenous proteins are found
in connective tissue such as skin or cell membranes. Fibrous proteins
are found in hair, muscle and connective tissue. Crystalline proteins
are exemplified by the lens of the eye and similar tissues. Enzymes are
proteins with specific chemical functions and mediate most of the
physiological processes of life. Several small polypeptides act as
hormones in tissue systems controlling different chemical or
physiological processes. Muscle protein is made of several forms of
polypeptides that allow muscular contraction and relaxation for physical
movement.
Properties
Proteins can also be characterized by their chemical reactions. Most
proteins are soluble in water, in alcohol, in dilute base or in various
concentrations of salt solutions. Proteins have the characteristic
coiled structure which is determined by the sequence of amino acids in
the primary polypeptide chain and the stereo configuration of the
radical groups attached to the alpha carbon of each amino acid. Proteins
are heat labile exhibiting various degrees of lability depending upon
type of protein, solution and temperature profile. Proteins can be
reversible or irreversible, denatured by heating, by salt concentration,
by freezing, by ultrasonic stress or by aging. Proteins undergo
characteristic bonding with other proteins in the so-called plastein
reaction and will combine with free aldyhyde and hydroxy groups of
carbohydrates to form Maillard type compounds.
Chemical Determination
The nitrogen content of most proteins found in animal, nut and grain
tissue is about 16 percent; therefore, protein content is commonly
expressed as nitrogen content × 6.25.
PROTEIN DIGESTION AND METABOLISM
Ingested proteins are first split into smaller fragments by pepsin in
the stomach or by trypsin or chymotrypsin from the pancreas. These
peptides are then further reduced by the action of carboxypeptidase
which hydrolyzes off one amino acid at a time beginning at the free
carboxyl end of the molecule or by aminopeptidase which splits off one
amino acid at a time beginning at the free amino end of the polypeptide
chain. The free amino acids released into the digestive system are then
absorbed through the walls of the gastro intestinal tract into the blood
stream where they are then resynthesized into new tissue proteins or are
catabolyzed for energy or for fragments for further tissue metabolism.
GROSS PROTEIN REQUIREMENTS
Gross protein requirements have been determined for a few species of
fish (see Table 1). Simulated whole egg protein component of test diets
contains an excess of indispensable amino acids. These diets were kept
approximately isocaloric by adjusting total protein plus digestible
carbohydrate components to a fixed amount as the protein diet treatments
were varied over the ranges tested. Tests in feeding fry, fingerling,
and yearling fish have shown that gross protein requirements are highest
in initial feeding fry and that they decrease as fish size increases. To
grow at the maximum rate, fry must have a diet in which nearly half of
the digestible ingredients consist of balanced protein; at 6-8 weeks
this requirement is decreased to about 40 percent of the diet for salmon
and trout and to about 35 percent of the diet for yearling salmonids
raised at standard environmental temperature (SET). Gross protein
requirements for young Catfish appear to be less than those for
salmonids. Initially feeding fry require that about 50 percent of the
digestible components of the ration be protein, and the requirement
decreases with size. Some feeding trials with salmon have indicated
direct relationships between changes in the protein requirements of
young fish and changes in water temperature. Chinook salmon in 7 C water
require about 40 percent whole egg protein for maximum growth; the same
fish in 15 C water require about 50 percent protein. Salmon, trout and
catfish can use more protein than required for maximum growth because of
efficiency in eliminating nitrogenous wastes in the form of soluble
ammonia compounds through the gill tissue directly into the water
environment. This system for eliminating nitrogen is more efficient than
that available to fowl and mammals. Fowl and mammals consume energy to
synthesize urea, uric acid, or other nitrogen compounds which are
excreted through the kidney tissue and expelled in urine. Digestible
carbohydrate and fat will spare excess protein in the diet as long as
the protein requirement for maximum growth is met.
