Directory UMM :Data Elmu:jurnal:A:Aquaculture:Vol185.Issue1-2.May2000:
Aquaculture 185 Ž2000. 101–120
www.elsevier.nlrlocateraqua-online
Growth and protein turnover in Atlantic salmon
žSalmo salar L. /; the effect of dietary protein level
and protein particle size
Harald Sveier a,) , Arnt Johan Raae b, Einar Lied c
a
NorAqua InnoÕation AS, N-4335 Dirdal, Norway
Institute of Molecular Biology, Høyteknologisenteret, N-5000 Bergen, Norway
Institute of Nutrition, Directorate of Fisheries, Box 185, N-5804 Bergen, Norway
b
c
Accepted 21 October 1999
Abstract
In modern fish feeds, the protein sources consist of denaturated finely ground ingredients.
From the literature, it has been reported that use of coarsely chopped, but not denaturated, fish as
the dietary protein source gave better growth performance and protein utilisation. Growth, feed
utilisation and protein turnover using two different fish meal particle sizes Žmicro- or coarsegrounded. at three dietary protein concentrations Ž30%, 35%, and 45%. were studied in individually tagged Atlantic salmon in a 3-month growth experiment. At the end of the experimental
period, 14C-L-lysine was injected intraperitonally and dorsal muscle samples were taken at 2- and
4-h post-injection. Incorporation of 14C-L-lysine into muscle protein, RNA, DNA and water
soluble protein was analysed from samples of muscle tissue. Only small effects on growth rate,
feed conversion rate, protein and energy retention, and nitrogen and fat digestion were found.
During the growth experiment, large individual variations in growth rates were observed, which
did not correlate to the initial body weight. The total RNA content expressed as RNA amount per
unit of DNA ŽRNA:DNA ratio. did not reflect the specific RNA activity, and individual growth
rate was not correlated to the specific RNA activity or RNA:DNA ratio and only poorly to the
relative incorporation rate of amino acids. Growth rate was, however, correlated to the relative
efficiency of protein synthesis. The results indicate that the protein catabolism is more important
)
Corresponding author. Tel.: q47-51-61-1700; fax: q47-51-61-6112.
E-mail address: hsv@noraqua.no ŽH. Sveier..
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 4 4 - 0
102
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
for net protein deposition and growth than protein anabolism. q 2000 Elsevier Science B.V. All
rights reserved.
Keywords: Fish meal; Particle size; RNA:DNA ratio; Protein synthesis; Specific RNA activity; Individual
growth rate
1. Introduction
Modern fish feeds comprise finely ground ingredients bonded together during the
gelatinisation of starch when exposed to mechanical shear, steam and pressure in the
extrusion process. The question has been raised on whether the physical state Že.g.,
particle size or friability. of food particles may affect growth and feed utilisation by
affecting the gastrointestinal transit time ŽJobling, 1986, 1988; dos Santos et al., 1993..
Sveier et al. Ž1999. examined the effect of feeding diets consisting of fish meal ground
into three different particle sizes at two different protein concentrations on growth, feed
utilisation and gastrointestinal transit time, and found no significant effects except for
the evacuation time in the stomach. Gastric emptying rate or solubility of the protein has
been shown to affect utilisation of dietary protein Ždos Santos and Jobling, 1991; Espe
and Lied, 1994; Espe et al., 1992, 1999; Schuhmacher et al., 1997, de la Higuera et al.,
1998.. Boirie et al. Ž1997. demonstrated that the rate of absorption of amino acids from
the gut from a casein or whey protein-based diets affects protein synthesis within the
whole body protein catabolism and oxidation of amino acids.
The major part of the weight increase is related to the deposition of protein. The
protein accretion is a balance between protein anabolism and catabolism. Protein
catabolism occurs through different mechanisms and there is no clear method for
estimating total protein catabolism in whole body or selected tissues ŽHoulihan et al.,
1993.. The extent of protein synthesis is, in principle, correlated to the amount of
ribosomal RNA ŽrRNA. which contributes approximately 85% of the total cellular RNA
species, available amino acids and it is under endocrine control. The concentration of
total ribonucleic acid ŽRNA. can be expressed as Žmg RNA. Žg tissue.y1 while Žmg
water soluble protein. Žmg RNA.y1 is an index for ribosomal activity. The RNA:DNA
ratio reflects the amount of RNA per cell and is a more reliable quantification compared
to the grams of tissue. The RNA content in white muscle tissue has been shown to be
directly related to protein synthesis and growth in a number of species ŽGoldspink and
Kelly, 1984; Houlihan et al., 1993; Valente et al., 1998.. The specific RNA activity and
the protein synthesis efficiency can be measured in animals using the flooding dose
method where the incorporation of a labelled amino acid into newly synthesised protein
is measured ŽGarlick et al., 1980.. The efficiency of RNA translation may change
significantly after a meal ŽLyndon, 1990.. Only a few studies have compared direct
protein synthesis measurement and RNA concentration in fish tissues ŽHoulihan et al.,
1993..
In the present work, an experiment with Atlantic salmon was performed in which the
dietary protein sources comprised of coarse- or micro-ground fish meal compared at
three different dietary protein levels. The aim of the study was to examine any dietary
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
103
effects of these factors on growth, protein turnover, specific RNA activity and feed
utilisation. Further, the individual variation and response on the same factors was
investigated.
2. Material and methods
2.1. Experimental conditions
The experiment was carried at NorAqua Innovation research station in Dirdal in the
Southwestern part of Norway. Atlantic salmon Ž Salmo salar L.. of the NLA ŽNorsk
Lakseavl, Kyrksæterøra, Norway. strain, hatched in February 1996, and smolted during
the autumn 1996 was used. Groups of 20 fish were adapted to the experimental
conditions for 4 weeks prior to the experiment. Initially, 18 fish averaging 336 " 41 g
Ž n s 324, 1 STD. were randomly assigned to 18 tanks with a water volume of 0.5 m3.
The six experimental diets were randomly assigned to the tanks using three replicates
per diet. Seawater Žsalinity 30.0 " 1.1‰. at 7.6 " 0.28C was used throughout the
experiment. Temperature and salinity were recorded daily, while the oxygen saturation
in the water outlet was recorded and adjusted weekly to ensure a minimum level of 7 mg
ly1 . The fish were exposed to continuous light from smoltification until the end of the
experiment.
The fish were fed in excess three times daily using automatic feeders for 96 days.
First meal from 2000 to 2100 h Ž20%., second meal from 0200 to 0300 h Ž20%. and
third meal from 0700 to 0800 h Ž60%.. A mechanical system for collecting uneaten feed
was installed at the outlet of each tank. The collection system was a moving mesh belt,
which removed the uneaten feed from the outlet water and into a collection box ŽSveier
et al., 1997..
2.2. Diet composition
The fish meals used in diet formulation were the same as that of Sveier et al. Ž1999..
The fish meals used were produced from the same batch of herring Ž Clupea harengus,
L.. fillet by-product by The Norwegian Herring Oil and Meal Industry Research
Institute, Bergen, Norway. After processing, the fish meal was milled and separated into
two parts by sieving. The batches consisted of coarse Ž3–5 mm. and micronised Ž78%,
0.3–0.1 mm. particle sizes. The chemical composition of the crude fish meal met the
specification of Norse-LT94 w as defined by the Norwegian Herring Oil and Meal
Industry Research Institute.
Each protein particle size was tested at three protein levels Ž30%, 35%, and 45%. at a
fixed oil concentration Ž27%.. The diets were produced as 9 mm extruded pellets, which
were crumbled and sieved to 5-mm pellet size. The extrusion process will, to some
extent, reduce the fish meal particle sizes. However, visual inspection of the pellets
showed large fish meal particles in the pellets when using coarse-ground fish meal.
Yttrium oxide was used as a marker in all diets for the determination of digestibility.
Diet composition data, results of chemical analyses and calculations are given in Table
1.
104
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Table 1
Dietary ingredients, chemical analysis and calculated values of the experimental diets
Coarse-ground
Micro-ground
30% protein 35% protein 45% protein 30% protein 35% protein 45% protein
Ingredients (g kg y 1)
Fish meal
Wheat flour
Suprex maize
Mono ammonium
phosphate
Capelin oil
ŽNorsamoil.
Premix a
Žvitamin, mineral,
betain, pigment.
Indicator ŽY2 O 3 .
Žmg kgy1 .
405
171
170
50
494
130
130
50
670
49
50
50
405
171
170
50
494
130
130
50
670
49
50
50
240
232
217
240
232
217
9.23
9.23
9.23
9.23
9.23
9.23
100
100
100
100
100
100
Chemical composition
(g kg y 1)
Protein Ž N =6.25.
Fat ŽSoxhlet.
Ash
Water
Rest b
304
241
65
78
312
389
263
79
31
238
456
269
110
58
107
301
280
69
93
257
389
287
79
72
176
445
282
105
61
107
Calculations
Gross energy ŽMJ kgy1 . c
Starch Žg kgy1 . d
22.1
216
23.7
165
22.6
216
23.5
165
23.3
63
23.5
63
a
Vitamins and minerals according to or higher than recommended by NRC Ž1993.. Betain Ž78.6%.:
0.128% of diet; Charphyll pink: 0.3581% of diet.
b
Rests NFEs100yŽproteinqfatqashqwater..
c
Calculated using the following values: Fat: 39.5 MJ kgy1 , Protein: 23.7 MJ kgy1 , Carbohydrate: 17.2 MJ
kgy1 .
d
Calculated based on the prescription using 57% starch in wheat flour and 70% starch in suprex maize.
