# Different Dietary Levels of Protein to Lipid Ratio Affected Digestive Efficiency, Skeletal Growth, and Muscle Protein in Rainbow Trout Families

Authors: Krisna Rungruangsak-Torrissen; Lars H. Stien; Britt S. Daae; Tone Vågseth; Grethe B. Thorsheim; Declan Tobin; Ossi Ritola
Journal: Scholarly Research Exchange
 (2009)

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## Abstract

Normal Protein (NP) and High Protein (HP) diets were provided to rainbow trout families from juvenile to maturity. Muscle protein concentration increased during growth and the protein to lipid (P/L) ratio was doubled at late stage with respect to the dietary P/L ratio. The HP-diet fish showed higher protein deposition in body and white muscle, and had lower condition factor due to protein deposition associated more with body length than body weight. Fish growth rates were decreased at maturation, and the HP-diet fish showed lower growth rate including the activity ratio of trypsin to chymotrypsin (T/C ratio) and feed efficiency. Trypsin and chymotrypsin specific activities were related to dietary protein levels, and the T/C ratio was related to intestinal weight and growth rate independent of the enzymes specific activity levels. The families with high growth capacity could double increase white muscle P/L ratio levels, compared to low and medium growth families, if they were fed on HP-diet. The digestive enzyme extracts from high growth families resulted in higher in vitro protein digestibility for all diets. The effect of dietary P/L ratio on digestion, skeletal growth (length) and the white muscle P/L ratio in fish with different growth capacities is illustrated.


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## Body

## 1. Introduction

One of the principal goals in  contemporary aquaculture is the dual optimisation of growth efficiency and protein content through a process of selective culture. The growth efficiency
of several fish species has been studied through variation of dietary protein
levels, and the results have shown not only the relationships between dietary
protein concentration and physiological parameters of consumption, growth, and
protein content in body or muscle, but also shown certain increased levels of
dietary protein limited consumption and growth of the fish recently, that is, [1–9]. Increased growth is usually not
associated with an increase in protein deposition rate, that is, [10–12], but rather an increase in lipid
deposition rate, that is, [8, 13]. Traditionally in aquaculture, dietary protein and
lipid are varied throughout the life cycle to optimise the economic return. In
the current work, we aim to understand a basic knowledge on long-term protein
growth by investigating the effects of maintaining dietary protein and lipid concentrations
throughout the fish life cycle on digestive efficiency and protein growth using
a unique combination of techniques recently described by
Rungruangsak-Torrissen’s research team [11, 12, 14]. In addition, we also aim
to study the potential for maximising protein growth efficiency in rainbow
trout (Oncorhynchus mykiss Walbaum)
families with differences in growth capacity by varying the concentrations of
dietary protein and lipid.The
current work was a part of a European Union project assessing the potential for
genetic selection of protein and growth efficiency traits in salmonids. The
work was performed in different families of rainbow trout divided into two
groups fed on two levels of dietary protein to lipid (P/L) ratio during a
two-year life span from juvenile to maturity, and subdivided according to
differences in growth capacities. Feed intake was measured by X-ray method
using diets labelled with radio-opaque markers [15]. The in vitro protein
digestibility of formulated fish feeds was determined using fish crude enzyme
extracts, as it has been correlated with the feed efficiency [11, 16] and the
chemical properties of dietary proteins, positively with the levels of reactive
sulphydryl group and negatively with the levels of disulphide bond and D-aspartic acid [16]. Fish digestive
efficiency [trypsin specific activity and the protease activity ratio of
trypsin to chymotrypsin (T/C ratio)] was measured because of their known
positive associations with fish growth rate [10–12, 14, 17, 18]. The effects of
dietary protein and P/L ratio levels on the protein and lipid compositions as
well as the P/L ratios of the body and white muscle were also determined. The
P/L ratio is important as both protein and lipid levels in the diets affect the
levels of both protein and lipid in fish where the variations may not be
observed if either protein or lipid level is studied alone. Although both
protein and lipid deposition are important but the in vitro lipid
digestibility was not determined because it has never been shown to associate
with feed efficiency and the quality of the lipid itself [19], and in vitro protein digestibility is the key factor for feed quality even in the herbivore
like mussel [19, 20]. This work was designed to illustrate the effects of dietary
P/L ratio levels on fish digestive ability and the quality of fish growth
performance (condition factor and composition of P/L ratio in the body and
white muscle) in families of rainbow trout with different growth capacities
throughout the life history.  The digestive efficiency was studied at the end of
the experiment at the late stage of life cycle. It is advantageous to use the
different methods simultaneously, as they could precisely provide practical
important biological informations between fish groups with differences in genetics (growth
capacity) and environmental impacts (diet quality) that could not be
explained by using growth parameter alone, especially when feeding regime were
not completely under control see [11, 12, 14].


