Protein turnover, amino acid profile and amino acid flux in juvenile shrimp Litopenaeus vannamei: effects of dietary protein source
1 Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen
AB24 2TZ, Scotland, UK
2 INVE Technologies, Oeverstraat 7, B-9200 Baasrode, Belgium
3 Department of Molecular and Cell Biology, University of Aberdeen, Polwarth
Building, Aberdeen AB25 5ZD, Scotland, UK
4 Laboratory of Aquaculture and Artemia Reference Centre, University of
Ghent, Rozier 44, B-9000 Gent, Belgium
* Author for correspondence (e-mail: e.mente{at}abdn.ac.uk)
Accepted 24 June 2002
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Summary |
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Key words: shrimp, Litopenaeus vannamei, growth, protein synthesis, diet, amino acid flux, casein, protein turnover
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Introduction |
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Attempts have been made to optimise the utilisation of these proteins by
determining the optimal dietary amino acid profile
(Cowey and Forster, 1971).
This optimal dietary amino acid profile will depend on the amino acid
requirement of an animal for protein synthesis and the use of individual amino
acids as energy substrates or for other purposes
(Ronnestad and Fyhn, 1993
).
Deshimaru and Shigeno (1972
)
suggested that the amino acid composition of the food should be very similar
to that of the animal's proteins. Ogata et al.
(1985
) measured the total
essential free amino acid concentrations of the European eel Anguilla
anguilla and noted that it was correlated with dietary protein content.
To minimize the effect of different sample pretreatment, the A/E ratio (the
concentration of each essential amino acid as a percentage of the
concentration of total essential amino acids, including tyrosine) was
calculated in some studies. Arai
(1981
) based diets for coho
salmon fry upon A/E ratios found in the whole-animal tissue of this species.
Penaflorida (1989
) used the
profile of essential amino acids of whole shrimp to calculate an essential
amino acid index (the nth root of the product of the ratios of each
essential amino acid in the feed to that of a reference protein). Changes in
the levels of free amino acids in tissue after a meal have been used as a
criterion for determining amino acid requirements, based on the hypothesis
that the concentration of an individual free amino acid will remain low until
its requirement has been met (Wilson,
1994
).
Protein turnover can be divided into its constituent processes, protein
synthesis, protein growth and protein degradation (reviewed by
Houlihan, 1991). At any
particular time, protein growth (kg, protein growth as a
percentage of the total protein mass) is the net balance between protein
synthesis (ks) and protein degradation
(kd), i.e.
kg=ks-kd (Millward
et al., 1975
,
1976
). Preliminary results
from invertebrates suggest that high specific growth rates may be achieved by
relatively low rates of protein turnover (equivalent to protein degradation in
growing animals). In order to improve our understanding of protein metabolism
in crustaceans, the amino acid flux model may be useful (Houlihan et al.,
1995a
,
b
). At the heart of this model
is the relationship between dietary amino acid intake, the free amino acid
pool and the protein pool, linked via protein synthesis, protein
degradation and amino acid metabolism (Houlihan et al.,
1995a
,
b
). The model allows ratios
between its components to be calculated.
The aims of this study were to determine the effects of replacing fish meal
protein with soybean or casein protein in practical diets for shrimp and to
determine the effects of the different diets on the rates of protein
metabolism in shrimps. Models of amino acid flux and protein turnover with the
above different diets were constructed using the protein synthesis data. Four
experiments were performed. Experiment 1 was mainly a methodological trial to
verify the validity of the method used by Garlick et al.
(1980) for measuring protein
synthesis in shrimps. Experiment 2 examined the effect of dietary protein
source on growth and protein turnover in shrimps. Experiment 3 examined
whole-animal protein synthesis after a meal and RNA:protein concentrations, to
investigate whether starvation and refeeding altered synthesis and RNA
concentrations. Experiment 4 measured free amino acid levels in tissues of
L. vannamei at various times following feeding. Free amino acid
concentrations in L. vannamei tail muscle and whole animals were
investigated with respect to dietary amino acids.
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Materials and methods |
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The experiments were set up as a randomized complete block design, with
five blocks containing one replicate of three diets
(Coutteau et al., 1995). Each
block consisted of an independent recirculating system with six, 301 dark grey
rectangular tanks (rearing units), a mechanical filter, a biological filter, a
carbon filter and a water reservoir (100 dm3) equipped with heaters
and aeration. Approximately 10% of the water in each tank was replaced every
day with fresh seawater.
At the start of the experiment a control group of 50 shrimps were weighed,
killed and kept frozen at -80°C for estimation of initial protein content.