2.3. Fish sampling and sample treatment
The fish was labelled intraperitoneally with a passive integrated transponder ŽPIT. tag
ŽBioSonics, Seattle, WA, USA. ŽCohen et al., 1989. 4 weeks before the experiment
started. Prior to the experiment, feed was withheld for 4 days. Three samples each of
five individuals were randomly removed and used for initial whole body analysis of fat
and protein. The remaining 18 fish per tank were then weighed individually, and the
corresponding PIT tag recorded. At the end of the experiment, faeces was stripped from
all fish according to Austreng Ž1978.. Feed was withheld for 4 days before all fish were
individual weighed and the corresponding PIT tag recorded. Six fish per tank were
sampled for whole body analysis of fat and protein and an additional six fish were used
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
105
for analyses of fat in cutlet according to the Norwegian quality cut ŽNQC, NS 9401,
1994. and measurement of body traits.
For measurement of fractional protein synthesis rate, a modification of Garlick’s
method was used ŽGarlick et al., 1980.. The remaining fish were fed for 2 weeks. From
each tank four to six fish were kept in ice water for 5 min before weighing and injected
intraperitonally with 2 mCi U 100 gy1 body weight 14 C-L-lysine ŽDu Pont de Nemours,
Belgium.. Two or three fish from each tank were killed by a blow on the head 2 or 4 h
after isotope injection. A muscle sample was removed from a segment of the epaxial
muscle under the dorsal fin and immediately frozen. Epaxial muscle tissue samples were
analysed for radioactivity in both trichloroacetic acid ŽTCA. soluble and non-soluble
fractions, and values obtained were used to calculate protein deposition and efficiency of
protein synthesis. The dose and sampling time were determined in a preliminary
experiment to detect the period of time where there was a linear incorporation of
14
C-L-lysine, using fish from the same batch as the experimental fish. The sample was
also used for analysis of RNA, DNA and water soluble protein.
All samples for chemical analysis were taken as pooled samples per tank except
RNA, DNA, soluble protein and 14 C-lysine which was analysed on an individual basis.
2.4. Chemical analyses
The diets were analysed in duplicate for dry matter, protein, fat and ash. Protein
Ž N = 6.25. in feeds was determined colorimetrically in micro Kjeldahl digests according
to Crooke and Simpson Ž1971.. Fat was determined gravimetrically after extraction with
ethyl acetate, dry matter after drying at 1058C for 24 h and ash after combustion at
5508C for 16 h. Protein in fish and faeces was analysed with a nitrogen gas analysator
ŽPerkin Elmer Series II Nitrogen analyzer 2410. according to the manufacturer’s
manual. Fat in the cutlet ŽNQC. and faeces was analysed gravimetrically after extraction
with ethyl acetate. Yttrium oxide ŽYt 2 O 3 . in feed and faeces was analysed using
ICP-MS by the Institute of Nutrition, Directorate of Fisheries, Bergen, Norway. RNA
and DNA were measured according to Boer Ž1975. and Raae et al. Ž1988. using
ethidium bromide. Water soluble protein in the same samples were measured essentially
by Biuret method ŽDawson et al., 1986..
Isotope activity was measured in muscle homogenates, in the precipitate and in the
supernatant after treatment with 20% TCA. The samples were solubilised in Soluene-350
overnight and counted in a scintillation counter in Hi Ionic Fluor according to Berge et
al. Ž1994..
2.5. Calculation and statistics
Specific growth rate ŽSGR. was calculated as:
SGR s 100 Ž lnW2 y lnW1 . ny1
where W1 and W2 are the initial and final weight, respectively, and n is the number of
days in the feeding period.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
106
Feed intake ŽFI. was calculated as ŽHelland et al., 1996.:
FI s Ž feed offered. y Ž feed collected. Ž corrected for dry matter loss .
= Ž collection efficiency.
y1
Feed conversion ratio ŽFCR. was calculated as:
FCR s FI Ž B2 q Bdead y B1 .
y1
where B1 and B2 are the biomass at the start and end, respectively, and Bdead is the
biomass of the dead fish.
Dressing out percentage ŽDOP. was calculated as:
ž
/
y1
DOP s 1 y Ž BWgutted BWungutted
. 100
where BWgutted and BWungutted are the weights of gutted and ungutted unbled fish,
respectively.
Productive protein value ŽPPV. was calculated as:
PPV s Ž P2 y P1 . PIy1
where P1 and P2 are estimates of protein content of the biomass at the start and end of
the experiment and PI is the protein intake.
Productive energy value ŽPEV. was calculated as:
PEV s Ž E2 y E1 . EIy1
where E1 and E2 are estimates of energy content of the biomass at the start and end of
the experiment, and EI is the energy intake.
Apparent digestibility ŽAD. was calculated as:
ž
AD s 100 1 y Ž Ifeed Nfaeces . Ž Ifaeces Nfeed .
y1
/
where Ifeed and Ifaeces are the concentrations of marker in the feed and faeces, and Nfeed
and Nfaeces are the nutrient concentrations in the feed and faeces, respectively ŽAustreng,
1978..
Protein synthesis efficiency was calculated as incorporation of 14 C-Lys into muscle
tissue protein according to Garlick et al. Ž1980. and Carter et al. Ž1993..
Fractional protein synthesis: K S s w SB Ž t 2. y S B Ž t1.rSAŽ t 2 y t1.xw100rŽ t 2 y t1.x. In
which S B Ž t 2. s protein bound isotope at 4 h, SB Ž t1. s protein bound isotope at 2 h,
SAŽ t 2 y t1. s mean pool of non-protein bound isotope Že.g., mainly free 14 C-L- Lys. in
the experimental period.
Protein growth rate Ž K g . s Žln P2 y ln P1 . ny1 = 100 where P1 and P2 are the
initial and final protein content, respectively, and n is the number of days in the feeding
period.
Coarse-ground
30% protein
35% protein
45% protein
Micro-ground
30% protein
35% protein
45% protein
Protein level
30% protein
35% protein
45% protein
Physical quality Micro-ground
Coarse-ground
Interactions
P-value
1
n Initial weight Žg. SGR Ž% dayy1 . FCR
PPV
3
3
3
3
3
3
6
6
6
9
9
0.45"0.05
0.48"0.04
0.36"0.00 1
0.44"0.011
0.43"0.01
0.56"0.04
0.39"0.01
0.46"0.03
0.35"0.04
0.44"0.05
0.44"0.041
0.62"0.00 1
0.42"0.02 Ž6. 0.47 B "0.02 Ž6.
0.36"0.02 Ž5. 0.44 B "0.03 Ž5.
0.43"0.02 Ž5. 0.58 A "0.03 Ž5.
0.39"0.02 Ž8.
0.50"0.02 Ž8.
0.41"0.02 Ž8.
0.49"0.02 Ž8.
n.s.
n.s.
342"4
339"10
330"9
349"6
326"5
334"7
346"5
333"5
332"5
336"4.1
337"4.1
n.s.
0.63"0.09
0.71"0.02
0.68"0.06
0.60"0.03
0.68"0.08
0.80"0.05
0.61"0.04
0.70"0.04
0.74"0.04
0.69"0.04
0.67"0.04
n.s.
1.09"0.08
1.05"0.031
0.80"0.01
1.10"0.05
1.07"0.01
0.81"0.041
1.10 B "0.04 Ž6.
1.06 B "0.04 Ž5.
0.80 A "0.04 Ž5.
0.99"0.04 Ž8.
0.98"0.04 Ž8.
n.s.
PEV
ADnitrogen Ž%.
ADfat Ž%.
85.8"0.54
86.0"0.89
87.2"0.48
86.9"1.40
87.5"0.42
88.4"0.35
86.3"0.51
86.8"0.51
87.8"0.51
87.6"0.41 Ž9.
86.3"0.41 Ž9.
n.s.
89.6"0.89
90.2"1.3
90.8"0.87
90.8"0.95
91.6"0.49
91.0"0.11
90.2"0.56 Ž6.
90.9"0.56 Ž6.
90.8"0.62 Ž5.
91.1"0.49 Ž8.
90.2"0.49 Ž9.
n.s.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Table 2
Growth and feed utilisation expressed as SGR, FCR, PPV, PEV, apparent digestibility of nitrogen and fat ŽADnitrogen and ADfat .. Values are presented for the ground
grades as mean"s.e., for protein level and physical quality as least squares means"SEM
ns 2. Significant differences within protein levels are denoted with capital letters and between protein levels with small letters.
107
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
108
Protein degradation rate Ž K d . s K S y K g ; Muscle mass s Žwhole body wet weight.
60% ŽLied, pers.med..; Protein synthesised: Ž K S .Žmuscle mass..
Protein catabolisms Protein anabolismy K d .
Deposited proteins Žmuscle mass.Ž K d ..
Relative protein synthesis efficiency: ŽDeposited proteinrprotein synthesised.100.
Relative amino acid incorporation rate per hour on tank level was calculated as:
Ž pmol
14
C lysine 4 h y pmol
14
C lysine 2 h . 2y1 .
For correlation of the individual relative amino acid incorporation rate with the
individual SGR, the relative amino acid incorporation per hour was calculated as:
Ž pmol
14
C lysine 2 h . 2y1 and Ž pmol
14
C lysine 4 h . 4y1 .
The specific RNA activity was calculated as:
Ž Relative amino acid incorporation rate per hour. RNAy1 .
All data related to feed levels were statistically processed by a general linear model
ŽGLM. procedure. The data were tested for interaction between protein level and protein
particle size; when no interaction effects was found, it was removed from the model.
Where significant Ž P - 0.05. differences were achieved a Tukey HSD multiple range
test was used to rank the means. All data are presented as average" SEM. Regression
analysis of individual data was performed using simple regression tested for best-fitted
model. Multiple regression was tested. The software Statgraphics, version 3.1 ŽStatistical
Graphics, Manugistics, MD, USA.. was used.
3. Results
3.1. Fish meal and diet quality
The chemical and biological quality of the fish meal used in the experiments met the
specifications of Norse-LT 94 w Ždefined by the Norwegian Herring Oil and Meal
Fig. 1. Initial wet weight of the fish and the specific growth rate after a 12 weeks growth period.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
109
Table 3
Body composition expressed as DOP, protein in whole fish, fat in cutlet ŽNQC. and change in body fat per
kilogram of growth. Values are present for the ground grades as mean"S.E., for protein level and physical
quality as mean"SEM
Coarse-ground
Micro-ground
Protein level
Physical quality
Interactions
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
Micro-ground
Coarse-ground
P-value
n
DOP Ž%.