## 2. Materials and Methods

### 2.1. Experimental Design

The experiment was performed on rainbow trout from an on-going selection programme
at the Finnish Game and Fisheries Research Institute,
Tervo, Finland. One-year old trout from 45 families selected were tagged
with PIT-transponders (Trovan Inc., Germany) and equally distributed into eight
3m3 tanks (340 fish per tank) at the end of January 2002. Further
details of the genetic design of the experiment have been described [21, 22].
From hatching to the initiation of the experiment, all fish were fed commercial
rainbow trout diets, Raisio Nutra Starter [52% crude
protein and 20% crude lipid (P/L ratio 2.6)] and Raisio Nutra Parr [50% crude
protein and 23% crude lipid (P/L ratio 2.2)] (Rehuraisio Inc., Finland) using
automatic feeders. In May 2002, two diet treatments were initiated. Four tanks
(average fish weight 141.2±2.2 g) were provided an
experimental normal protein (NP-) diet, and the other four (average fish weight
143.9±2.2 g) were provided an experimental high protein (HP-) diet. The
experimental NP- and HP-diets were manufactured by Rehuraisio Inc., Finland. The
diets were formulated using standard ingredients for rainbow trout in Finnish
aquaculture, consisting of fish meal, fish oil, wheat meal, wheat starch, and
supplemented for vitamins and minerals according to the National Research
Council [23]. The compositions of the experimental diets are shown in Table 1.
To avoid restricted growth, a slight over-feeding regime was applied. In June
2002, the fish were transferred to larger tanks (20 m3) and reared
through until the end of the experiment in November 2003. Fish density was
maintained below 20 kg m−3. Oxygen
concentration in the water was maintained above 6 mg L−1. The
fish experienced ambient seasonal temperature regimes during the experimental
period, fluctuating from below 5C° during winters to around 20C°
in summers. Individual fish weight and fork length were recorded in May and
September 2002, and in May, September, and November 2003. Individual feed
consumption was measured using the X-ray method [15] involving both NP- and HP-diets labelled with radio-opaque markers (more details in [21]). Nine estimates
of feed intake were  calculated during the three-week X-ray sessions performed
during May-June 2002, September-October 2002, and
September 2003. Feed efficiency was estimated, as weight gained per weight dry
feed consumed in each session, and averaged across all three sessions. To
estimate white muscle and whole body compositions, samples were collected in
November 2002 and November 2003.Table 1
Compositions
on wet weight basis of the experimental diets, normal protein (NP) and high
protein (HP) diets.
Experimental dietsMoisture [%]Crude protein (P) [%]Crude lipid (L) [%]P/L ratioCrude fibre [%]Ash [%]Nitrogen free extract [%]Phosphorus [%]Energy [kJg−1]NP (3 mm)2.144.930.51.50.737.415.11.2123.6NP (6 mm)1.544.630.31.50.727.715.91.2726.0NP (7 mm)4.039.533.41.21.006.715.40.9625.9HP (3 mm)2.356.420.72.70.449.611.11.5525.6HP
(6 mm)1.756.320.62.70.489.511.91.5424.0HP
(7 mm)7.249.423.82.11.308.310.01.1823.3Of the 45 families, 16
families were sorted with representatives of both diets
available for each family selected. Three different growth capacity groups were divided, 6 families for high
(H), 5 families for medium (M), and 5 families for low (L) growth capacity,
based on the body weight measurements recorded in
September 2003 (altogether 120 individuals). At the end of the experiment in November 2003,
pyloric caeca samples were collected (3–5 fish per family per diet). Intestinal, gonad, and carcass weights were measured. In vitro protein
digestibility of the experimental feeds and fish digestive efficiency were analysed, using the pyloric caecal enzyme extracts. Only
these 120 fish were studied in the current work. As thermal growth coefficient
[24] did not
provide different interpretations from specific growth rate (SGR) in this
experiment, the simple SGR was chosen and calculated [25]:(1)SGR(%day−1)=100[eg−1], where g=(lnW2−lnW1)/(t2−t1), and W2= Weight at day t2,W1= Weight at day t1.The condition factor was calculated [26](2)Conditionfactor=100×W/L3, where W = Live body weight (g) and L = Body fork length (cm).


### 2.2. Determinations of White Muscle and Body Composition

About
10 g of epaxial white muscle was dissected from individual fish in November
2002 and November 2003. The remainder of the fish was used for determination of
whole body composition. All tissue samples were stored at−80C° and homogenised
before analyses. The compositions of protein, lipid, and dry matter were
estimated for the white muscle and whole body. The white muscle in November
2002 and the whole body samples in November 2003 were determined using FMA2001
Milk Analyser (Miris AB, Sweden) with mid-infrared transmittance, after
homogenisation in standard solvent Mirasolve with Losmixer (Miris AB, Sweden).
Due to later technical complications, the body composition in November 2002 and
the white muscle composition in November 2003 were analysed using an INFRATEC
Food & Feed Analyser (Tecator, Sweden) with near-infrared transmittance. To
calibrate between the methods, approximately 50 samples were coanalysed by
both analysers (more details in [27]).The results from INFRATEC
Food & Feed Analyser were estimated using The Unscrambler,
software for multivariate data analysis (CAMO Process AS, Norway) with
multiregression models standardised by the values from 50 white muscle samples
and 50 whole body samples analysed chemically. The moisture content of the
standards was determined by drying a known amount (about 3 g) of homogenised
sample at 105C° for 24 hours to constant weight. The lipid concentration
was determined from the dry matter by extracting with 15 mL ethylacetate for 60
minutes on a mixing plate, and the lipid-ethylacetate extract was subsequently
dried gradually at 65–105C°, cooled in
desiccator, and then weighed [20, 28–30]. The protein concentration of
the standards was determined using TRIzol reagent (Life Technologies, NY, USA) to obtain complete
solubilisation of protein, as described in Rungruangsak-Torrissen [11] modified
from Sunde et al. [10, 18].Protein
deposition (protein growth) was expressed as the composition ratio of protein
to lipid (P/L ratio) in the body and white muscle.