Table 1 - Estimated Dietary Protein Requirement of Certain Fish
|
Species |
Crude protein level in diet for optimal growth
(g/kg) |
| Rainbow trout (Salmo gairdneri) |
400-460 |
| Carp (Cyprinus carpio) |
380 |
| Chinook salmon (Oncorhynchus tshawytscha) |
400 |
| Eel (Anguilla japonica) |
445 |
| Plaice (Pleuronectes platessa) |
500 |
| Gilthead bream (Chrysophrys aurata) |
400 |
| Grass carp (Ctenopharyngodon idella) |
410-430 |
| Brycon sp. |
356 |
| Red sea bream (Chrysophrys major) |
550 |
| Yellowtail (Seriola quinqueradiata) |
550 |
Basically the fish must be given a diet containing graded levels of
high quality protein and energy and adequate balances of essential fatty
acids, vitamins and minerals over a prolonged period. From the resulting
dose/response curve the protein requirement is usually obtained by an
Almquist plot. These differences in apparent protein requirement are
thought to be due to differences in culture techniques and diet
composition.
The relatively high dietary protein levels required for maximal
growth of certain fish such as grass carp, Ctenopharyngodon
idella, and Brycon spp. are surprising as these fish are
omnivorous. Brycon spp. are grown on unwanted fruit and other
plant material of low protein content and under these conditions there
is presumably a substantial contribution to their protein intake from a
natural food chain.
Protein requirement of eurythaline fish such as the rainbow trout,
Salmo gairdneri, and the coho salmon, Oncorhynchus kisutch,
reared in water of salinity 20 ppt is about the same as the requirement
in freshwater. No data are available for the protein requirement of
these species in full strength sea water.(35 ppt).
Amino
Acids
The amino acids are the building blocks of proteins; about 23 amino
acids have been isolated from natural proteins. Ten of these are
indispensable for fish. The animal is incapable of synthesizing
indispensable amino acids and must therefore obtain these from the diet.
Essential and Non-essential Amino Acids
Salmon, trout and channel catfish fed diets devoid of arginine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan or valine failed to grow. These same fish
fed diets devoid of other L-amino acids grew as well as fish receiving
all 18 amino acids tested. The nitrogen component in the test diets was
made up of 18 L-amino acids in the pattern found in whole egg protein.
All fish on test recovered rapidly when the missing amino acid was
replaced in the diet. The slope of the growth curve of the recovery
group was identical with that of fish receiving the complete amino acid
test diet.
Dispensable amino acids tested were alanine, aspartic acid, cystine,
glutamic acid, glycine, proline, serine, and tyrosine. These amino acids
were found to be not essential for the growth of salmon, trout and
channel catfish.
Quantitative studies on the requirements of the 10 indispensable
amino acids used a casein-gelatin mixture supplemented with crystalline
L-amino acids. The test diet had an amino acid pattern of 40 percent
whole egg protein for the nitrogen component. Experiments conducted with
carp and eel showed a similar lack of growth when an indispensable amino
acid was absent from the diet.
Essential Amino Acids and Protein Quality
If the essential amino acid requirements of fish are known, it should
be possible to meet these needs in culture systems in a number of ways
from different food proteins or combinations of food proteins.
Phenylalanine is spared by tyrosine. It is not known to be chemically
modified nor rendered unavailable by the harsh conditions to which
feedstuff proteins are normally subjected during processing. Measurement
of phenylalanine in proteins is uncomplicated so that the provision and
evaluation of phenylalanine in proteins in practical diets presents
little difficulty.
Lysine is a basic amino acid. In addition to the
a -amino acid group normally bound in peptide linkage, it also
contains a second, a -amino group. This
a -amino group must be free and reactive,
otherwise the lysine, although chemically measurable, will not be
biologically available. During the processing of feedstuff proteins the
a -amino group of lysine may react with
non-protein molecules present in the feedstuff to form additional
compounds that render the lysine biologically unavailable.
Methionine is spared by cystine. However, measurement of the
methionine content of feed proteins is not easy as the amino acid is
subject to oxidation during processing. After processing, methionine may
be present as such or as the sulphoxide or as the sulphone. The
sulphoxide may be formed from methionine during acid hydrolysis of the
feed protein prior to measurement of its any-no acid composition. Acid
hydrolysis of proteins before analysis disturbs the original equilibrium
between the two compounds so that the composition of the hydrolysate no
longer reflects that of the protein. In determining the methionine
content of pure proteins, oxidation of the amino acid to methionine
sulphone is normally quantitative. In the case of feed proteins,
however, this will not reveal how much methionine or methionine
sulphoxide was present in the protein prior to performate oxidation and
hydrolysis.
Methionine sulphoxide may have some biological value for fish which
may have some capability of reconverting it to methionine and thus
partially make up for some of the methionine oxidized during processing.