Protein end
Ž%. in whole
fish
NQC Ž% fat
in cutlet.
Change in
body fat per
kilogram
of growth
3
3
3
3
3
3
6
6
6
9
9
10.6"1.1
11.4"0.2
11.2"0.2
10.4"0.4
10.6"0.8
10.3"0.9
10.5"0.5
11.0"0.5
10.7"0.5
10.4"0.4
11.1"0.4
n.s.
16.9"0.1
16.7"0.1
17.2"0.2
16.1"0.4
16.6"0.1
16.7"0.2
16.5"0.1
16.6"0.1
17.0"0.1
16.5 A "0.1
16.9 B "0.1
n.s.
9.8"0.6
9.6"0.4
8.7"0.6
10.4"0.6
9.9"0.3
10.3"0.3
10.1"0.3
9.8"0.3
9.5"0.3
10.2"0.3
9.4"0.3
n.s.
13.5"3.1
9.8"0.6
8.5"2.0
15.1"0.9
11.6"1.7
12.3"1.2
14.3"1.2
10.7"1.2
10.4"1.2
10.6"1.0
13.0"1.0
n.s.
Significant differences within protein levels are denoted with capital letters and between protein levels with
small letters.
Industry Research Institute, Bergen, Norway. except for slightly lower protein and
higher ash content due to the higher bone content from the herring by-product used as
Table 4
Nucleotide levels in epaxial muscle tissue of Atlantic salmon fed different experimental diets. Values are
presented for the ground grades as mean"s.e., for protein level and physical quality as least squares
means"SEM Ž n.
Coarse-ground
Micro-ground
Protein level
Physical quality
Interactions
a
ns 2.
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
Micro-ground
Coarse-ground
P-value
n
DNA Žmg g
muscley1 .
RNA:DNA
ratio
Water soluble
protein:RNA
Žmg mgy1 .
Specific RNA
activity
Žpmol h mgy1 .
3
3
3
3
3
3
6
6
6
9
9
5.7"1.3
7.1"0.8
5.6"0.4
7.9"1.5
7.1"1.3
6.1"0.1
6.8"0.7
7.1"0.7
5.8"0.7
7.0"0.6
6.2"0.6
n.s.
7.3"2.5
4.2"0.5
7.2"1.7
2.9"0.5
3.5"0.9
5.7"1.1
5.1"1.0
3.8"1.0
6.5"1.0
4.0"0.8
6.2"0.8
n.s.
4.1"0.7
8.3"3.0
5.5"3.0 a
6.1"1.1
4.7"2.4 a
2.9"1.2
5.1"1.3 Ž6.
6.7"1.5 Ž5.
4.0"1.5 Ž5.
4.7"1.1 Ž8.
5.8"1.1 Ž8.
n.s.
0.04"0.04
0.05"0.02
0.04"0.01
0.14"0.08
0.05"0.02
0.04"0.05
0.09"0.019
0.05"0.019
0.04"0.019
0.08"0.015
0.04"0.015
n.s.
110
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Fig. 2. Ža. Correlation between RNA concentration in muscle expressed as RNA:DNA ratio and the specific
RNA activity measured as the incorporation of 14 C-L-lysine into muscle protein per unit RNA Žpmol mgy1 .
Ž Y s1Žy17.71q10.74 X .y1 , r s 0.575, P s 0.019.. Žb. Specific RNA activity vs. SGR Ž% dayy1 .. All data
on tank levels.
the raw material source. The experimental diets showed no major difference between
calculated and analysed values ŽTable 1..
3.2. Growth and feed utilisation
Growth and feed utilisation data are presented in Table 2. Fish body weight increased
by about 90% on average during the experiment. SGR and protein retention ŽPPV. data
were not affected by dietary protein level or fish meal particle size. FCR and energy
retention ŽPEV. data were significantly affected by dietary protein level, but not fish
Fig. 3. Ža. Specific RNA activity Žpmol mgy1 . vs. SGR Ž% dayy1 . achieved using individual data. Žb.
RNA:DNA ratio vs. specific RNA activity Žpmol mgy1 . using individual data Ž Y s 0.277y0.186 X q
0.041 X 2 y0.0028 X 3 , r s 0.785, P s 0.000.. Žc. Water soluble protein:RNA vs. specific RNA activity Žpmol
mgy1 . using individual data Ž Y s 29.819 X 0.553 , r s 0.807, P s 0.000..
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
111
112
Table 5
Incorporation of amino acids in terms of 14 C-L-lysine in epaxial muscle tissue of Atlantic salmon fed experimental diets. Values are presented for the ground grades as
mean"s.e., for protein level and physical quality as mean"SEM
Coarse-ground
30% protein
35% protein
45% protein
Micro-ground
30% protein
35% protein
45% protein
Protein level
30% protein
35% protein
45% protein
Physical quality Micro-ground
Coarse-ground
Interactions
P-value
3
3
3
3
3
3
6
6
6
9
9
40"3
39"1
44"5
43"4
48"4
40"6
41"3
44"3
41"3
43"2
40"2
n.s.
45"4
48"5
49"1
50"4
50"4
51"3
48"3
49"3
53"3
52"2
47"2
n.s.
0.25"0.02 2
1.4"0.7
1.26"0.03 2
3.0"0.3
2.0"0.5 2
0.9"0.3
1.6"0.3
1.7"0.3
1.1"0.3
2.0"0.3 a
1.0"0.3 b
0.02
Significant differences between physical qualities are denoted with small letters.
1
Percentage of injected activity.
2
ns 2.
3
ns8.
1.24"0.06 2
7.5"2.8
4.1"1.0
14.8"2.2
6.8"1.4 2
3.7"1.9
5.0"1.5 3
7.1"1.5 3
3.9"1.3
8.4"1.2 a,3
4.3"1.2 b,3
0.01
0.71"0.10 2
6.8"2.8
3.5"1.0
14.3"2.2
4.0"2.4
3.0"1.8
7.5"1.6 2
5.4"1.5
3.3"1.5
7.1"1.2
3.7"1.3 3
n.s.
29"25
14"14
18"12
3.2"0.8
9"5
32"36
16"9
11"9
27"9
17"7
21"7
n.s.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
n Activity in
Activity in
Relative amino
Fractional protein Protein degradation Relative protein
muscle tissue
muscle tissue
acid incorporation
synthesis rate
rate Ž K d , % dayy1 . synthesis efficiency
1
1
y1
y1
y1
Ž% retained of
2 h post-injection 4 h post-injection rate Žpmol g h . Ž K S , % day .
synthesised.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
113
meal particle size. The higher the dietary protein Žand energy. level, the lower the FCR
value observed. Digestion of nitrogen and fat was not affected by protein level or fish
meal particle size. There was no correlation between initial weight and SGR achieved in
this experiment ŽFig. 1..
3.3. Body traits
The body trait data are listed in Table 3. Whole body protein content was not
influenced by the protein level, but there were significantly higher protein levels in the
group fed the coarse-ground fish meal. Whole body fat deposition was not affected by
the protein level or fish meal particle size. DOP and fat in cutlet ŽNQC. showed no
significant effect of protein level or fish meal particle size.
Fig. 4. Ža. Relative protein synthesis efficiency Ž%. vs. SGR Ž% dayy1 . Ž Y s 0.542q0.068 ln X, r s 0.741,
P s 0.001.. Žb. Relative amino acid incorporation rate Žpmol Žg t.y1 . vs. SGR Ž% dayy1 . Ž Y s 0.827y0.144 X,
r sy0.333, P s 0.002..
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
114
3.4. Nucleic acids and protein turnoÕer
The amount of RNA expressed as the RNA:DNA ratio and specific RNA activity was
not influenced by fish meal particle size or protein level ŽTable 4.. There was a
correlation between specific RNA activity and RNA:DNA ratio ŽFig. 2a. until the
RNA:DNA ratio reached a level of about 4. Beyond an RNA:DNA ratio of 4, there were
only minor changes. There was no correlation between the specific RNA activity and
SGR ŽFig. 2b..
Regression analysis using the individual data showed no correlation between specific
RNA activity and SGR ŽFig. 3a.. However, a high correlation between specific RNA
activity and RNA:DNA ratio up to an RNA:DNA ratio of about 3 ŽFig. 3b. was
observed. A high correlation between the specific RNA activity and water soluble
protein:RNA ratio as an index of ribosomal activity was found ŽFig. 3c..
3.5.
14
C-lysine incorporation and protein turnoÕer
The relative efficiency of protein synthesis measured as incorporation of 14 C-L-Lys,
was not influenced by protein level or the fish meal particle size ŽTable 5.. There was,
however, a high correlation with growth ŽFig. 4a.. Dietary protein level did not affect
the incorporation of 14 C-L-Lys into muscle, but there was a significant effect on the
physical qualities of the fish meal ŽTable 5.. Using data from individual fish, a negative
correlation between SGR and 14 C-L-Lys incorporation rate was observed ŽFig. 4b..
4. Discussion
In the present experiment, normal growing fish, which were offered feed in excess,
was studied. Although the overall growth performance may be considered as acceptable,
a large variation in individual growth rate was observed, which did not correlate to the
initial weight ŽFig. 1.. Normally, a negative correlation between SGR and fish size
would be expected; fish with a low SGR may therefore be considered as slow growers.
A large variation in individual growth rate have also been reported by McCarthy et al.
Ž1992. and Houlihan et al. Ž1993.. More differentiated information may therefore be
generated when analysis is done on an individual level in addition to tank or treatment
level.