### 2.3. Determination of Digestive Efficiency

Pyloric caeca sampled from 120 individuals
were analysed for trypsin specific activity and protease activity ratio of
trypsin to chymotrypsin (T/C ratio), as indicators for digestive efficiency
that have been related with fish growth rate [10–12, 14, 16, 18]. All samples were kept frozen at −80C° prior to analyses. The samples were
homogenised and the enzymes were extracted using 1 mM HCl. After centrifugation
at 15000×g for 60 minutes at 4C°,
the supernatants were kept frozen at −80C° until analyses. Trypsin and
chymotrypsin activities were determined, using benzoyl-L-arginine-p-nitroanilide as
trypsin substrate and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as chymotrypsin substrate, according to
Rungruangsak-Torrissen [11] based on Sunde et al. [10, 18] and
Rungruangsak-Torrissen et al. [16]. The concentration of protein in the crude
enzyme extract was determined by the Lowry method [31] using the Bio-Rad DC
(Detergent Compatible) Protein Assay (Bio-Rad Laboratories, Calif, USA). The
enzyme specific activities were expressed as μmol p-nitroaniline produced h−1 mg
protein−1. The
protease activity ratio of trypsin to chymotrypsin (T/C ratio) was directly calculated
from each individual, and the slope T/C ratio was obtained from the regression
between trypsin (y-axis) and chymotrypsin (x-axis) specific activities at each
sampling period [32].


### 2.4. In Vitro Digestibility Study

The in
vitro protein digestibility of
experimental feeds was performed according to Rungruangsak-Torrissen [11],
modified from Rungruangsak-Torrissen et al. [16] and Bassompierre et al. [33].
Due to limited amount of pyloric caecal sample collected from each fish, the
pyloric caecal crude enzyme extracts were pooled per family per diet (16
samples for each diet) after determination of the trypsin and chymotrypsin
specific activities in each individual. The pooled crude enzyme extracts were
dialysed using a Pierce Slide-A-Lyzer Dialysis Cassette (Pierce Chemical Company, Ill,
USA) and concentrated using an Amicon Ultra-15 centrifugal filter
device 10 K NMWL (Millipore Corporation, Mass, USA) before use. In
vitro protein digestibility was
measured in a commercial control (C) diet [Raisio Royal Response with 47% crude
protein and 23% crude lipid (P/L ratio 2.0), Rehuraisio Inc., Finland] and the
experimental NP (40% crude protein with P/L ratio 1.2) and HP (49% crude
protein with P/L ratio 2.1) diets (7 mm, see Table 1). In vitro protein digestibility of control C-diet, and
experimental NP-diet and HP-diet were performed using crude enzyme extracts
from the 16 families selected, divided into low (n=34), medium (n=36), and high (n=41) growth capacity family groups.Digestion of a known
amount of feed (about 20 mg in 40 mL of 50 mM phosphate buffer pH 8.2 and 0.2 mL of 0.5% chloramphenicol) by enzyme extract (0.5 mL) was performed in
triplicate at 15C° for 24 hours [11] for each diet and each dialysed
pooled crude enzyme extract. The reactive amino groups of peptides produced
after digestion were determined using the trinitrobenzene sulphonic acid (TNBS)
assay and calculated using DL-alanine as a standard, as
described in Rungruangsak-Torrissen [11], modified from Bassompierre et al. [33] based on Ihekoronye [34]. The in vitro protein digestibility value was expressed as μmol DL-alanine equivalent liberated
reactive amino group of cleaved peptides per mg feed sample after standardising
trypsin activity in each dialysed pooled crude enzyme extract to 300 μmol p-nitroaniline produced h−1mL−1.


### 2.5. Statistical Analysis

The statistical
procedures were performed using the SAS software package (v. 8.02 for Windows, SAS Institute Inc., NC, USA). Relationships, on
an individual basis, between different diets, families, and growth capacity
groups were studied using the general linear model (glm) procedure. This analysis included individual
continuous variables (covariates: e.g., white muscle P/L ratio or SGR). In cases
where class-by-covariate interaction was significant, the analysis was broken
down and performed per class (e.g., per diet or per growth capacity group). All
results are given as the least squaresmeans±standard error. Differences between means were
tested by t-test. The
difference was set at 95% significance level.