A total of 120 healthy juveniles were selected randomly, blotted dry in tissue
paper, weighed (224±0.01 mg wet mass, 51.58 mg dry mass) to the nearest
mg and transferred to the experimental tanks (rearing units). Each
experimental tank contained eight randomly chosen juveniles. The experimental
tank conditions were kept constant: temperature 27±1°C, photoperiod
12h:12h light:dark, salinity 30; [NH4+] and
[NO2-] never exceeded 0.5 and 0.4p.p.m, respectively.
The water circulation provided adequate levels of dissolved oxygen. The
experimental conditions remained stable throughout the experiment and were
within the limits normally considered acceptable for the growth and survival
of penaeid shrimps (Wickins,
1976
). Shrimps were hand-fed 12% of their body mass twice a day at
9:00 h and 17:00 h for a period of 28 days. Feeding rate was adjusted every
week. Faeces and any uneaten feed were siphoned daily prior to the first
feeding. After the 28th day, experiments 1-3 were conducted (see Experimental
design).
For measurement of the free amino acid (FAA) concentrations at various times following feeding (Experiment 4), four rearing units were used (in two separate blocks, block 6 and 7) each containing ten shrimps. The shrimps were starved for 1 week, then those in two tanks were fed diet 1 and in the other two tanks diet 3, for 14 days. The shrimps were offered food ad libitum once per day.
Experimental diets
The diets were prepared at the Artemia Reference Centre, according to a
method and formulation modified by Teshima et al.
(1982). A mixture of fish
meal, squid and shrimp powder (INVE Aquaculture, Belgium) was used to make the
protein source for diet 1 (45% protein), whilst for diet 2, half the
fish/squid/shrimp meal was replaced by soybean meal (45.2% protein) (INVE
Aquaculture, Belgium) (Table
1). Diet 3 was a casein-based microbound diet (powdered diet with
carrageenan as a binder) (44.5% protein). The three experimental diets were
designed to be isonitrogenous and isoenergetic. Diets were analysed for crude
protein using the Kjeldahl procedure
(Williams, 1984
). Total lipids
were extracted according to the method of Folch et al.
(1957
), modified for
freshwater invertebrates by Herbes and Allen
(1983
). Moisture and ash
content were estimated by difference in mass after 24 h in a drying oven at
60°C (moisture) and 6 h in a muffling furnace at 600°C (ash). Total
carbohydrates were obtained by adding the percentage values determined for
moisture, crude protein, lipid content and ash and subtracting the total from
100 (Tacon, 1990
). The
carbohydrates, lipids and protein were multiplied by their respective fuel
value (kal g-1) and the sum obtained defined the energy value of
the feed (Halver, 1989
). The
solubility of the diets was estimated from the loss of total dry matter after
rotating 0.25 g of the diet for 5 min and 60 min in 50 ml of distilled water,
centrifugation for 35 min at 3100 g and discarding the supernatants.
The water stability of diets 1, 2 and 3 varied from 69%, 74% and 69% after 5
min to 63%, 69% and 63%, respectively, after 60 min of emersion in deionized
water (Camara, 1994
).
|
Experimental design
Validation of the protein synthesis measurement (Experiment 1)
At the end of the experiment, individual shrimps (1-2 g wet mass) were
randomly selected and fractional rates of tail-muscle protein synthesis were
measured using the flooding-dose method. After an incorporation period of 10,
30, 60 and 120 min, eight shrimps were killed at each time point and samples
were taken and analysed as described below.
Protein turnover (Experiment 2)
At the end of the experiment, fractional rates of whole-animal protein
synthesis were measured in eight shrimps (final masses 1-2 g,
Fig. 2) randomly selected from
each of the three diets. These shrimps were fasted for 1 day before the
measurements. After an incorporation period of 1 h (see results for Experiment
1), samples of tail muscle and the remaining whole animal were taken and
analyzed as described below.
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Protein synthesis levels after a meal (Experiment 3)
On day 30, immediately before feeding (t=0) and 1, 2 and 4h after
feeding, fractional rates of whole-animal protein synthesis were measured in
five shrimps (at each time point) fed diet 1 using a flooding dose of
3H-phenylalanine. These shrimps were fasted for 24h before the
measurements. Five shrimps were denied food for 6 days (starved group). At the
end of the incorporation time, all the shrimps were killed, frozen in liquid
nitrogen and stored in -80°C until analysis as described below.
Amino acid levels after a meal (Experiment 4)
After 7 days without food, a group of ten shrimps (1.2 g) were removed,
killed and used as the prefeeding t=0 group. The remaining 30 shrimps
were fed normally and groups of five shrimps fed diet 1 and another five fed
diet 3, selected at random, were removed at 4, 9 and 24h after feeding. Each
shrimp was individually netted, removed and killed, and samples of tail muscle
were quickly dissected out. Tail-muscle tissue and the whole animal samples
were frozen in liquid nitrogen and stored at -80°C until analysis.