The SGR was slightly, although not significantly, lower in fish fed the 30% protein
diet as compared to those fed the 35% and 45% protein diets. Sveier et al. Ž1999. found
no such effect using the same high and low dietary protein levels, but they used larger
fish Žinitial weight of about 700 g.. This may indicate that 35% dietary protein for
salmon weighing 300 g or higher is sufficient to support maximum growth. The lack of
enhanced growth in salmon fed the coarse-ground fish meal is in agreement with Sveier
et al. Ž1999., but not with dos Santos et al. Ž1993.. They reported a slightly negative
impact on growth rate and a significantly negative effect on FCR when using reduced
food particle size.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
115
The feed conversion rate is in accordance with results previously reported for salmon
of a similar body size ŽJuell et al., 1994; Aksnes, 1995; Sveier et al., 1999.. Dietary
protein level, but not fish meal particle size, significantly affected FCR. It may be
argued whether this is a dietary energy effect or due to a change in the protein:carbohydrate ratio. Hillestad and Johnsen Ž1994., Aksnes Ž1995. and Wathne Ž1995. have
previously demonstrated a reduced FCR with increased dietary energy levels. Using the
same low and high protein diets as in the present experiment, Sveier et al. Ž1999.
showed that the higher FCR found in the low protein group was mainly caused by a
lower dietary energy level.
The lack of effect on the PPV of the coarse-ground fish meal as compared to a finely
ground meal disagrees with the findings of dos Santos et al. Ž1993.. Sveier et al. Ž1999.
found no effect on the PPV using different protein levels or fish meal particle sizes. dos
Santos et al. Ž1993. used moist pellets in which the protein source was not denaturated.
Denaturation of the protein molecule destroys the tertiary protein structure and exposes
the polypeptide chain for more efficient protolytic degradation. This may be one reason
for the lack of agreement between the results found in the present study and those of dos
Santos et al. Ž1993..
The fat content of all experimental diets were the same. The protein sparing effect of
energy is related to fat. PEV was significantly higher in the high protein group as
compared to the two other dietary protein levels. This may indicate that the high protein
diet contained more energy than needed to support maximum growth. The higher energy
level in the diet was not fully compensated with decreasing FCR. The fish did not fully
compensate for the lower dietary protein level with increased feed intake. As long as
SGR did not correlate to protein intake, the results showed a protein sparing effect of
starch. This is similar to the results obtained by Hemre et al. Ž1995..
Nitrogen or fat digestibility was not influenced by fish meal particle size or fish meal
level. Sveier et al. Ž1999. reported a reduced digestibility of fat in the coarse meal as
compared to the micro-ground fish meal. Aksnes Ž1995. and Hemre et al. Ž1995. found a
significant reduction in fat digestibility, but not in protein digestibility when the starch
level was 22% or higher. Hemre et al. Ž1995., however, used smaller fish held at a lower
water temperature.
Whole body protein level was significantly higher in the fish fed the coarse-ground
fish meal as compared to those fed the micro-ground fish meal. This may indicate a
higher protein retention although the PPV value was not significantly different. The
reason for this is unclear. DOP, whole body fat deposition, and fat in cutlet, were not
influenced by dietary treatment. Although the fish nearly doubled their body weights, a
12-week growth period is too short a period to expect significant changes in body traits
when feeding diets with relative small differences in energy content.
4.1. Protein turnoÕer
Protein turnover is the process of protein synthesis and breakdown. In the present
study, aspects on the protein turnover have been looked into on an individual andror
group level by using four different methods, weight increase, specific RNA activity,
116
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
water soluble protein:RNA ratio, and relative efficiency of protein synthesis. Net protein
deposited is the difference between protein anabolism and catabolism. The extent of
protein synthesis or anabolism correlates to the amount of active RNA species in the
cell. In addition, the cellular content of amino acids and factors regulating gene activity
Že.g., endocrine factors. affects protein synthesis. The quantity of RNA or the RNA:DNA
ratio reflects the gene transcription capacity of the cells. The total amount of RNA may
be difficult to standardise while the RNA:DNA ratio refers to the RNA content per cell,
as the cellular DNA content is constant. The amount of RNA has been used as an index
of growth in fish larvae ŽRaae et al., 1988. and in several fish species ŽBulow, 1987;
Houlihan et al., 1993.. Large differences in the efficiency of RNA content, as correlated
to growth, have been reported ŽHoulihan, 1991..
In the present study, protein synthesis has been measured as the incorporation rate of
14
C-L-Lys into muscle protein. Some assumptions have been made in the calculation of
protein synthesis. Specific activity of lysine was not analysed in the present experiment,
but as lysine is absolutely essential, it is not transaminated into other amino acids
ŽJungermann and Møhler, 1980.. Excess lysine may, however, be metabolised into
carbon containing metabolites, which may be reutilized for dispensable amino acids
synthesis. This may be incorporated into protein and thereby give an overestimation of
the lysine incorporation in the TCA precipitate. Any 14 C containing metabolic products
of 14 C-Lys may be overlooked, consider the short time from injection to sampling
Žmaximum 4 h.. Owen et al. Ž1999. have summarised the assumptions for using labeled
amino acid incorporation as a measurement for protein synthesis: Ž1. that the presence of
the high intracellular concentration of a single amino acid does not itself affect the rate
of protein synthesis; Ž2. that the labeled amino acid equilibrates rapidly with the
intercellular free pool; Ž3. that the enrichment of the intercellular free poll remains
elevated and stable over the incorporation time or shows a slow linear decline; and Ž4.
that the enrichment of body protein with the labelled amino acids is linear over the
incorporation time. All this assumptions have been taken into consideration when
modifying the flooding dose method described by Garlick et al. Ž1980..
On treatmentrgroup level, no significant effects of protein level or physical qualities
on the nucleotide and protein turnover parameters examined were found except for the
amino acid incorporation rate. On the other hand, there was no effect on growth rate,
which should be the net result of any change in nucleotide and protein turnover ŽBulow,
1987; Houlihan et al., 1993; de la Higuera et al., 1998; Valente et al., 1998..
The RNA:DNA ratio as an indicator of protein synthesis potential and growth may be
argued. Yang and Dick Ž1993., Bergeron and Boulhic Ž1994. and Mclaughlin et al.
Ž1995. found a poor correlation between RNA:DNA ratio and growth. On the other
hand, Sveier and Raae Ž1992., Foster et al. Ž1993., Grant Ž1996., and Rooker and Holt
Ž1996. found a positive correlation. Mclaughlin et al. Ž1995. concluded that it was
difficult to measure any short time affect on growth using the RNA:DNA ratio.
In the present experiment, the specific RNA activity did not change when the
quantity of RNA per cell ŽRNA:DNA ratio. exceeded a certain level ŽFigs. 2a and 3b..
This indicates that the fish under normal feeding and rearing conditions had an overall
poor correlation between specific RNA activity and total RNA available. There was,
however, a high correlation between specific ribosomal activity and water soluble
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
117
protein:RNA in the cell ŽFig. 3c. which clearly shows the correlation between the two
methods for measurement of protein synthesis. Goldspink and Kelly Ž1984. used
RNA:water soluble protein as an index of the ribosomal capacity for protein synthesis.
The results from the present experiment support their results. Growth or weight increase
is, however, not necessarily correlated with the specific RNA activity or ribosomal
capacity as shown in the present study.
The specific RNA activity ŽFigs. 2b and 3a. gave only poor or no correlation with
SGR, but the relative efficiency of protein synthesis correlated well with SGR ŽFig. 4a..
The results showed that with increasing protein synthesis efficiency, the increase in SGR
was reduced ŽFig. 4a.. This indicates that an increase in relative protein synthesis
efficiency was mainly caused by a decrease in protein catabolism. Carter et al. Ž1993.
found a positive correlation between RNA content and growth using fewer fish.
Sveier et al. Ž1999. reported a slower gastric evacuation time when using coarseground compare to micro-ground fish meal Ž t50 s 14.8 and 10.8 h, respectively.. The
uptake of 14 C-L-lysine after intraperitonal injection may be considered as fast, 50% of
the lysine was found in the muscle tissue 4-h post-injection ŽTable 5.. The significantly
higher relative amino acid incorporation rate found using micro-grounded fish meal
ŽTable 5. therefore might be due to a better relationship between the supply of amino
acids arising from the digested meal and the intraperitonal injection of lysine into the
muscle cells. This is supported by de la Higuera et al. Ž1998., who found a significant
affect of dietary amino acid absorption pattern on protein synthesis rate.
The data from individual fish of relative amino acid incorporation rate into muscle
protein showed a weak negative correlation with SGR ŽFig. 4b.. This indicates that no
systematic correlation exists between protein synthesis measured over a period of 2 h
and growth rate measured over 12 weeks in a fish population. The lack of correlation
indicates that it is the protein catabolism and not the protein anabolism that is
controlling the growth rate in fish. In the method used for measuring the amino acid
incorporation rate, the 14 C-L-Lys was not given in excess. This implicates that the
incorporation rate measured was a result of the given state of nutrition, genetical and
endocrinological status of the fish, and not the maximum incorporation rate possible to
achieve. In our opinion, the method used is suitable to detect any treatment effects.
Large variation in feed intake andror protein turnover within a fish population has
been reported by McCarthy et al. Ž1992. and Houlihan et al. Ž1993.. Protein synthesis
parameters as nucleotides or the flooding dose method done over a short period of time
may therefore not be a good parameter for measurement of long-term growth.
5. Conclusion
Use of large fish meal particles in extruded diets for Atlantic salmon did not have any
affect on growth rate and feed utilisation in the present study. There were only poor
correlations between specific RNA activity and the total amount of RNA in the muscle.
Long-term SGR did not correlate with short-term measurement of specific RNA
activities. In normal growing fish, large individual variations in amino acid incorporation rates were found; this variation did not correlate to the specific growth rate. The
118
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
present experiment indicates that growth rate is mainly controlled by protein catabolism
and to a lesser degree by protein anabolism.
Acknowledgements
Marinne Kaland Gjesdal and Leif Pedersen are thanked for taking care of the
experimental fish in a conscientious way. The skilled analytical assistance of Edel Erdal,
Anita Birkenes and Bjørn Olav Kvamme is greatly appreciated. The authors are grateful
to Marit Espe for valuable discussions and comments on the manuscript.