## 2.1. Experimental Design

The experiment was performed on rainbow trout from an on-going selection programme
at the Finnish Game and Fisheries Research Institute,
Tervo, Finland. One-year old trout from 45 families selected were tagged
with PIT-transponders (Trovan Inc., Germany) and equally distributed into eight
3m3 tanks (340 fish per tank) at the end of January 2002. Further
details of the genetic design of the experiment have been described [21, 22].
From hatching to the initiation of the experiment, all fish were fed commercial
rainbow trout diets, Raisio Nutra Starter [52% crude
protein and 20% crude lipid (P/L ratio 2.6)] and Raisio Nutra Parr [50% crude
protein and 23% crude lipid (P/L ratio 2.2)] (Rehuraisio Inc., Finland) using
automatic feeders. In May 2002, two diet treatments were initiated. Four tanks
(average fish weight 141.2±2.2 g) were provided an
experimental normal protein (NP-) diet, and the other four (average fish weight
143.9±2.2 g) were provided an experimental high protein (HP-) diet. The
experimental NP- and HP-diets were manufactured by Rehuraisio Inc., Finland. The
diets were formulated using standard ingredients for rainbow trout in Finnish
aquaculture, consisting of fish meal, fish oil, wheat meal, wheat starch, and
supplemented for vitamins and minerals according to the National Research
Council [23]. The compositions of the experimental diets are shown in Table 1.
To avoid restricted growth, a slight over-feeding regime was applied. In June
2002, the fish were transferred to larger tanks (20 m3) and reared
through until the end of the experiment in November 2003. Fish density was
maintained below 20 kg m−3. Oxygen
concentration in the water was maintained above 6 mg L−1. The
fish experienced ambient seasonal temperature regimes during the experimental
period, fluctuating from below 5C° during winters to around 20C°
in summers. Individual fish weight and fork length were recorded in May and
September 2002, and in May, September, and November 2003. Individual feed
consumption was measured using the X-ray method [15] involving both NP- and HP-diets labelled with radio-opaque markers (more details in [21]). Nine estimates
of feed intake were  calculated during the three-week X-ray sessions performed
during May-June 2002, September-October 2002, and
September 2003. Feed efficiency was estimated, as weight gained per weight dry
feed consumed in each session, and averaged across all three sessions. To
estimate white muscle and whole body compositions, samples were collected in
November 2002 and November 2003.Table 1
Compositions
on wet weight basis of the experimental diets, normal protein (NP) and high
protein (HP) diets.
Experimental dietsMoisture [%]Crude protein (P) [%]Crude lipid (L) [%]P/L ratioCrude fibre [%]Ash [%]Nitrogen free extract [%]Phosphorus [%]Energy [kJg−1]NP (3 mm)2.144.930.51.50.737.415.11.2123.6NP (6 mm)1.544.630.31.50.727.715.91.2726.0NP (7 mm)4.039.533.41.21.006.715.40.9625.9HP (3 mm)2.356.420.72.70.449.611.11.5525.6HP
(6 mm)1.756.320.62.70.489.511.91.5424.0HP
(7 mm)7.249.423.82.11.308.310.01.1823.3Of the 45 families, 16
families were sorted with representatives of both diets
available for each family selected. Three different growth capacity groups were divided, 6 families for high
(H), 5 families for medium (M), and 5 families for low (L) growth capacity,
based on the body weight measurements recorded in
September 2003 (altogether 120 individuals). At the end of the experiment in November 2003,
pyloric caeca samples were collected (3–5 fish per family per diet). Intestinal, gonad, and carcass weights were measured. In vitro protein
digestibility of the experimental feeds and fish digestive efficiency were analysed, using the pyloric caecal enzyme extracts. Only
these 120 fish were studied in the current work. As thermal growth coefficient
[24] did not
provide different interpretations from specific growth rate (SGR) in this
experiment, the simple SGR was chosen and calculated [25]:(1)SGR(%day−1)=100[eg−1], where g=(lnW2−lnW1)/(t2−t1), and W2= Weight at day t2,W1= Weight at day t1.The condition factor was calculated [26](2)Conditionfactor=100×W/L3, where W = Live body weight (g) and L = Body fork length (cm).


## 2.2. Determinations of White Muscle and Body Composition

About
10 g of epaxial white muscle was dissected from individual fish in November
2002 and November 2003. The remainder of the fish was used for determination of
whole body composition. All tissue samples were stored at−80C° and homogenised
before analyses. The compositions of protein, lipid, and dry matter were
estimated for the white muscle and whole body. The white muscle in November
2002 and the whole body samples in November 2003 were determined using FMA2001
Milk Analyser (Miris AB, Sweden) with mid-infrared transmittance, after
homogenisation in standard solvent Mirasolve with Losmixer (Miris AB, Sweden).
Due to later technical complications, the body composition in November 2002 and
the white muscle composition in November 2003 were analysed using an INFRATEC
Food & Feed Analyser (Tecator, Sweden) with near-infrared transmittance. To
calibrate between the methods, approximately 50 samples were coanalysed by
both analysers (more details in [27]).The results from INFRATEC
Food & Feed Analyser were estimated using The Unscrambler,
software for multivariate data analysis (CAMO Process AS, Norway) with
multiregression models standardised by the values from 50 white muscle samples
and 50 whole body samples analysed chemically. The moisture content of the
standards was determined by drying a known amount (about 3 g) of homogenised
sample at 105C° for 24 hours to constant weight. The lipid concentration
was determined from the dry matter by extracting with 15 mL ethylacetate for 60
minutes on a mixing plate, and the lipid-ethylacetate extract was subsequently
dried gradually at 65–105C°, cooled in
desiccator, and then weighed [20, 28–30]. The protein concentration of
the standards was determined using TRIzol reagent (Life Technologies, NY, USA) to obtain complete
solubilisation of protein, as described in Rungruangsak-Torrissen [11] modified
from Sunde et al. [10, 18].Protein
deposition (protein growth) was expressed as the composition ratio of protein
to lipid (P/L ratio) in the body and white muscle.


## 2.3. Determination of Digestive Efficiency

Pyloric caeca sampled from 120 individuals
were analysed for trypsin specific activity and protease activity ratio of
trypsin to chymotrypsin (T/C ratio), as indicators for digestive efficiency
that have been related with fish growth rate [10–12, 14, 16, 18]. All samples were kept frozen at −80C° prior to analyses. The samples were
homogenised and the enzymes were extracted using 1 mM HCl. After centrifugation
at 15000×g for 60 minutes at 4C°,
the supernatants were kept frozen at −80C° until analyses. Trypsin and
chymotrypsin activities were determined, using benzoyl-L-arginine-p-nitroanilide as
trypsin substrate and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as chymotrypsin substrate, according to
Rungruangsak-Torrissen [11] based on Sunde et al. [10, 18] and
Rungruangsak-Torrissen et al. [16]. The concentration of protein in the crude
enzyme extract was determined by the Lowry method [31] using the Bio-Rad DC
(Detergent Compatible) Protein Assay (Bio-Rad Laboratories, Calif, USA). The
enzyme specific activities were expressed as μmol p-nitroaniline produced h−1 mg
protein−1. The
protease activity ratio of trypsin to chymotrypsin (T/C ratio) was directly calculated
from each individual, and the slope T/C ratio was obtained from the regression
between trypsin (y-axis) and chymotrypsin (x-axis) specific activities at each
sampling period [32].