Measurements of growth rates, protein synthesis rates and RNA
concentrations
Whole-animal specific growth rates (SGR), expressed as a percentage
increase in body mass per day, were calculated for days 0-28 using the growth
rate equation of Ricker
(1979):
![]() | (1) |
Samples (100 mg) of the whole bodies were weighed and homogenised in 0.2
mol l-1 perchloric acid (PCA), and centrifuged, releasing
intracellular `free' (i.e. unbound) amino acids into the PCA-soluble fraction.
Following separation of this fraction by centrifugation, the supernatants were
retained for phenylalanine analysis as described by Houlihan et al.
(1995a,b
).
Protein content was determined (Lowry et
al., 1951
) as modified by Schacterle and Pollock
(1973
) after solubilisation in
0.3 mol l-1 NaOH. Whole-animal RNA content was measured, following
extraction, using the orcinol method
(Mejbaum, 1939
), comparing the
samples against known RNA standard (Type IV, calf liver, Sigma) concentrations
determined spectrophotometrically (absorbance at 665 nm, using quartz cuvettes
and a Perkin Elmer LamdaUV/vis spectrophotometer) and expressed as RNA:protein
concentration (µg RNA mg-1 protein). This was used as an
indication of the animal's capacity for protein synthesis. The concentrations
of phenylalanine in intracellular free-pools, protein pellets and the solution
injected into the animals were measured, using a fluorometric assay following
enzymatic conversion of the phenylalanine to ß-phenylethylamine (PEA)
(Houlihan et al., 1995a
).
Specific activities of the PEA from the intracellular free-pools, protein
pellets and injected solution were measured using a scintillation counter.
Fractional rates of protein synthesis (ks), the percentage
of the protein mass synthesised per day (% day-1), were estimated
using the flooding-dose method equation
(Garlick et al., 1980
;
Houlihan et al., 1988
):
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Amino acid profiles
The amino acid composition of the whole bodies was measured using the
method outlined in Lyndon et al.
(1993). Briefly, 200 mg
samples, taken from the tail muscle and whole bodies, were homogenised in 4 ml
absolute ethanol plus 100 µl aqueous norleucine (2.5 µmol
ml-1). The homogenate was centrifuged to pellet the precipitated
proteins. Duplicate samples of the supernatant were then taken to measure the
free-pool amino acid composition. A subsample of 100 µl was dried down
under vacuum and reconstituted in 100 µl 0.1 mol l-1 HCl and
filtered, and a 5 µl sample was taken for amino acid analysis, using an
Applied Biosystems 420A amino acid analyser. By this method, cysteine was only
20-30% recoverable and was therefore not quantified. A validation experiment
was conducted to check the solubility of the amino acids in 95% ethanol. Known
amounts of hydroxyproline, asparagine, glutamine, taurine, tryptophan and
norleucine were added to the supernatant described, at a concentration range
(over 1 nmol) known to be within the linear range of the amino acid analyser.
Recovery was 100%. The amounts of protein-bound amino acids (PAA) of the
tissues were determined after hydrolysis in 6 mol l-1 HCl
(Finn et al., 1995
) on samples
that had been extracted for FAA as described above. Tryptophan is destroyed by
acid hydrolysis, so separate samples of the tissues were prepared by alkaline
hydrolysis for analysis of the tryptophan content. Briefly, 50-75 mg samples,
taken from the tail muscle and whole bodies, were hydrolysed for 22h (4.2 mol
l-1 NaOH, 110°C) and filtered. 2.5 ml of filtrate sample with 1
ml internal standard solution were dried at 40°C. The residue was further
dissolved in 2.5 ml acetic acid and 10 µl of each of the samples were
analysed in a Kontron 450 data system analyser. The same procedure was
followed for analysing the diets.
Calculations
Amino acid results were expressed as µmoles of amino acid per gram of
diet (µmol g-1) and as grams per 100 g determined amino acid for
protein. The essential amino acid (A/E) ratio
(Arai, 1981) of each essential
amino acid (EAA) was calculated as the percentage of the total EAA. The
essential amino acid index (EAAI) of the two diets was determined from the
formula:
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Amino acid flux
In order to present a metabolic model for protein and amino acid metabolism
for the two diets, the model of Millward and Rivers
(1988) was used. The models
used here describe the amino acid flux for a 1.25 g and a 1.12 g L.
vannamei offered diets 1 and 3, at 28°C, at a consumption rate 4% of
the body mass (Gopal and Raj,
1990
). The assimilation efficiencies were assumed to be 90% for
marine meal protein and 91% for casein
(Fenucci et al., 1982
;
Dall, 1992
;
D'Abramo et al., 1997
).