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www.elsevier.nlrlocateraqua-online
Growth and protein turnover in Atlantic salmon
žSalmo salar L. /; the effect of dietary protein level
and protein particle size
Harald Sveier a,) , Arnt Johan Raae b, Einar Lied c
a
NorAqua InnoÕation AS, N-4335 Dirdal, Norway
Institute of Molecular Biology, Høyteknologisenteret, N-5000 Bergen, Norway
Institute of Nutrition, Directorate of Fisheries, Box 185, N-5804 Bergen, Norway
b
c
Accepted 21 October 1999
Abstract
In modern fish feeds, the protein sources consist of denaturated finely ground ingredients.
From the literature, it has been reported that use of coarsely chopped, but not denaturated, fish as
the dietary protein source gave better growth performance and protein utilisation. Growth, feed
utilisation and protein turnover using two different fish meal particle sizes Žmicro- or coarsegrounded. at three dietary protein concentrations Ž30%, 35%, and 45%. were studied in individually tagged Atlantic salmon in a 3-month growth experiment. At the end of the experimental
period, 14C-L-lysine was injected intraperitonally and dorsal muscle samples were taken at 2- and
4-h post-injection. Incorporation of 14C-L-lysine into muscle protein, RNA, DNA and water
soluble protein was analysed from samples of muscle tissue. Only small effects on growth rate,
feed conversion rate, protein and energy retention, and nitrogen and fat digestion were found.
During the growth experiment, large individual variations in growth rates were observed, which
did not correlate to the initial body weight. The total RNA content expressed as RNA amount per
unit of DNA ŽRNA:DNA ratio. did not reflect the specific RNA activity, and individual growth
rate was not correlated to the specific RNA activity or RNA:DNA ratio and only poorly to the
relative incorporation rate of amino acids. Growth rate was, however, correlated to the relative
efficiency of protein synthesis. The results indicate that the protein catabolism is more important
)
Corresponding author. Tel.: q47-51-61-1700; fax: q47-51-61-6112.
E-mail address: hsv@noraqua.no ŽH. Sveier..
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 4 4 - 0
102
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
for net protein deposition and growth than protein anabolism. q 2000 Elsevier Science B.V. All
rights reserved.
Keywords: Fish meal; Particle size; RNA:DNA ratio; Protein synthesis; Specific RNA activity; Individual
growth rate
1. Introduction
Modern fish feeds comprise finely ground ingredients bonded together during the
gelatinisation of starch when exposed to mechanical shear, steam and pressure in the
extrusion process. The question has been raised on whether the physical state Že.g.,
particle size or friability. of food particles may affect growth and feed utilisation by
affecting the gastrointestinal transit time ŽJobling, 1986, 1988; dos Santos et al., 1993..
Sveier et al. Ž1999. examined the effect of feeding diets consisting of fish meal ground
into three different particle sizes at two different protein concentrations on growth, feed
utilisation and gastrointestinal transit time, and found no significant effects except for
the evacuation time in the stomach. Gastric emptying rate or solubility of the protein has
been shown to affect utilisation of dietary protein Ždos Santos and Jobling, 1991; Espe
and Lied, 1994; Espe et al., 1992, 1999; Schuhmacher et al., 1997, de la Higuera et al.,
1998.. Boirie et al. Ž1997. demonstrated that the rate of absorption of amino acids from
the gut from a casein or whey protein-based diets affects protein synthesis within the
whole body protein catabolism and oxidation of amino acids.
The major part of the weight increase is related to the deposition of protein. The
protein accretion is a balance between protein anabolism and catabolism. Protein
catabolism occurs through different mechanisms and there is no clear method for
estimating total protein catabolism in whole body or selected tissues ŽHoulihan et al.,
1993.. The extent of protein synthesis is, in principle, correlated to the amount of
ribosomal RNA ŽrRNA. which contributes approximately 85% of the total cellular RNA
species, available amino acids and it is under endocrine control. The concentration of
total ribonucleic acid ŽRNA. can be expressed as Žmg RNA. Žg tissue.y1 while Žmg
water soluble protein. Žmg RNA.y1 is an index for ribosomal activity. The RNA:DNA
ratio reflects the amount of RNA per cell and is a more reliable quantification compared
to the grams of tissue. The RNA content in white muscle tissue has been shown to be
directly related to protein synthesis and growth in a number of species ŽGoldspink and
Kelly, 1984; Houlihan et al., 1993; Valente et al., 1998.. The specific RNA activity and
the protein synthesis efficiency can be measured in animals using the flooding dose
method where the incorporation of a labelled amino acid into newly synthesised protein
is measured ŽGarlick et al., 1980.. The efficiency of RNA translation may change
significantly after a meal ŽLyndon, 1990.. Only a few studies have compared direct
protein synthesis measurement and RNA concentration in fish tissues ŽHoulihan et al.,
1993..
In the present work, an experiment with Atlantic salmon was performed in which the
dietary protein sources comprised of coarse- or micro-ground fish meal compared at
three different dietary protein levels. The aim of the study was to examine any dietary
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
103
effects of these factors on growth, protein turnover, specific RNA activity and feed
utilisation. Further, the individual variation and response on the same factors was
investigated.
2. Material and methods
2.1. Experimental conditions
The experiment was carried at NorAqua Innovation research station in Dirdal in the
Southwestern part of Norway. Atlantic salmon Ž Salmo salar L.. of the NLA ŽNorsk
Lakseavl, Kyrksæterøra, Norway. strain, hatched in February 1996, and smolted during
the autumn 1996 was used. Groups of 20 fish were adapted to the experimental
conditions for 4 weeks prior to the experiment. Initially, 18 fish averaging 336 " 41 g
Ž n s 324, 1 STD. were randomly assigned to 18 tanks with a water volume of 0.5 m3.
The six experimental diets were randomly assigned to the tanks using three replicates
per diet. Seawater Žsalinity 30.0 " 1.1‰. at 7.6 " 0.28C was used throughout the
experiment. Temperature and salinity were recorded daily, while the oxygen saturation
in the water outlet was recorded and adjusted weekly to ensure a minimum level of 7 mg
ly1 . The fish were exposed to continuous light from smoltification until the end of the
experiment.
The fish were fed in excess three times daily using automatic feeders for 96 days.
First meal from 2000 to 2100 h Ž20%., second meal from 0200 to 0300 h Ž20%. and
third meal from 0700 to 0800 h Ž60%.. A mechanical system for collecting uneaten feed
was installed at the outlet of each tank. The collection system was a moving mesh belt,
which removed the uneaten feed from the outlet water and into a collection box ŽSveier
et al., 1997..
2.2. Diet composition
The fish meals used in diet formulation were the same as that of Sveier et al. Ž1999..
The fish meals used were produced from the same batch of herring Ž Clupea harengus,
L.. fillet by-product by The Norwegian Herring Oil and Meal Industry Research
Institute, Bergen, Norway. After processing, the fish meal was milled and separated into
two parts by sieving. The batches consisted of coarse Ž3–5 mm. and micronised Ž78%,
0.3–0.1 mm. particle sizes. The chemical composition of the crude fish meal met the
specification of Norse-LT94 w as defined by the Norwegian Herring Oil and Meal
Industry Research Institute.
Each protein particle size was tested at three protein levels Ž30%, 35%, and 45%. at a
fixed oil concentration Ž27%.. The diets were produced as 9 mm extruded pellets, which
were crumbled and sieved to 5-mm pellet size. The extrusion process will, to some
extent, reduce the fish meal particle sizes. However, visual inspection of the pellets
showed large fish meal particles in the pellets when using coarse-ground fish meal.
Yttrium oxide was used as a marker in all diets for the determination of digestibility.
Diet composition data, results of chemical analyses and calculations are given in Table
1.
104
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Table 1
Dietary ingredients, chemical analysis and calculated values of the experimental diets
Coarse-ground
Micro-ground
30% protein 35% protein 45% protein 30% protein 35% protein 45% protein
Ingredients (g kg y 1)
Fish meal
Wheat flour
Suprex maize
Mono ammonium
phosphate
Capelin oil
ŽNorsamoil.
Premix a
Žvitamin, mineral,
betain, pigment.
Indicator ŽY2 O 3 .
Žmg kgy1 .
405
171
170
50
494
130
130
50
670
49
50
50
405
171
170
50
494
130
130
50
670
49
50
50
240
232
217
240
232
217
9.23
9.23
9.23
9.23
9.23
9.23
100
100
100
100
100
100
Chemical composition
(g kg y 1)
Protein Ž N =6.25.
Fat ŽSoxhlet.
Ash
Water
Rest b
304
241
65
78
312
389
263
79
31
238
456
269
110
58
107
301
280
69
93
257
389
287
79
72
176
445
282
105
61
107
Calculations
Gross energy ŽMJ kgy1 . c
Starch Žg kgy1 . d
22.1
216
23.7
165
22.6
216
23.5
165
23.3
63
23.5
63
a
Vitamins and minerals according to or higher than recommended by NRC Ž1993.. Betain Ž78.6%.:
0.128% of diet; Charphyll pink: 0.3581% of diet.
b
Rests NFEs100yŽproteinqfatqashqwater..
c
Calculated using the following values: Fat: 39.5 MJ kgy1 , Protein: 23.7 MJ kgy1 , Carbohydrate: 17.2 MJ
kgy1 .
d
Calculated based on the prescription using 57% starch in wheat flour and 70% starch in suprex maize.
2.3. Fish sampling and sample treatment
The fish was labelled intraperitoneally with a passive integrated transponder ŽPIT. tag
ŽBioSonics, Seattle, WA, USA. ŽCohen et al., 1989. 4 weeks before the experiment
started. Prior to the experiment, feed was withheld for 4 days. Three samples each of
five individuals were randomly removed and used for initial whole body analysis of fat
and protein. The remaining 18 fish per tank were then weighed individually, and the
corresponding PIT tag recorded. At the end of the experiment, faeces was stripped from
all fish according to Austreng Ž1978.. Feed was withheld for 4 days before all fish were
individual weighed and the corresponding PIT tag recorded. Six fish per tank were
sampled for whole body analysis of fat and protein and an additional six fish were used
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
105
for analyses of fat in cutlet according to the Norwegian quality cut ŽNQC, NS 9401,
1994. and measurement of body traits.