## 2.4. In Vitro Digestibility Study

The in
vitro protein digestibility of
experimental feeds was performed according to Rungruangsak-Torrissen [11],
modified from Rungruangsak-Torrissen et al. [16] and Bassompierre et al. [33].
Due to limited amount of pyloric caecal sample collected from each fish, the
pyloric caecal crude enzyme extracts were pooled per family per diet (16
samples for each diet) after determination of the trypsin and chymotrypsin
specific activities in each individual. The pooled crude enzyme extracts were
dialysed using a Pierce Slide-A-Lyzer Dialysis Cassette (Pierce Chemical Company, Ill,
USA) and concentrated using an Amicon Ultra-15 centrifugal filter
device 10 K NMWL (Millipore Corporation, Mass, USA) before use. In
vitro protein digestibility was
measured in a commercial control (C) diet [Raisio Royal Response with 47% crude
protein and 23% crude lipid (P/L ratio 2.0), Rehuraisio Inc., Finland] and the
experimental NP (40% crude protein with P/L ratio 1.2) and HP (49% crude
protein with P/L ratio 2.1) diets (7 mm, see Table 1). In vitro protein digestibility of control C-diet, and
experimental NP-diet and HP-diet were performed using crude enzyme extracts
from the 16 families selected, divided into low (n=34), medium (n=36), and high (n=41) growth capacity family groups.Digestion of a known
amount of feed (about 20 mg in 40 mL of 50 mM phosphate buffer pH 8.2 and 0.2 mL of 0.5% chloramphenicol) by enzyme extract (0.5 mL) was performed in
triplicate at 15C° for 24 hours [11] for each diet and each dialysed
pooled crude enzyme extract. The reactive amino groups of peptides produced
after digestion were determined using the trinitrobenzene sulphonic acid (TNBS)
assay and calculated using DL-alanine as a standard, as
described in Rungruangsak-Torrissen [11], modified from Bassompierre et al. [33] based on Ihekoronye [34]. The in vitro protein digestibility value was expressed as μmol DL-alanine equivalent liberated
reactive amino group of cleaved peptides per mg feed sample after standardising
trypsin activity in each dialysed pooled crude enzyme extract to 300 μmol p-nitroaniline produced h−1mL−1.


## 2.5. Statistical Analysis

The statistical
procedures were performed using the SAS software package (v. 8.02 for Windows, SAS Institute Inc., NC, USA). Relationships, on
an individual basis, between different diets, families, and growth capacity
groups were studied using the general linear model (glm) procedure. This analysis included individual
continuous variables (covariates: e.g., white muscle P/L ratio or SGR). In cases
where class-by-covariate interaction was significant, the analysis was broken
down and performed per class (e.g., per diet or per growth capacity group). All
results are given as the least squaresmeans±standard error. Differences between means were
tested by t-test. The
difference was set at 95% significance level.


## 3. Results

### 3.1. Fish Growth and Feed Efficiency

Due to some
uncertain labelling, only 111 out of 120 fish were used for the analyses.
Paired comparison indicated that the HP-diet fish had lower condition factor than
the NP-diet fish during the entire experimental period (P<.05; Figure 1). The differences in condition factor were
enhanced during autumn to winter, especially the last period from September to
November 2003 (P<.01; Figure 1 and Table 2). This was due to differences in body length between
the diet treatments (P<.03; Table 2). There were no significant differences in body
weight between the two dietary groups although the HP-diet fish seemed to have
a relatively higher body weight than the NP-diet fish before maturation (P>.05). During maturation in the last two months, fish growth
rates were reduced in both dietary groups, and the HP-diet fish showed a higher
decrease in SGR (P<.001) with the body weight changed (P>.05) from
a relatively higher in September 2003 to a relatively lower in November 2003,
compared to the NP-diet fish (Figure 1 and Table 2). The HP-diet fish also had
lower feed efficiency (FE) than the NP-diet fish (P<.05; Table 2). Individual feed intake in
September 2003 was not correlated with FE but showed a positive correlation
with SGR during the last two months (R2=0.11, P<.05).Table 2
Different biological parameters in rainbow trout fed the
experimental normal protein (NP) and high protein (HP) diets, in vitro protein
digestibility was expressed asμmol DL-alanine
equivalent liberated reactive amino group of cleaved peptides per mg feed after
standardising trypsin activity in each dialysed pooled crude enzyme extract to
300 μmol p-nitroaniline
produced h−1 mL−1, and
the values with asterisk (*) are significantly different (P<.05).
ParametersNP-diet group (n=56)HP-diet group (n=55)Initial weight in May 2002 [g]140±5148±5September 2003Weight [g]2120±552151±44Length [cm]47.9±0.37*49.0±0.34*Condition factor [g cm−3]1.91±0.02*1.82±0.02*November 2003Final weight [g]2622±732560±60Final length [cm]51.0±0.451.6±0.4Final condition factor [gcm−3]1.96±0.03*1.85±0.03*Specific growth rate (Sept–Nov 2003) [%day−1]0.50±0.02*0.40±0.02*Feed efficiency [daily-gain in g per g feed-intake]Average of all three X-ray sessions1.73±0.13*1.31±0.09*Trypsin specific activity [μmol p-nitroaniline
h−1 mg protein−1]28.7±2.2*41.0±4.5*low growth capacity group30.1±4.5 (n=17)39.3±7.9 (n=17)medium growth capacity group27.7±3.3 (n=17)40.2±7.0 (n=19)high growth capacity group28.3±3.7 (n=22)43.4±8.8 (n=19)Chymotrypsin specific activity [μmol p-nitroaniline
h−1 mg protein−1]102±9*143±12*Low growth capacity group108±19 (n=17)139±20 (n=17)Medium growth capacity group103±17 (n=17)138±15 (n=19)High growth capacity group96±13* (n=22)151±24* (n=19)Protease activity ratio of trypsin to chymotrypsin
(T/C ratio)0.32±0.02*0.27±0.01*Low growth capacity group0.31±0.02 (n=17)0.25±0.02 (n=17)Medium growth capacity group0.33±0.04 (n=17)0.28±0.03 (n=19)High growth capacity group0.32±0.03 (n=22)0.29±0.03 (n=19)In vitro digestibility [μmol DL-alanine
equivalent mg feed−1]Control diet4.25±0.45  (n=16)4.61±0.69 (n=16)NP–diet4.24±0.45 (n=16)4.80±0.75 (n=16)HP–diet4.92±0.48 (n=16)5.49±0.80 (n=16)In vitro digestibility regardless of diet typeLow growth capacity group4.32±0.44 (n=15)3.85±0.48 (n=15)Medium growth capacity group4.17±0.39 (n=15)3.91±0.43 (n=15)High growth capacity group (P<.06)4.85±0.37 (n=18)6.77±0.49 (n=18)Intestinal weight [g]340±12*279±9*Gonad weight [g]60±772±7Carcass weight [g]2191±602196±50Proximate composition [% on wet weight basis]Muscle protein22.9±0.4*24.8±0.4*Muscle lipid8.6±0.3*6.3±0.3*Muscle ratio of protein to lipid (P/L ratio)2.92±0.14*4.44±0.22*Body protein15.7±0.07*16.4±0.07*Body lipid22.1±0.17*20.2±0.19*Body ratio of protein to lipid (P/L ratio)0.71±0.01*0.82±0.01*Figure 1
Weight and condition factor of
rainbow trout fed normal protein (NP) and high protein (HP) diets during the
entire experimental period. The values with asterisk (*) are significantly
different (P<.05) between the dietary groups.There
were relationships between body weight and carcass weight, and between body
weight and intestinal weight (R2=0.49–0.98, P<.0001). The
relationships between SGR with carcass weight and between SGR with intestinal
weight were also observed (R2=0.08–0.29, P<.0001). This
was consistent for both dietary treatments. However, intestinal weight was
lower in the HP-diet fish (P<.0001; Table 2), due to reduction in
lipid deposits around the intestine (observed visually). No differences in
carcass and gonad weights (Table 2) were observed between the two dietary
groups.Maturation
rate seemed to be higher in the HP-diet than the NP-diet fish in all growth
capacity groups, varied from 64.7–84.2% in the HP-diet fish compared
to 52.9–70.6% in the NP-diet fish.