Nitrogenous excretion was taken as 0.9 mg N g-1 day-1
(Wickins, 1985
). The nitrogen
protein content was calculated from the N content using a conversion factor of
5.85 (Gnaiger and Bitterlich,
1984
) and a protein equivalent of amino acid nitrogen was
calculated assuming that there are 9 mmol amino acids per g protein
(Houlihan et al., 1995c
).
Faecal amino acid losses were calculated as consumption minus absorption. The
total free amino acid (FAA) and protein concentrations were taken from direct
measurements of whole-animal tissues for both diets. Data of the fractional
rates of protein synthesis, protein growth and protein breakdown were used to
calculate the respective flux components.
Statistical analysis
All values are means ± S.E.M. and differences present at 5% level
(P<0.05) were considered significant. Data were compared by
Student's t-test, analysis of variance (ANOVA) followed, where
applicable, by Tukey's or Scheffe's multiple-comparison tests
(Zar, 1996). The
Fmax test for homogeneity of variances was used to
determine whether the assumption of equal variance was met. Pearson
correlation coefficients between dietary and tissue amino acid composition
were calculated according to Zar
(1996
).
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Results |
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Experiment 2
At the start of the experiment there were no significant differences in
means or range of initial body mass between shrimps fed the three diets
(ANOVA, P>0.05). There were no significant differences between the
final mean masses of shrimps fed diets 1 and 2 over the first 14 days and 24
days (ANOVA, P>0.05) (Fig.
2). However, the mean final masses of shrimps fed diet 3 on days
14 and 25 were significantly lower than the mean final masses of shrimps fed
diets 1 and 2 (ANOVA, P<0.05). The growth performance of shrimps
fed diet 1 (Table 2) was not
significantly different from those fed diet 2, but was significantly different
from those fed diet 3 (ANOVA, P<0.05). Survival rates of shrimps
were 98% (diet 1) to 93% (diet 2) (Table
2). Shrimps fed diet 3 had a significantly lower survival rate
than shrimps fed diets 1 or 2. The effect of diet quality on whole-animal
protein turnover is summarized in Table
2. Whole-animal fractional rates of protein synthesis
(ks, % day-1), were not significantly different
between diets 1, 2 and 3, but tended to be lower for diet 3 (ANOVA,
P>0.05). However, diet 3 had statistically significant lower
fractional protein growth rate (kg, % day-1)
and statistically significant higher fractional protein breakdown
(kd, % day-1) compared to diets 1 and 2 (ANOVA,
P<0.005).
|
The efficiency with which synthesised protein was retained as protein growth in the whole animal was similar for all the diets (80-94%; Table 2). A significant correlation was found between ks and kg for individual shrimps fed diet 1 (y=0.3379x+6.5468, r2=0.65, N=8), indicating that shrimps with a higher protein synthesis were more efficient in retained growth protein (P<0.05).
Experiment 3
The mean whole-animal fractional protein synthesis rates over the first 4 h
following feeding of diet 1 was 15% day-1. 1 h after feeding the
absolute protein synthesis rates of whole animals tended to increase, although
this was not statistically significant
(Fig. 3A), compared to those
starved for 24 h and 6 days. Fractional protein synthesis rates 2 h after
feeding were higher than in shrimps starved for 24 h and for 6 days, and
significantly different from those in shrimps 4 h after feeding. In the 6-day
and 24 h starved groups the low ks was accompanied by high
RNA:protein concentrations.
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The RNA:protein concentration increased significantly 2 h and 4 h after
feeding compared with the 6-day starvation group
(Fig. 3B). RNA:protein
concentration of the 6-day starved shrimps was not significantly different
from those starved for 24 h. RNA activity (kRNA) increased
significantly 4 h after feeding (ANOVA; P<0.05). The relationship
between ks and RNA:protein after feeding was significant
[y=-21.465+2.001xRNA:protein, N=5 (at each time point
for each diet), r2=0.48, P<0.05]. The slope of
the line (not shown) corresponds to the amount of protein synthesised per unit
RNA (ksx10/RNA)
(Millward et al., 1973). Thus
the increase in the slope after feeding indicates that there is an increase in
ks/RNA. A meal brings a significant increase in RNA
activity.
Experiment 4
The total FAA (EAA + NEAA) concentrations in the tail muscle and
whole-animal tissue of the unfed group were compared with those in the three
groups (4, 9 and 24 h after feeding) of shrimps fed diets 1 and 3
(Table 3). Generally, the mean
concentrations of total and non-essential whole-animal FAA for shrimps fed
diets 1 and 3 were not significantly different compared with the unfed
shrimps. However, with diet 3, 9 h after feeding there was a statistically
significant increase in the concentration of the essential FAA in the whole
animal (ANOVA; P<0.05). There were no statistically significant
differences between the mean concentrations of total, essential and
non-essential tail muscle FAA for the two dietary treatments following
feeding. A two-way analysis of variance showed that diet has a major effect on
the total essential amino acid concentration in the tail muscle
(P<0.03). The total EAA and NEAA concentrations in the tail muscle
and whole-animal tissues of the unfed group and those 24 h after feeding were
not significantly different from each other in any of the tissues.