For measurement of fractional protein synthesis rate, a modification of Garlick’s
method was used ŽGarlick et al., 1980.. The remaining fish were fed for 2 weeks. From
each tank four to six fish were kept in ice water for 5 min before weighing and injected
intraperitonally with 2 mCi U 100 gy1 body weight 14 C-L-lysine ŽDu Pont de Nemours,
Belgium.. Two or three fish from each tank were killed by a blow on the head 2 or 4 h
after isotope injection. A muscle sample was removed from a segment of the epaxial
muscle under the dorsal fin and immediately frozen. Epaxial muscle tissue samples were
analysed for radioactivity in both trichloroacetic acid ŽTCA. soluble and non-soluble
fractions, and values obtained were used to calculate protein deposition and efficiency of
protein synthesis. The dose and sampling time were determined in a preliminary
experiment to detect the period of time where there was a linear incorporation of
14
C-L-lysine, using fish from the same batch as the experimental fish. The sample was
also used for analysis of RNA, DNA and water soluble protein.
All samples for chemical analysis were taken as pooled samples per tank except
RNA, DNA, soluble protein and 14 C-lysine which was analysed on an individual basis.
2.4. Chemical analyses
The diets were analysed in duplicate for dry matter, protein, fat and ash. Protein
Ž N = 6.25. in feeds was determined colorimetrically in micro Kjeldahl digests according
to Crooke and Simpson Ž1971.. Fat was determined gravimetrically after extraction with
ethyl acetate, dry matter after drying at 1058C for 24 h and ash after combustion at
5508C for 16 h. Protein in fish and faeces was analysed with a nitrogen gas analysator
ŽPerkin Elmer Series II Nitrogen analyzer 2410. according to the manufacturer’s
manual. Fat in the cutlet ŽNQC. and faeces was analysed gravimetrically after extraction
with ethyl acetate. Yttrium oxide ŽYt 2 O 3 . in feed and faeces was analysed using
ICP-MS by the Institute of Nutrition, Directorate of Fisheries, Bergen, Norway. RNA
and DNA were measured according to Boer Ž1975. and Raae et al. Ž1988. using
ethidium bromide. Water soluble protein in the same samples were measured essentially
by Biuret method ŽDawson et al., 1986..
Isotope activity was measured in muscle homogenates, in the precipitate and in the
supernatant after treatment with 20% TCA. The samples were solubilised in Soluene-350
overnight and counted in a scintillation counter in Hi Ionic Fluor according to Berge et
al. Ž1994..
2.5. Calculation and statistics
Specific growth rate ŽSGR. was calculated as:
SGR s 100 Ž lnW2 y lnW1 . ny1
where W1 and W2 are the initial and final weight, respectively, and n is the number of
days in the feeding period.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
106
Feed intake ŽFI. was calculated as ŽHelland et al., 1996.:
FI s Ž feed offered. y Ž feed collected. Ž corrected for dry matter loss .
= Ž collection efficiency.
y1
Feed conversion ratio ŽFCR. was calculated as:
FCR s FI Ž B2 q Bdead y B1 .
y1
where B1 and B2 are the biomass at the start and end, respectively, and Bdead is the
biomass of the dead fish.
Dressing out percentage ŽDOP. was calculated as:
ž
/
y1
DOP s 1 y Ž BWgutted BWungutted
. 100
where BWgutted and BWungutted are the weights of gutted and ungutted unbled fish,
respectively.
Productive protein value ŽPPV. was calculated as:
PPV s Ž P2 y P1 . PIy1
where P1 and P2 are estimates of protein content of the biomass at the start and end of
the experiment and PI is the protein intake.
Productive energy value ŽPEV. was calculated as:
PEV s Ž E2 y E1 . EIy1
where E1 and E2 are estimates of energy content of the biomass at the start and end of
the experiment, and EI is the energy intake.
Apparent digestibility ŽAD. was calculated as:
ž
AD s 100 1 y Ž Ifeed Nfaeces . Ž Ifaeces Nfeed .
y1
/
where Ifeed and Ifaeces are the concentrations of marker in the feed and faeces, and Nfeed
and Nfaeces are the nutrient concentrations in the feed and faeces, respectively ŽAustreng,
1978..
Protein synthesis efficiency was calculated as incorporation of 14 C-Lys into muscle
tissue protein according to Garlick et al. Ž1980. and Carter et al. Ž1993..
Fractional protein synthesis: K S s w SB Ž t 2. y S B Ž t1.rSAŽ t 2 y t1.xw100rŽ t 2 y t1.x. In
which S B Ž t 2. s protein bound isotope at 4 h, SB Ž t1. s protein bound isotope at 2 h,
SAŽ t 2 y t1. s mean pool of non-protein bound isotope Že.g., mainly free 14 C-L- Lys. in
the experimental period.
Protein growth rate Ž K g . s Žln P2 y ln P1 . ny1 = 100 where P1 and P2 are the
initial and final protein content, respectively, and n is the number of days in the feeding
period.
Coarse-ground
30% protein
35% protein
45% protein
Micro-ground
30% protein
35% protein
45% protein
Protein level
30% protein
35% protein
45% protein
Physical quality Micro-ground
Coarse-ground
Interactions
P-value
1
n Initial weight Žg. SGR Ž% dayy1 . FCR
PPV
3
3
3
3
3
3
6
6
6
9
9
0.45"0.05
0.48"0.04
0.36"0.00 1
0.44"0.011
0.43"0.01
0.56"0.04
0.39"0.01
0.46"0.03
0.35"0.04
0.44"0.05
0.44"0.041
0.62"0.00 1
0.42"0.02 Ž6. 0.47 B "0.02 Ž6.
0.36"0.02 Ž5. 0.44 B "0.03 Ž5.
0.43"0.02 Ž5. 0.58 A "0.03 Ž5.
0.39"0.02 Ž8.
0.50"0.02 Ž8.
0.41"0.02 Ž8.
0.49"0.02 Ž8.
n.s.
n.s.
342"4
339"10
330"9
349"6
326"5
334"7
346"5
333"5
332"5
336"4.1
337"4.1
n.s.
0.63"0.09
0.71"0.02
0.68"0.06
0.60"0.03
0.68"0.08
0.80"0.05
0.61"0.04
0.70"0.04
0.74"0.04
0.69"0.04
0.67"0.04
n.s.
1.09"0.08
1.05"0.031
0.80"0.01
1.10"0.05
1.07"0.01
0.81"0.041
1.10 B "0.04 Ž6.
1.06 B "0.04 Ž5.
0.80 A "0.04 Ž5.
0.99"0.04 Ž8.
0.98"0.04 Ž8.
n.s.
PEV
ADnitrogen Ž%.
ADfat Ž%.
85.8"0.54
86.0"0.89
87.2"0.48
86.9"1.40
87.5"0.42
88.4"0.35
86.3"0.51
86.8"0.51
87.8"0.51
87.6"0.41 Ž9.
86.3"0.41 Ž9.
n.s.
89.6"0.89
90.2"1.3
90.8"0.87
90.8"0.95
91.6"0.49
91.0"0.11
90.2"0.56 Ž6.
90.9"0.56 Ž6.
90.8"0.62 Ž5.
91.1"0.49 Ž8.
90.2"0.49 Ž9.
n.s.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Table 2
Growth and feed utilisation expressed as SGR, FCR, PPV, PEV, apparent digestibility of nitrogen and fat ŽADnitrogen and ADfat .. Values are presented for the ground
grades as mean"s.e., for protein level and physical quality as least squares means"SEM
ns 2. Significant differences within protein levels are denoted with capital letters and between protein levels with small letters.
107
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
108
Protein degradation rate Ž K d . s K S y K g ; Muscle mass s Žwhole body wet weight.
60% ŽLied, pers.med..; Protein synthesised: Ž K S .Žmuscle mass..
Protein catabolisms Protein anabolismy K d .
Deposited proteins Žmuscle mass.Ž K d ..
Relative protein synthesis efficiency: ŽDeposited proteinrprotein synthesised.100.
Relative amino acid incorporation rate per hour on tank level was calculated as:
Ž pmol
14
C lysine 4 h y pmol
14
C lysine 2 h . 2y1 .
For correlation of the individual relative amino acid incorporation rate with the
individual SGR, the relative amino acid incorporation per hour was calculated as:
Ž pmol
14
C lysine 2 h . 2y1 and Ž pmol
14
C lysine 4 h . 4y1 .
The specific RNA activity was calculated as:
Ž Relative amino acid incorporation rate per hour. RNAy1 .
All data related to feed levels were statistically processed by a general linear model
ŽGLM. procedure. The data were tested for interaction between protein level and protein
particle size; when no interaction effects was found, it was removed from the model.
Where significant Ž P - 0.05. differences were achieved a Tukey HSD multiple range
test was used to rank the means. All data are presented as average" SEM. Regression
analysis of individual data was performed using simple regression tested for best-fitted
model. Multiple regression was tested. The software Statgraphics, version 3.1 ŽStatistical
Graphics, Manugistics, MD, USA.. was used.
3. Results
3.1. Fish meal and diet quality
The chemical and biological quality of the fish meal used in the experiments met the
specifications of Norse-LT 94 w Ždefined by the Norwegian Herring Oil and Meal
Fig. 1. Initial wet weight of the fish and the specific growth rate after a 12 weeks growth period.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
109
Table 3
Body composition expressed as DOP, protein in whole fish, fat in cutlet ŽNQC. and change in body fat per
kilogram of growth. Values are present for the ground grades as mean"S.E., for protein level and physical
quality as mean"SEM
Coarse-ground
Micro-ground
Protein level
Physical quality
Interactions
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
Micro-ground
Coarse-ground
P-value
n
DOP Ž%.