### 3.2. Digestive Proteases in the Pyloric Caeca

At
maturity in November 2003, rainbow trout fed on HP-diet had higher specific
activities of the protease enzymes trypsin (P<.02) and chymotrypsin (P<.01), and lower T/C ratio (P<.03), than those fed on NP-diet (Table 2).
When the fish were grouped according to family growth capacity, the same trends
were also observed within each growth capacity group, but the difference was
only significant in the chymotrypsin specific activity between the fish fed HP-
and NP-diets in the high growth capacity families (P<.05; Table 2).Trypsin
(P<.002; Figure 2(a)) and chymotrypsin (P<.003; Figure
2(b)) specific activities were inversely correlated with the intestinal weight.
In contrast, the T/C ratio was positively correlated with the intestinal weight
(P<.03; Figure 2(c)). Similarly, the specific activities of trypsin
(R2=0.05, P<.002) and chymotrypsin (R2=0.04, P<.05) were inversely correlated with body weight, and the
T/C ratio showed a trend of positive correlation with fish body weight although
insignificant (data not shown). Among the protease parameters measured, only
the specific activity of trypsin was inversely correlated with SGR (R2=0.04, P<.04) during
maturation, regardless of dietary type (data not shown). Within each dietary
group, these relationships were not significant, except for the negative
correlation in the HP-diet group between intestinal weight and trypsin specific
activity (P<.03; Figure 2(a)) and
between final weight and trypsin specific activity (R2=0.09, P<.03). The observations of similarity in slope T/C ratio
(Figure 3) between the HP-diet (0.222) and the NP-diet (0.212) groups, and the
lack of the relationship between trypsin specific activity and the T/C ratio
(see details in [12, 14]) indicated that the fish were at a similar steady growth
phase at sampling at the end of the experiment.Relationships
between intestinal weight with trypsin specific activity [a],
chymotrypsin specific activity [b], and protease activity ratio of
trypsin to chymotrypsin (T/C ratio) [c]. The protease specific
activities were expressed asμmol p-nitroaniline
produced h−1 mg protein−1.
(a)(b)(c)Figure 3
Relationships
between trypsin (T) and chymotrypsin (C) specific activities showing the slope
T/C ratios (bold values) 	 of rainbow trout fed
normal protein (NP) and high protein (HP) diets at sampling at the end of the
experiment in November 2003.