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The changes in individual essential free amino acid concentrations after feeding are shown in Fig. 4. There was considerable variation in the profile of individual amino acids in the tail-muscle free pool and whole-animal over 24 h after feeding. Following feeding, there were significant increases in the concentrations of arginine, histidine, isoleucine, leucine, threonine and valine in whole animals fed diet 1 (Fig. 4B). In contrast, in whole animals fed diet 3 there were significant increases only in isoleucine, leucine and valine concentrations (Fig. 4C,D). Following feeding, the concentrations of valine, isoleucine, leucine and threonine increased significantly in the tail muscle for both dietary treatments. Arginine was the most abundant amino acid in the tail muscle and in whole animals. In both diet groups the most abundant EAAs in the tail-muscle free pool tended to be arginine, lysine, leucine and valine. In each tissue, tryptophan concentration was the lowest among the EAA and its level remained stable after feeding.
|
The concentrations of non-essential FAA in the white muscle and whole animal remained stable following feeding, and individual FAA exhibited few significant changes. Glycine, alanine, proline were the most abundant non-essential FAAs. In the tail muscle, asparagine, glycine, ornithine, hydroxyproline and tyrosine concentrations were significantly higher at 4 h after feeding. Although the total non-essential FAA concentrations in the whole animal did not change significantly following feeding diet 1, the concentration of six non-essential FAAs showed significant changes (P<0.05, Table 4). The concentrations of five NEFAAs changed significantly in shrimps after feeding diet 3 (P<0.05, Table 4). Taurine, tyrosine and alanine concentrations increased in tail muscle and reached a plateau after feeding, whilst concentrations of serine and asparagine peaked before decreasing again. Glycine was the most abundant free amino acid in animals fed with either diet, followed by alanine, proline and arginine. Taurine in whole animals decreased significantly after feeding with both diets. Generally, non-essential amino acids were found in the free form in larger amounts than essential amino acids.
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Amino acid profile of whole-animal protein
The amino acid profiles of the whole-animal protein from animals fed the
two diets did not differ significantly with respect to their essential amino
acid concentrations. However, when the amino acid compositions of the diets
were compared with the animals' protein, significant differences were found
for most of the non-essential amino acids in shrimps fed diet 3 (alanine,
asparagine, glycine, glutamine, serine and proline; ANOVA; P<0.05)
compared with the amino acid profiles in those fed diet 1. The essential
protein amino acid composition of whole-animal and tail-muscle protein of
shrimps fed diet 1 exhibited a higher correlation (whole animals,
r=0.88; tail muscle, r=0.97, P<0.01) than of
those fed diet 3 (whole animals, r=0.83; tail muscle,
r=0.86, P<0.05). Fig.
5 shows the relationship between the amino acid patterns of diets
1 and 3 and those in protein of whole animals fed these diets. The
relationship for diet 1 is close to the ideal line. The deviations from diet 3
for asparagine, alanine, threonine and arginine suggest a deficiency of these
amino acids in this diet. The assimilation rate of all amino acids between the
two diets was assumed to be the same.
|
To minimize the effects of different sample pretreatment and hydrolysing agents, the A/E ratio was calculated (Table 5). The A/E ratio for arginine in diet 3 appears to be unsatisfactory. The A/E ratio of L. vannamei fed diet 1 showed a decrease in methionine compared with P. monodon and P. japonicus (Table 5). The A/E ratio (Table 6) of the various animal protein sources generally showed higher arginine, lysine and methionine A/E ratios than those derived from plant sources. The common limiting amino acid for prawn diets utilising either animal or plant protein sources is arginine (Table 6). Only shrimp, squid meals and diet 1 contained arginine levels close to the ideal, confirming the observation that these are the best protein sources for L. vannamei.
|
|
Using the EAAI (Table 6),
based on the method of Penaflorida
(1989), a protein material was
assumed to be good quality with an EAAI of 0.90 or greater, to be useful when
it is approximately 0.80, and to be inadequate when it is below 0.70. Shrimp
meal, squid meal, white fish meal, diet 1 and tuna meal were the best protein
sources, with an EAAI of 0.98, 0.96, 0.96, 0.95 and 0.92, respectively.
Soybean meal was also a good quality source, while casein and diet 3 are
useful sources and the sweet potato meal is inadequate.