Protein end
Ž%. in whole
fish
NQC Ž% fat
in cutlet.
Change in
body fat per
kilogram
of growth
3
3
3
3
3
3
6
6
6
9
9
10.6"1.1
11.4"0.2
11.2"0.2
10.4"0.4
10.6"0.8
10.3"0.9
10.5"0.5
11.0"0.5
10.7"0.5
10.4"0.4
11.1"0.4
n.s.
16.9"0.1
16.7"0.1
17.2"0.2
16.1"0.4
16.6"0.1
16.7"0.2
16.5"0.1
16.6"0.1
17.0"0.1
16.5 A "0.1
16.9 B "0.1
n.s.
9.8"0.6
9.6"0.4
8.7"0.6
10.4"0.6
9.9"0.3
10.3"0.3
10.1"0.3
9.8"0.3
9.5"0.3
10.2"0.3
9.4"0.3
n.s.
13.5"3.1
9.8"0.6
8.5"2.0
15.1"0.9
11.6"1.7
12.3"1.2
14.3"1.2
10.7"1.2
10.4"1.2
10.6"1.0
13.0"1.0
n.s.
Significant differences within protein levels are denoted with capital letters and between protein levels with
small letters.
Industry Research Institute, Bergen, Norway. except for slightly lower protein and
higher ash content due to the higher bone content from the herring by-product used as
Table 4
Nucleotide levels in epaxial muscle tissue of Atlantic salmon fed different experimental diets. Values are
presented for the ground grades as mean"s.e., for protein level and physical quality as least squares
means"SEM Ž n.
Coarse-ground
Micro-ground
Protein level
Physical quality
Interactions
a
ns 2.
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
30% protein
35% protein
45% protein
Micro-ground
Coarse-ground
P-value
n
DNA Žmg g
muscley1 .
RNA:DNA
ratio
Water soluble
protein:RNA
Žmg mgy1 .
Specific RNA
activity
Žpmol h mgy1 .
3
3
3
3
3
3
6
6
6
9
9
5.7"1.3
7.1"0.8
5.6"0.4
7.9"1.5
7.1"1.3
6.1"0.1
6.8"0.7
7.1"0.7
5.8"0.7
7.0"0.6
6.2"0.6
n.s.
7.3"2.5
4.2"0.5
7.2"1.7
2.9"0.5
3.5"0.9
5.7"1.1
5.1"1.0
3.8"1.0
6.5"1.0
4.0"0.8
6.2"0.8
n.s.
4.1"0.7
8.3"3.0
5.5"3.0 a
6.1"1.1
4.7"2.4 a
2.9"1.2
5.1"1.3 Ž6.
6.7"1.5 Ž5.
4.0"1.5 Ž5.
4.7"1.1 Ž8.
5.8"1.1 Ž8.
n.s.
0.04"0.04
0.05"0.02
0.04"0.01
0.14"0.08
0.05"0.02
0.04"0.05
0.09"0.019
0.05"0.019
0.04"0.019
0.08"0.015
0.04"0.015
n.s.
110
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
Fig. 2. Ža. Correlation between RNA concentration in muscle expressed as RNA:DNA ratio and the specific
RNA activity measured as the incorporation of 14 C-L-lysine into muscle protein per unit RNA Žpmol mgy1 .
Ž Y s1Žy17.71q10.74 X .y1 , r s 0.575, P s 0.019.. Žb. Specific RNA activity vs. SGR Ž% dayy1 .. All data
on tank levels.
the raw material source. The experimental diets showed no major difference between
calculated and analysed values ŽTable 1..
3.2. Growth and feed utilisation
Growth and feed utilisation data are presented in Table 2. Fish body weight increased
by about 90% on average during the experiment. SGR and protein retention ŽPPV. data
were not affected by dietary protein level or fish meal particle size. FCR and energy
retention ŽPEV. data were significantly affected by dietary protein level, but not fish
Fig. 3. Ža. Specific RNA activity Žpmol mgy1 . vs. SGR Ž% dayy1 . achieved using individual data. Žb.
RNA:DNA ratio vs. specific RNA activity Žpmol mgy1 . using individual data Ž Y s 0.277y0.186 X q
0.041 X 2 y0.0028 X 3 , r s 0.785, P s 0.000.. Žc. Water soluble protein:RNA vs. specific RNA activity Žpmol
mgy1 . using individual data Ž Y s 29.819 X 0.553 , r s 0.807, P s 0.000..
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
111
112
Table 5
Incorporation of amino acids in terms of 14 C-L-lysine in epaxial muscle tissue of Atlantic salmon fed experimental diets. Values are presented for the ground grades as
mean"s.e., for protein level and physical quality as mean"SEM
Coarse-ground
30% protein
35% protein
45% protein
Micro-ground
30% protein
35% protein
45% protein
Protein level
30% protein
35% protein
45% protein
Physical quality Micro-ground
Coarse-ground
Interactions
P-value
3
3
3
3
3
3
6
6
6
9
9
40"3
39"1
44"5
43"4
48"4
40"6
41"3
44"3
41"3
43"2
40"2
n.s.
45"4
48"5
49"1
50"4
50"4
51"3
48"3
49"3
53"3
52"2
47"2
n.s.
0.25"0.02 2
1.4"0.7
1.26"0.03 2
3.0"0.3
2.0"0.5 2
0.9"0.3
1.6"0.3
1.7"0.3
1.1"0.3
2.0"0.3 a
1.0"0.3 b
0.02
Significant differences between physical qualities are denoted with small letters.
1
Percentage of injected activity.
2
ns 2.
3
ns8.
1.24"0.06 2
7.5"2.8
4.1"1.0
14.8"2.2
6.8"1.4 2
3.7"1.9
5.0"1.5 3
7.1"1.5 3
3.9"1.3
8.4"1.2 a,3
4.3"1.2 b,3
0.01
0.71"0.10 2
6.8"2.8
3.5"1.0
14.3"2.2
4.0"2.4
3.0"1.8
7.5"1.6 2
5.4"1.5
3.3"1.5
7.1"1.2
3.7"1.3 3
n.s.
29"25
14"14
18"12
3.2"0.8
9"5
32"36
16"9
11"9
27"9
17"7
21"7
n.s.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
n Activity in
Activity in
Relative amino
Fractional protein Protein degradation Relative protein
muscle tissue
muscle tissue
acid incorporation
synthesis rate
rate Ž K d , % dayy1 . synthesis efficiency
1
1
y1
y1
y1
Ž% retained of
2 h post-injection 4 h post-injection rate Žpmol g h . Ž K S , % day .
synthesised.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
113
meal particle size. The higher the dietary protein Žand energy. level, the lower the FCR
value observed. Digestion of nitrogen and fat was not affected by protein level or fish
meal particle size. There was no correlation between initial weight and SGR achieved in
this experiment ŽFig. 1..
3.3. Body traits
The body trait data are listed in Table 3. Whole body protein content was not
influenced by the protein level, but there were significantly higher protein levels in the
group fed the coarse-ground fish meal. Whole body fat deposition was not affected by
the protein level or fish meal particle size. DOP and fat in cutlet ŽNQC. showed no
significant effect of protein level or fish meal particle size.
Fig. 4. Ža. Relative protein synthesis efficiency Ž%. vs. SGR Ž% dayy1 . Ž Y s 0.542q0.068 ln X, r s 0.741,
P s 0.001.. Žb. Relative amino acid incorporation rate Žpmol Žg t.y1 . vs. SGR Ž% dayy1 . Ž Y s 0.827y0.144 X,
r sy0.333, P s 0.002..
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
114
3.4. Nucleic acids and protein turnoÕer
The amount of RNA expressed as the RNA:DNA ratio and specific RNA activity was
not influenced by fish meal particle size or protein level ŽTable 4.. There was a
correlation between specific RNA activity and RNA:DNA ratio ŽFig. 2a. until the
RNA:DNA ratio reached a level of about 4. Beyond an RNA:DNA ratio of 4, there were
only minor changes. There was no correlation between the specific RNA activity and
SGR ŽFig. 2b..
Regression analysis using the individual data showed no correlation between specific
RNA activity and SGR ŽFig. 3a.. However, a high correlation between specific RNA
activity and RNA:DNA ratio up to an RNA:DNA ratio of about 3 ŽFig. 3b. was
observed. A high correlation between the specific RNA activity and water soluble
protein:RNA ratio as an index of ribosomal activity was found ŽFig. 3c..
3.5.
14
C-lysine incorporation and protein turnoÕer
The relative efficiency of protein synthesis measured as incorporation of 14 C-L-Lys,
was not influenced by protein level or the fish meal particle size ŽTable 5.. There was,
however, a high correlation with growth ŽFig. 4a.. Dietary protein level did not affect
the incorporation of 14 C-L-Lys into muscle, but there was a significant effect on the
physical qualities of the fish meal ŽTable 5.. Using data from individual fish, a negative
correlation between SGR and 14 C-L-Lys incorporation rate was observed ŽFig. 4b..
4. Discussion
In the present experiment, normal growing fish, which were offered feed in excess,
was studied. Although the overall growth performance may be considered as acceptable,
a large variation in individual growth rate was observed, which did not correlate to the
initial weight ŽFig. 1.. Normally, a negative correlation between SGR and fish size
would be expected; fish with a low SGR may therefore be considered as slow growers.
A large variation in individual growth rate have also been reported by McCarthy et al.
Ž1992. and Houlihan et al. Ž1993.. More differentiated information may therefore be
generated when analysis is done on an individual level in addition to tank or treatment
level.
The SGR was slightly, although not significantly, lower in fish fed the 30% protein
diet as compared to those fed the 35% and 45% protein diets. Sveier et al. Ž1999. found
no such effect using the same high and low dietary protein levels, but they used larger
fish Žinitial weight of about 700 g.. This may indicate that 35% dietary protein for
salmon weighing 300 g or higher is sufficient to support maximum growth. The lack of
enhanced growth in salmon fed the coarse-ground fish meal is in agreement with Sveier
et al. Ž1999., but not with dos Santos et al. Ž1993.. They reported a slightly negative
impact on growth rate and a significantly negative effect on FCR when using reduced
food particle size.