### 3.3. White Muscle and Body Compositions

SGR
correlated positively with lipid concentration (P<.0001) in the white muscle, and negatively
with moisture (P<.0001) and protein (P<.02) concentrations (Figure 4). This resulted in an inverse
relationship between SGR and the composition ratio of protein to lipid (P/L
ratio) in the white muscle (P<.001; Figure 4). Within each dietary group, the relationships
were significant only in the NP-diet fish (P<.02), except for protein concentration that no correlations were
observed in both dietary groups (Figure 4).Figure 4
Relationships
between specific growth rate (SGR) with white muscle composition and white
muscle ratio of protein to lipid (P/L ratio). Regardless of dietary group, the
regressions arey=−4.0641x+72.166 (R2=0.1579,n=111, P<.0001 for moisture), y=−5.0925x+26.137 (R2=0.0553,n=111, P<.02 for protein), y=6.1719x+4.636 (R2=0.1444,n=111, P<.0001 for lipid) and y=−3.4688x+5.2283 (R2=0.1053,n=111, P<.001 for P/L ratio).At successive
measurements, protein concentration increased significantly in the white muscle
(P<.0001) from November 2002 (20.50±0.08%) to November 2003
(24.34±0.12%). Lipid concentration (7.48±0.19 vs 7.71±0.08%), P/L
ratio (3.53±0.09 vs 3.62±0.05) also increased, albeit
insignificant. Moisture concentration, on the other hand, decreased (P<.0001) from November 2002 (72.12±0.21%) to November 2003 (70.14±0.05%). At the end of the experiment, the moisture concentration in the white
muscle was significantly higher in fish fed on HP-diet than those fed on
NP-diet (71.0±0.2 vs 69.7±0.2%, P<.0001). The concentration
of protein (P<.004) and the P/L ratio (P<.0001) in the white muscle were also higher and the lipid
concentration was lower (P<.0001) in the fish fed the HP-diet than
the NP-diet (Table 2). According to the dietary P/L ratio levels in the HP (2.1–2.7) and the NP (1.2–1.5) diets (Table 1), rainbow trout
at late stage seemed to double the P/L ratios in the white muscle (HP-diet
fish: 4.44±0.22; NP-diet fish: 2.92±0.14) (Table 2) with respect to the P/L
ratio of the diet consumed.For
whole body composition, SGR also showed positive correlation with lipid
concentration (R2=0.09, P<.002), and negative
correlation with protein concentration (R2=0.10, P<.001), resulting in an inverse relationship with the P/L ratio (R2=0.11, P<.001). Similar to white muscle composition, whole body
concentrations of protein (November 2002: 9.94±0.14%; November 2003: 16.04±0.06%), lipid (17.98±0.08 vs 21.14±0.16%), and P/L ratio (0.56±0.01 vs
0.77±0.01) increased between successive measurement periods (P<.0001). At the end of the experiment, fish fed on HP-diet had higher
concentrations of whole body protein (16.44±0.07 vs 15.65±0.07%) and P/L
ratio (0.82±0.01 vs 0.71±0.01), and lower concentration of whole body lipid
(20.15±0.19 vs 22.10±0.17%) than the NP-diet fish (P<.0001;
Table 2). Moreover, there was a significant inverse relationship between body
P/L ratio and the condition factor, regardless of dietary group (P<.0003; Figure 5). Within the dietary groups, the relationship was significant
only in the NP-diet fish (P<.004; Figure 5).Figure 5
Relationship
between body P/L ratio and condition factor.Among different
composition parameters studied, white muscle protein concentration was the only
factor that significantly correlated with feed efficiency, with a negative
relationship (R2=0.11, P<.05), regardless of
dietary type.


### 3.4. In Vitro Digestibility and Comparisons between Different Growth Capacity Groups

Low growth
capacity families had lower final weight (2253±63 g) than those from medium
(2684±63 g) and high (2791±85 g) growth capacities (P<.0001). Within each growth capacity group,
no differences in weight (Figure 6(a)) and the T/C ratio (Figure 6(b)) were
observed between the fish fed NP-diet and HP-diet. Feed efficiency was
significantly different between the two dietary treatments only in the medium
growth capacity families (P<.01; Figure 6(c)). The P/L ratio in the white muscle was higher in
the HP-diet than the NP-diet fish in all growth groups (P<.02; Figure 6(d)).
Interestingly in the high growth families, the white muscle P/L ratio of the
HP-diet fish increased 80% more than the NP-diet fish, whereas the increase was
only 40% in the low and medium growth families (P<.03; Figure 6(d)).Final body
weight [a], protease activity ratio of trypsin to chymotrypsin (T/C
ratio) [b], feed efficiency [c], and white muscle composition
ratio of protein to lipid (P/L ratio) [d], in groups of rainbow trout
with low, medium, and high growth capacity, subdivided according to feeding
normal protein (NP) or high protein (HP) diet. The values with different superscripts are significantly different (P<.05).
(a)(b)(c)(d)The digestive ability
was different among different growth capacity groups (Figure7).  The enzyme extracts from families with high
growth capacity resulted in relatively higher in vitro protein digestibility
values for C-diet (P<.05), NP-diet (P<.05), and HP-diet (P>.05) than those from low and medium
growth capacity families (Figure 7). Paired comparison indicated significantly
higher in vitro protein digestibility of the HP-diet than the C-diet and
the NP-diet, regardless of the enzyme extracts used (P<.0001).
Within each growth capacity family, a tendency of higher in vitro protein
digestibility was observed in the enzyme extracts from fish fed HP-diet, albeit
insignificant (Table 2). A significant inverse relationship between in vitro protein digestibility values and SGR (R2=0.18, P<.02) was observed at maturity in November 2003, regardless of dietary
type.Figure 7
In vitro protein digestibility of control
(C), normal protein (NP), and high protein (HP) diets, among rainbow trout with
low, medium, and high growth capacities. The values with different superscripts
are significantly different (P<.05).