Amino acid flux
The daily amino acid fluxes of L. vannamei fed either diet 1 or
diet 3 are shown in Fig. 6. The
two groups were assumed to have consumed 0.22 mmol amino acid equivalents for
diet 1 or 0.21 mmol amino acid equivalents for diet 3. The amount of amino
acid that could be partitioned into protein synthesis were 59% and 57% of the
consumed amino acids for diets 1 and 3, respectively, which represents 52% and
75%, respectively, of the FAA pool. The amount of recycled amino acids derived
from protein degradation of the protein pool was estimated to be 2.12% and 7%,
respectively, for the two diets. The nitrogenous products excreted from the
free amino acid pool were calculated to be 32% for the group fed diet 1
compared with 63% for those fed diet 3. It was estimated from the measured
growth rates that the percentage of growth relative to consumption was 55% for
diet 1 and 43% for diet 3. The FAA pools were calculated as 53% and 38%,
respectively, of the protein pool.
|
![]() |
Discussion |
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The effect of diet quality on growth and protein synthesis rates
Several studies have examined the relationship between growth and diet
quality for Penaeus sp. Squid meal is known to be a good protein
source for prawns (Kitabayashi et al.,
1971; Deshimaru and Shigeno,
1972
; Fenucci et al.,
1980
). Both fish meal and soybean meal are regarded as high
quality protein and are highly digestible by shrimps; the apparent protein
digestibility of soybean meal is 90% and of fishmeal, 80.7% (Akiyama, 1989).
Akiyama et al. (1992
) also
reported that soybean meal has the best protein profile of all plant sources.
Replacement of 50% of the fish meal and shrimp meal by soybean produced higher
growth rates and better feed-conversion ratios in P. californiensis
(Colvin and Brand, 1977
). In
Macrobrachium rosenbergii diets, soybean meal was successfully used
to replace fish meal and shrimp meal
(Balazs and Ross, 1976
).
Fenucci et al. (1980
) replaced
50% of squid meal with a purified soy protein and obtained better growth,
survival and feed conversion ratios in P. setiferus and P.
stylioritis, while Akiyama
(1988
) indicated that soybean
meal at a level of 20 to 50% can replace fish and shrimp head meals without
affecting growth and survival of P. schmitti, P. setiferus and L.
vannamei. In contrast, Forster and Beard
(1973
) observed a growth
reduction of Palaemon serratus when all dietary fish meal was
replaced by soybean meal.
The effect of diet quality on in vivo rates of protein synthesis
has been little studied in crustaceans, although there are some studies in
fish (McCarthy, 1993; Carter
et al.,
1993a
,b
).
Despite the development of the flooding-dose method and the current interest
in alternative protein sources for shrimp diets, there have been few studies
on the effect of diet quality and protein turnover in juvenile shrimp. The
rate of muscle (tail) protein synthesis measured at 27°C in this study
(1.26% day-1) was higher than that measured for Carcinus
maenas carpopodite extensor muscle at a lower temperature (15°C,
1.15% day-1) El Haj and
Houlihan, 1987
) and for H. americanus claw muscle (0.385
day-1) (El Haj et al.,
1996
). Hewitt
(1992
) reports similar muscle
protein synthesis rates (0.9-1.4% day-1) in P. esculentus
(5 g wet mass) to those obtained in the present study at 30°C.
Protein turnover
In the present study, whole-animal synthesis rates were 5-9.75%
day-1, depending on the diet and the growth conditions. Fractional
protein-specific growth rate was higher in the shrimps fed the fish meal diet
(diet 1) than those fed the casein protein diet (diet 3). Shrimps fed the fish
meal diet and the 50% replacement diet with soybean meal showed enhanced
retention of protein via a decline in protein degradation, while the
casein diet decreased growth rate through an increase in protein degradation.
The mortality rate was also high with the casein diet. Therefore casein is
likely to be a poor protein source for crustaceans
(Lim et al., 1979;
Deshimaru, 1982
). Experiment 4
investigated whether a dietary amino acid balance is responsible for the
difference in protein turnover observed with the casein diet. The results show
that the low poor performance of the casein diet may be due to its low content
of arginine, an essential amino acid for protein synthesis.
In this study the efficiency of retention of synthesised protein
[(whole-animal growthx100/whole-animal synthesis),
kg/ks] was found to be as high as 94%
(diets 1, 2) and 81.8% (diet 3), which is much higher than in fish (trout
Oncorhynchus mykiss, 35-69%; plaice Pleuronectes platessa,
51%; salmon Salmo salar, 32%; cod Gadus morhua, 42%)
(Houlihan et al., 1995b).
Octopus vulgaris achieved high growth rates and very high retention
efficiencies by increasing the level of protein synthesis in combination with
very low rates of degradation (Houlihan et
al., 1990
). In Mytilus edulis, Hawkins
(1985
) found protein retention
efficiencies as high as 92%. Thus, protein turnover rates in invertebrates may
be much lower than in fish and mammals.