Methods have recently been reported for measurement of methionine in
proteins using an iodoplatinate reagent before and after reduction with
titanium trichloride, to give values for both methionine and the
sulphoxide in the original protein. A method for measuring methionine
specifically by cyanogen bromide cleavage has also been described. Both
methods remain to be independently assessed. Microbiological assay of
methionine in feed proteins is a valuable tool although there is the
danger that oxides of methionine may differ in their activity for
micro-organisms and misrepresent values.
QUANTITATIVE REQUIREMENTS OF AMINO ACID
Quantitative requirements by salmonids for the ten indispensable
amino acids were determined by feeding linear increments of one amino
acid at a time in a test diet containing an amino acid profile identical
with whole egg protein except for the amino acid tested. Replicate
groups of fish were fed the diet treatments until gross differences
appeared in the growth of test lots. An Almquist plot of growth response
indicated the level of amino acids required for maximum growth under
those specific test conditions. Diets were designed to contain protein
at or slightly below the optimum protein requirement for that species
and test condition to assure maximum utilization of the limiting amino
acid. A comparison of the requirements for the ten indispensable amino
acids between species is shown in Table 2.
A recent innovation has been the use in test diets of proteins
relatively deficient in a given essential amino acid. Thus combinations
of fishmeal and zein have been used in test diets to define the
requirement of rainbow trout for arginine. Diets containing different
relative amounts of casein and gelatin showed that an increase in the
level of protein-bound arginine from 11 to 17 g/kg resulted in a
significant increase in the growth of channel catfish.
Table 2 Amino Acid Requirements of Seven Animals 1/
|
Amino acid |
Eel fingerling |
Carp fry |
Channel catfish |
Chinook salmon fingerling |
Chick |
Young Pig |
Rat |
| Arginine |
3.9 (1.7/42) |
4.3 (1.65/38.5) |
|
6.0 (2.4/40) |
6.1 (1.1/18) |
1.5 (0.2/13) |
1.0 (0.2/19) |
| Histidine |
1.9 (0.8/42) |
|
|
1.8 (0.7/40) |
1.7 (0.3/18) |
1.5 (0.2/13) |
2.1 (0.4/19) |
| Isoleucine |
3.6 (1.5/42) |
2.6 (1.0/38.5) |
|
2.2 (0.9/41) |
4.4 (0.8/18) |
4.6 (0.6/13) |
3.9 (0.5/13) |
| Leucine |
4.1 (1.7/42) |
3.9 (1.5/38.5) |
|
3.9 (1.6/41) |
6.7 (1.2/18) |
4.6 (0.6/13) |
4.5 (0.9/19) |
| Lysine |
4.8 (2.0/42) |
|
5.1 (1.23/24.0) |
5.0 (2.0/40) |
6.1 (1.1/18) |
4.7 (0.65/13) |
5.4 (1.0/19) |
| Methionine 2/ |
4.5 (2.1/42) 3/ |
3.1 (1.2/38.5) |
2.3 (0.56/24.0) |
4.0 (1.6/40)3/ |
4.4 (0.8/18) |
3.0 (0.6/20) |
3.0 (0.6/20) |
| Phenylalanine 4/ |
|
|
|
5.1 (2.1/41)5/ |
7.2 (1.3/18) |
3.6 (0.45/13) |
5.3 (0.9/17) |
| Threonine |
3.6 (1.5/42) |
|
|
2.2 (0.9/40) |
3.3 (0.6/18) |
3.0 (0.4/13) |
3.1 (0.2/19) |
| Tryptophan |
1.0 (0.4/42) |
|
|
0.5 (0.2/40) |
1.1 (0.2/18) |
0.8 (0.2/25) |
1.0 (0.2/19) |
| Valine |
3.6 (1.5/42) |
|
|
3.2 (1.3/40) |
4.4 (0.8/18) |
3.1 (0.4/13) |
3.1 (0.4/13) |
1/ Expressed as percent of dietary protein. In
parentheses, the numerators are requirements as percent of dry diet,
and the denominators are percent total protein in the diet
2/ In the absence of cystine
3/ Methionine plus cystine
4/ In the absence of tyro sine
5/ Phenylalanine plus tyrosine
(Adapted from: National Research Council, 1977)
Arginine requirement of rainbow trout has been determined from a
conventional dose/response (growth) curve and also by measuring the
tissue (blood and muscle) levels of free arginine in groups of trout
given increasing amounts of dietary arginine. After the dietary
requirement of the trout for arginine has been met, any further increase
in arginine intake led to an increase in the concentration of free
arginine in blood and muscle. Good agreement was obtained between the
two methods.