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
115
The feed conversion rate is in accordance with results previously reported for salmon
of a similar body size ŽJuell et al., 1994; Aksnes, 1995; Sveier et al., 1999.. Dietary
protein level, but not fish meal particle size, significantly affected FCR. It may be
argued whether this is a dietary energy effect or due to a change in the protein:carbohydrate ratio. Hillestad and Johnsen Ž1994., Aksnes Ž1995. and Wathne Ž1995. have
previously demonstrated a reduced FCR with increased dietary energy levels. Using the
same low and high protein diets as in the present experiment, Sveier et al. Ž1999.
showed that the higher FCR found in the low protein group was mainly caused by a
lower dietary energy level.
The lack of effect on the PPV of the coarse-ground fish meal as compared to a finely
ground meal disagrees with the findings of dos Santos et al. Ž1993.. Sveier et al. Ž1999.
found no effect on the PPV using different protein levels or fish meal particle sizes. dos
Santos et al. Ž1993. used moist pellets in which the protein source was not denaturated.
Denaturation of the protein molecule destroys the tertiary protein structure and exposes
the polypeptide chain for more efficient protolytic degradation. This may be one reason
for the lack of agreement between the results found in the present study and those of dos
Santos et al. Ž1993..
The fat content of all experimental diets were the same. The protein sparing effect of
energy is related to fat. PEV was significantly higher in the high protein group as
compared to the two other dietary protein levels. This may indicate that the high protein
diet contained more energy than needed to support maximum growth. The higher energy
level in the diet was not fully compensated with decreasing FCR. The fish did not fully
compensate for the lower dietary protein level with increased feed intake. As long as
SGR did not correlate to protein intake, the results showed a protein sparing effect of
starch. This is similar to the results obtained by Hemre et al. Ž1995..
Nitrogen or fat digestibility was not influenced by fish meal particle size or fish meal
level. Sveier et al. Ž1999. reported a reduced digestibility of fat in the coarse meal as
compared to the micro-ground fish meal. Aksnes Ž1995. and Hemre et al. Ž1995. found a
significant reduction in fat digestibility, but not in protein digestibility when the starch
level was 22% or higher. Hemre et al. Ž1995., however, used smaller fish held at a lower
water temperature.
Whole body protein level was significantly higher in the fish fed the coarse-ground
fish meal as compared to those fed the micro-ground fish meal. This may indicate a
higher protein retention although the PPV value was not significantly different. The
reason for this is unclear. DOP, whole body fat deposition, and fat in cutlet, were not
influenced by dietary treatment. Although the fish nearly doubled their body weights, a
12-week growth period is too short a period to expect significant changes in body traits
when feeding diets with relative small differences in energy content.
4.1. Protein turnoÕer
Protein turnover is the process of protein synthesis and breakdown. In the present
study, aspects on the protein turnover have been looked into on an individual andror
group level by using four different methods, weight increase, specific RNA activity,
116
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
water soluble protein:RNA ratio, and relative efficiency of protein synthesis. Net protein
deposited is the difference between protein anabolism and catabolism. The extent of
protein synthesis or anabolism correlates to the amount of active RNA species in the
cell. In addition, the cellular content of amino acids and factors regulating gene activity
Že.g., endocrine factors. affects protein synthesis. The quantity of RNA or the RNA:DNA
ratio reflects the gene transcription capacity of the cells. The total amount of RNA may
be difficult to standardise while the RNA:DNA ratio refers to the RNA content per cell,
as the cellular DNA content is constant. The amount of RNA has been used as an index
of growth in fish larvae ŽRaae et al., 1988. and in several fish species ŽBulow, 1987;
Houlihan et al., 1993.. Large differences in the efficiency of RNA content, as correlated
to growth, have been reported ŽHoulihan, 1991..
In the present study, protein synthesis has been measured as the incorporation rate of
14
C-L-Lys into muscle protein. Some assumptions have been made in the calculation of
protein synthesis. Specific activity of lysine was not analysed in the present experiment,
but as lysine is absolutely essential, it is not transaminated into other amino acids
ŽJungermann and Møhler, 1980.. Excess lysine may, however, be metabolised into
carbon containing metabolites, which may be reutilized for dispensable amino acids
synthesis. This may be incorporated into protein and thereby give an overestimation of
the lysine incorporation in the TCA precipitate. Any 14 C containing metabolic products
of 14 C-Lys may be overlooked, consider the short time from injection to sampling
Žmaximum 4 h.. Owen et al. Ž1999. have summarised the assumptions for using labeled
amino acid incorporation as a measurement for protein synthesis: Ž1. that the presence of
the high intracellular concentration of a single amino acid does not itself affect the rate
of protein synthesis; Ž2. that the labeled amino acid equilibrates rapidly with the
intercellular free pool; Ž3. that the enrichment of the intercellular free poll remains
elevated and stable over the incorporation time or shows a slow linear decline; and Ž4.
that the enrichment of body protein with the labelled amino acids is linear over the
incorporation time. All this assumptions have been taken into consideration when
modifying the flooding dose method described by Garlick et al. Ž1980..
On treatmentrgroup level, no significant effects of protein level or physical qualities
on the nucleotide and protein turnover parameters examined were found except for the
amino acid incorporation rate. On the other hand, there was no effect on growth rate,
which should be the net result of any change in nucleotide and protein turnover ŽBulow,
1987; Houlihan et al., 1993; de la Higuera et al., 1998; Valente et al., 1998..
The RNA:DNA ratio as an indicator of protein synthesis potential and growth may be
argued. Yang and Dick Ž1993., Bergeron and Boulhic Ž1994. and Mclaughlin et al.
Ž1995. found a poor correlation between RNA:DNA ratio and growth. On the other
hand, Sveier and Raae Ž1992., Foster et al. Ž1993., Grant Ž1996., and Rooker and Holt
Ž1996. found a positive correlation. Mclaughlin et al. Ž1995. concluded that it was
difficult to measure any short time affect on growth using the RNA:DNA ratio.
In the present experiment, the specific RNA activity did not change when the
quantity of RNA per cell ŽRNA:DNA ratio. exceeded a certain level ŽFigs. 2a and 3b..
This indicates that the fish under normal feeding and rearing conditions had an overall
poor correlation between specific RNA activity and total RNA available. There was,
however, a high correlation between specific ribosomal activity and water soluble
H. SÕeier et al.r Aquaculture 185 (2000) 101–120
117
protein:RNA in the cell ŽFig. 3c. which clearly shows the correlation between the two
methods for measurement of protein synthesis. Goldspink and Kelly Ž1984. used
RNA:water soluble protein as an index of the ribosomal capacity for protein synthesis.
The results from the present experiment support their results. Growth or weight increase
is, however, not necessarily correlated with the specific RNA activity or ribosomal
capacity as shown in the present study.
The specific RNA activity ŽFigs. 2b and 3a. gave only poor or no correlation with
SGR, but the relative efficiency of protein synthesis correlated well with SGR ŽFig. 4a..
The results showed that with increasing protein synthesis efficiency, the increase in SGR
was reduced ŽFig. 4a.. This indicates that an increase in relative protein synthesis
efficiency was mainly caused by a decrease in protein catabolism. Carter et al. Ž1993.
found a positive correlation between RNA content and growth using fewer fish.
Sveier et al. Ž1999. reported a slower gastric evacuation time when using coarseground compare to micro-ground fish meal Ž t50 s 14.8 and 10.8 h, respectively.. The
uptake of 14 C-L-lysine after intraperitonal injection may be considered as fast, 50% of
the lysine was found in the muscle tissue 4-h post-injection ŽTable 5.. The significantly
higher relative amino acid incorporation rate found using micro-grounded fish meal
ŽTable 5. therefore might be due to a better relationship between the supply of amino
acids arising from the digested meal and the intraperitonal injection of lysine into the
muscle cells. This is supported by de la Higuera et al. Ž1998., who found a significant
affect of dietary amino acid absorption pattern on protein synthesis rate.
The data from individual fish of relative amino acid incorporation rate into muscle
protein showed a weak negative correlation with SGR ŽFig. 4b.. This indicates that no
systematic correlation exists between protein synthesis measured over a period of 2 h
and growth rate measured over 12 weeks in a fish population. The lack of correlation
indicates that it is the protein catabolism and not the protein anabolism that is
controlling the growth rate in fish. In the method used for measuring the amino acid
incorporation rate, the 14 C-L-Lys was not given in excess. This implicates that the
incorporation rate measured was a result of the given state of nutrition, genetical and
endocrinological status of the fish, and not the maximum incorporation rate possible to
achieve. In our opinion, the method used is suitable to detect any treatment effects.
Large variation in feed intake andror protein turnover within a fish population has
been reported by McCarthy et al. Ž1992. and Houlihan et al. Ž1993.. Protein synthesis
parameters as nucleotides or the flooding dose method done over a short period of time
may therefore not be a good parameter for measurement of long-term growth.
5. Conclusion
Use of large fish meal particles in extruded diets for Atlantic salmon did not have any
affect on growth rate and feed utilisation in the present study. There were only poor
correlations between specific RNA activity and the total amount of RNA in the muscle.
Long-term SGR did not correlate with short-term measurement of specific RNA
activities. In normal growing fish, large individual variations in amino acid incorporation rates were found; this variation did not correlate to the specific growth rate. The
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H. SÕeier et al.r Aquaculture 185 (2000) 101–120
present experiment indicates that growth rate is mainly controlled by protein catabolism
and to a lesser degree by protein anabolism.
Acknowledgements
Marinne Kaland Gjesdal and Leif Pedersen are thanked for taking care of the
experimental fish in a conscientious way. The skilled analytical assistance of Edel Erdal,
Anita Birkenes and Bjørn Olav Kvamme is greatly appreciated. The authors are grateful
to Marit Espe for valuable discussions and comments on the manuscript.
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