Fractional rates of whole-animal protein synthesis in these experiments
were found to peak 4 h after a meal. A stimulation in ks 3
h after a meal was reported for rainbow trout
(McMillan and Houlihan, 1988).
Protein synthesis rates also peaked 3 h after feeding in the shore crab
Carcinus maenas (Houlihan et al.,
1990
). In the present study, the fractional rates of protein
synthesis of the 6-day starved group was not significantly different from
those of after 24 h starvation. Protein synthesis rates are closely correlated
with growth rates and RNA: protein concentration
(Houlihan et al., 1988
), and
it is clear that the rate of protein synthesis relative to the RNA
concentration can be radically elevated after a meal
(Houlihan et al., 1990
). The
arrival of a meal brings a significant increase in the RNA activity
(kRNA). In this study the RNA activity followed the same
pattern as the protein synthesis (elevated at 2 h and 4 h after a meal).
Free amino acids in shrimps
The concentration of free amino acids (FAA) in most crustaceans is higher
than that in vertebrate tissues
(Claybrook, 1983), possibly
for osmoregulatory reasons (Awapara,
1962
; Claybrook,
1983
). The FAA profile for L. vannamei (intermoult stage)
is similar to that of P. kerathurus
(Torres, 1973
). The FAA
pattern in L. vannamei is also broadly similar to those in other
crustaceans and its concentration is comparable with those in Carcinus
maenas and Palaemon xiphias
(D'aniello, 1980
;
Claybrook, 1983
;
Dall and Smith, 1987
). In the
present study, however, there were some notable differences between the FAA
concentrations in whole animal and in tail-muscle tissue
(Watts, 1968
). The A/E ratio
for arginine in diet 3 (Table
5) would appear to be unsatisfactory, implying that adequate
arginine was not provided at the protein level in diet 3. In the present work,
tryptophan was consistently present at the lowest concentration of all amino
acids. Lyndon et al. (1992
,
1993
) found a significant
increase in tryptophan levels in white muscle 12 h after a meal in cod, and
correlated this increase with the protein synthesis rates at around this time.
Tryptophan is a candidate amino acid that limits the rate of protein
synthesis.
The present study demonstrated that although there was some variation in
the free pool concentration of individual amino acids, the total level of
essential and non-essential amino acids in the tail-muscle free pool remained
stable over 24 h for both diet groups. This suggests that intracellular amino
acid pools are not determined by passive movements of amino acids, but rather
are regulated by active transmembrane transport. The consequence of this is
that the tissue free pools are to some extent defended against sudden changes
in concentration, although clearly feeding does cause an increase, although
insignificant, in the tail-muscle free pools of both diets (4 h post-feeding).
Previous work showed peak concentrations of plasma amino acids 12 h after
feeding in pellet-fed rainbow trout
(Walton and Wilson, 1986) and
whole sandeel-fed Atlantic cod (Lyndon et
al., 1993
). We obtained similar results for whole-animal levels in
shrimps fed diet 1; however, when fed diet 3, there was a significant increase
9 h after feeding, which is difficult to explain, but may be due to the
combined effects of starvation and the inadequate nature of the diet. It has
been suggested that the changes observed in refeeding studies would probably
decrease with regular feeding, which would result in a more continuous supply
of absorbed amino acids to the body tissue
(Lyndon, 1990
).
Ratios of amino acids
The ratios of bound to free amino acids vary for the individual amino
acids, and are also specific for organs and, to some extent, for species
(Simon, 1989). The amino acid
pattern of the whole animal is mainly determined by the pattern of body
proteins, and changes in the free amino acid pattern are of negligible
influence. The results of the present study show that the concentrations of
free amino acids after a single meal are lower in relative amount than those
of protein-bound amino acids (Fig.
7), except for alanine, proline and glycine. Thus, free amino
acids that are precursors for protein synthesis and substrates for oxidation
appear to turn over at very high rates. In addition, the present study shows
that there is a low protein turnover in shrimps and that high growth rates are
achieved through efficient retention of synthesized proteins. Use of the
whole-animal amino-acid profile of an animal to determine its dietary protein
requirement was first considered by Philipps and Brockway
(1956
) in rainbow trout
(Salmo gairdneri) and by Deshimaru and Shigeno
(1972
) in crustaceans (P.
japonicus). A high correlation was assumed between the dietary profile of
essential amino acids and that in the whole-animal protein. Penaflorida
(1989
) used the profile of
essential amino acids of whole shrimp to calculate an essential amino acid
index (EAAI). However, when formulating diets, the EAAI should be supported by
feeding trials and digestibility tests to determine the incorporation of these
protein sources. In the present study, diet 1 was the best protein source with
an EAAI of 0.95, while diet 3 was useful with an EAAI of 0.85. Diet 3 had low
growth rates and low fractional protein synthesis rates, which is consistent
with the amino acid profile results.