The data shown in Table 2 suggest that real differences exist between
fish species in their requirement for certain amino acids. This leads to
difficulties in formulating the protein component of practical diets for
those species whose amino acid requirements are not yet known. A
possible solution is to use, for each amino acid, the highest level
required by any of those species for which data is available. The need
for further quantitative data on the amino acid requirements of fish,
especially those actually or potentially useful as farm animals, is
obvious.
SUPPLEMENTING DIETS WITH AMINO ACIDS
One solution to the use of proteins that are relatively deficient in
one or more amino acids is to supplement the protein with appropriate
amounts of the amino acid needed in practical diets. Fish appear to
utilize free amino acids at various degrees of efficiency.
Young carp, Cyprinus carpio, were shown to be unable to
grow on diets in which the protein component (casein, gelatin) was
replaced by a mixture of amino acids similar in overall composition. A
trypsin hydrolyzate of casein was equally ineffective. However, if a
diet containing free amino acids as the protein component is carefully
neutralized with NaOH to pH 6.5-6.7 then some growth of young carp does
occur. This growth was markedly inferior to that occurring on a
comparable casein diet under the same conditions.
Channel catfish are also unable to utilize free amino acids given as
supplements to deficient proteins. When soybean meal was substituted
isonitrogenously for menhaden meal, growth and feed efficiency of
channel catfish were substantially reduced. Addition of free methionine,
cystine or lysine, the most limiting amino acids, to these
soy-substituted diets did not enhance weight gain.
Raising the arginine level of catfish diets from 11 to 17 g/kg by
isonitrogenous substitution of gelatin for casein enhanced weight gain
significantly but the addition of free arginine, cystine, tryptophan or
methionine to casein had little effect on growth or food conversion.
Salmonids are able to utilize free amino acids for growth. A
zein-gelatin diet supplemented with lysine and trytophan was shown to be
markedly superior to an unsupplemented zein-gelatin diet for rainbow
trout when weight gain and protein utilization were used as criteria.
Several investigators have demonstrated the potential of
supplementing amino acid deficient proteins with limiting amino acids in
diets for salmonids. Casein supplemented with six amino acids produced
feed conversion ratios with Atlantic salmon similar to those obtained
when an isolated fish protein was used as the dietary protein source.
Soybean meal supplemented with five or more amino acids (including
methionine and lysine) was a superior protein source to soybean meal
alone for rainbow trout. Single additions of methionine and lysine did
not, however, improve the value of soybean meal. These results suggest
that the amino acid spectrum of the isolated fish protein they used may
possibly approximate the amino acid requirement of rainbow trout. The
nutritional value of a soy protein isolate could be enhanced by
supplementing it with the first limiting amino acid; i.e., methionine.
Diets containing, as protein component, fishmeal, meat and bone meal,
and yeast and soybean meal could be improved by supplementing with
cystine (10 g/kg) and tryptophan (5 g/kg) together. Fishmeal can be
entirely replaced without a reduction in food conversion rate in diets
for rainbow trout by a mixture of poultry by-product meal and feather
meal together with 17 g lysine HCL/kg, 4.8 g DL-methionine/kg, and 1.44
g DL-tryptophan/kg.
REFERENCES
Cowey, C.B. and J.R. Sargent, 1972 Fish nutrition. Adv.Mar.Biol.,
10:383-492
Cowey, C.B., 1979 Protein and amino acid requirements of finfish.
In Finfish nutrition and fishfeed technology, edited by J.E. Halver
and K. Tiews. Proceedings of a World Symposium sponsored by EIFAC/FAO,
ICES and IUNS, Hamburg, 20-23 June, 1978. Schr.Bundesforschungsanst.Fisch.,Hamb.,
(14/15)vol. 1:3-16
Mertz, E.T., 1972 The protein and amino acid needs. In Fish
nutrition, edited by J.E. Halver. New York, Academic Press, pp. 106-43
National Research Council, 1977 Subcommittee on Warmwater Fishes,
Nutrient requirements of warmwater fishes. Washington, D.C., National
Academy of Sciences (Nutrient requirements of domestic animals) 78 p.