|
Amino acid profile in the diets and in shrimps
In this study, for both diet 1 and 3 treatments, the percentages of
essential amino acids and total amino acids were found to be greater than
those reported by Farmanfarmaian and Lauterio
(1980), who fed M.
rosenbergii juveniles a commercial feed, and by Reed and D'Abramo
(1989
), who fed two standard
reference diets. The amino acid profile of the whole L. vannamei was
compared with those of the two diets. When compared to the whole-animal amino
acid profile, diet 1 was low in isoleucine, phenylalanine and valine, while
diet 3 was low in arginine, isoleucine, phenylalanine and threonine
(Table 5). The contributions of
asparagine and alanine were lower in diet 3 compared with diet 1
(Fig. 5). The amino acid
profile of diet 3 is rather imbalanced, which is consistent with the long-term
growth results. No significant correlation was found between diet amino-acid
profile and either whole-animal or tail-muscle free essential amino acids, up
to 24 h post-feeding. Most of the amino acids were abundant in diets but were
poorly represented in the whole animal after feeding. Thus, the FAA patterns
in the whole animal and tail muscle seem to be relatively stable. Reed and
D'Abramo (1989
) also found no
correlation between dietary amino acid composition and the concentrations of
FAA in the tissues (tail muscle and whole animal) of juvenile prawns M.
rosenbergii. Other studies have found that the plasma FAA pattern shows
the best correlation with the dietary amino acid pattern in fish 12 h after
food intake (Nose, 1972
;
Ogata, 1986
;
Lyndon et al., 1993
). There
are no differences apparent when comparing the protein amino acid profiles of
this study to those of other Penaeid sp.
(Table 4). However, increasing
arginine and lysine and decreasing isoleucine, leucine, valine and threonine
levels with growth stage in L. vannamei and P. japonicus has
been observed (Table 5). It is
usually accepted that the amino acid profiles of the whole-animal protein
differ little between and within species
(Wilson and Poe, 1985
;
Wilson, 1994
;
Ramseyer and Garling,
1994
).
Amino acid flux model
The amino acid flux models suggest a high food-conversion efficiency
(growth/intake) compared to the range of values between 20% in juvenile and
48% in adult fish (Houlihan et al.,
1995b), though it is not as high as the 63% found in larval
herring (Houlihan et al.,
1995b
) and 93% found in turbot larvae
(Conceição et al.,
1997b
). These high values apply to fast-growing larval fish, where
high protein efficiencies are expected. Estimates of protein conversion
efficiency in the present study should be treated with caution, since the
estimated amino acid consumption is based on literature values. Shrimp have a
nibbling feeding habit (Bordner and
Conklin, 1981
), and consumption measurements are not widely
available in the literature due to the difficulties in measuring food and
protein intake. Although there are a number of uncertainties, the amino acid
model in this study does point to differences between the two experimental
diets.
The amino acid flux diagram (Fig.
6) is comparable to the one published for larval herring
(Houlihan et al., 1995b). The
size of the FAA pool is 53% of the protein pool for diet 1 and 38% for diet 3,
compared to 29% in herring larvae and 2.3% in rainbow trout (wet mass 250 g).
This means that, on a daily basis, the dietary amino acid intake is 88% (diet
1) of the whole animal's FAA pool. This might imply that the arrival of the
ingested amino acids would have a relatively small effect on the FAA pool
composition in diet 1, while in diet 3 the daily dietary amino acid intake
represents almost the whole-animal FAA pool. In addition, protein synthesis
will remove on a daily basis the equivalent of 52% (diet 1) or 75% (diet 3) of
the FAA pool. We know that free pools remain relatively constant following a
meal (present study; Houlihan et al.,
1993
). Protein degradation will return to the FAA pool 4% (diet 1)
or 18.75% (diet 3) of its size. An amount equivalent to 60% of the absorbed
amino acid was incorporated into protein for diet 1 and 47% for diet 3,
respectively. The retention of synthesised protein was 92% (diet 1) and 75%
(diet 3). Amino acid losses were 32% (diet 1) and 63% (diet 3) of the FAA.
Thus, it appears that in shrimps there is a little scope for recycling of
essential amino acids if these become limiting in the diet. The present study
suggests that high growth rates seem to involve a reduction in the turnover of
proteins while amino acid losses appear to be high. The high amino-acid
oxidation may be related to the high FAA concentration. Lower protein turnover
may conserve energy for growth (or other processes), as protein turnover is
responsible for a large fraction of the energy budget
(Conceição, 1997a); however, high protein turnover allows fast
response of the organism to environmental/disease stress, through the
synthesis of specific enzymes and other proteins (Conceição,
1997b).
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References |
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