### 3.5. Analysis of Covariance

The continuous
variable white muscle P/L ratio was included as covariate in the models in
order to study its effect on growth together with fish growth capacity group or
family fed on the different diets. Due to class-by-covariate interaction being
significant (P<.05), the
statistical analysis was performed separately per class. The effects on the
growth parameters (final weight, condition factor, and SGR) by growth capacity
group and muscle P/L-ratio, and by family and muscle P/L-ratio, were studied
separately for each dietary group (Table 3). Differences in fish growth
capacity significantly affected final body weight for both diets but did not
affect condition factor and SGR. Family influenced fish SGR for both diets, and
the growth of the fish fed on NP-diet was highly affected by family and muscle
P/L ratio compared to the HP-diet fish. The results clearly indicated that the
differences in fish growth capacity among different families and the level of
P/L ratio in the white muscle affected fish growth, and their effects varied
according to the dietary P/L ratio.Table 3
Analysis of covariance showing the effects of fish growth capacity
(Group) or family and white muscle ratio of protein to lipid (P/L ratio) as
covariate, on final weight, condition factor, and specific growth rate (SGR)
during September–November 2003. Due to class-by-covariate interaction was
significant (P<.05); the
statistical analysis was performed separately for the experimental normal
protein (NP) and high protein (HP) diets. Probabilities with significant
effects are shown by the P values <.05 (bold values).
(a)Covariance effectdfFinal weightCondition factorSGRNPHPNPHPNPHPGroup20.00030.00220.41510.84530.97030.7316Muscle P/L ratio10.00120.17760.00330.71620.01250.1689Family150.00380.0961<0.00010.16800.00790.0465Muscle P/L ratio10.00310.10330.00890.45140.00670.2924(b)NPHPGroupFinal weightR2=0.389(P<.0001)R2=0.224(P<.005)Condition factorR2=0.186(P<.015)R2=0.013(P=.883)SGRR2=0.117(P=.089)R2=0.066(P=.321)FamilyFinal weightR2=0.606(P<.0005)R2=0.407(P=.107)Condition factorR2=0.725(P<.0001)R2=0.370(P=.195)SGRR2=0.560(P<.0025)R2=0.467(P<.035)In order to study the association
between SGR, dietary quality, and digestive abilities of different growth
capacity groups, the continuous variable SGR was included as covariate in the
models. Similarly, the class-by-covariate interaction was significant (P<.05); thus, the statistical analysis was also performed separately per
class. The effects on specific activities of trypsin and chymotrypsin, T/C
ratio, and in vitro protein digestibility of diet, by dietary P/L ratio
and SGR were studied for each growth capacity group (Table 4). The results
indicated an effect of the diet on the T/C ratio of the low growth capacity
group. There was an indication of the associations between SGR and the specific
activities of trypsin and chymotrypsin. Trypsin specific activity was affected
in the high growth capacity group, whereas chymotrypsin specific activity was
affected in the low growth capacity group. There was an association between SGR
and in vitro protein digestibility of the experimental diets in the high
growth capacity group.Table 4
Analysis
of covariance showing the effects of dietary P/L ratio level (Diet) and
specific growth rate (SGR) during September–November 2003 as covariate, on
the expression of trypsin and chymotrypsin (expressed asμmol p-nitroaniline h−1 mg protein−1), the activity ratio of trypsin to
chymotrypsin (T/C ratio), and in vitro protein digestibility (expressed as μmol DL-alanine equivalent per
mg feed after standardising trypsin activity to 300 μmol p-nitroaniline
produced  h−1mL−1). Due to class-by-covariate interaction was
significant (P < .05); the statistical analysis was performed
separately for low (L), medium (M), and high (H) growth capacity groups.
Probabilities with significant effects are shown by the P values <.05 (bold values).
(a)Covariance effectdfTrypsinChymotrypsinT/C ratioIn vitro digestibilityLMHLMHLMHLMHDiet10.17960.32760.49050.10330.36470.20350.04580.48640.44710.32860.69380.4804SGR10.10590.10550.03200.01740.08540.09050.97050.18260.74240.44030.48070.0403(b)LMHTrypsinR2=0.111(P=.163)R2=0.139(P=.085)R2=0.173(P<.03)ChymotrypsinR2=0.205(P<.035)R2=0.147(P=.078)R2=0.169(P<.03)T/C ratioR2=0.131(P=.123)R2=0.092(P=.213)R2=0.015(P=.746)In vitro digestibilityR2=0.041(P=.523)R2=0.016(P=.764)R2=0.163(P<.035)The
analyses of covariance (Tables3 and 4) indicated the associations between
dietary P/L ratio, fish digestive ability (trypsin and chymotrypsin specific
activity, T/C ratio, in vitro protein digestibility), white muscle P/L
ratio, growth parameters (fish weight, condition factor, SGR), and variations
in growth capacity of different family memberships.


## 3.1. Fish Growth and Feed Efficiency

Due to some
uncertain labelling, only 111 out of 120 fish were used for the analyses.
Paired comparison indicated that the HP-diet fish had lower condition factor than
the NP-diet fish during the entire experimental period (P<.05; Figure 1). The differences in condition factor were
enhanced during autumn to winter, especially the last period from September to
November 2003 (P<.01; Figure 1 and Table 2). This was due to differences in body length between
the diet treatments (P<.03; Table 2). There were no significant differences in body
weight between the two dietary groups although the HP-diet fish seemed to have
a relatively higher body weight than the NP-diet fish before maturation (P>.05). During maturation in the last two months, fish growth
rates were reduced in both dietary groups, and the HP-diet fish showed a higher
decrease in SGR (P<.001) with the body weight changed (P>.05) from
a relatively higher in September 2003 to a relatively lower in November 2003,
compared to the NP-diet fish (Figure 1 and Table 2). The HP-diet fish also had
lower feed efficiency (FE) than the NP-diet fish (P<.05; Table 2). Individual feed intake in
September 2003 was not correlated with FE but showed a positive correlation
with SGR during the last two months (R2=0.11, P<.05).Table 2
Different biological parameters in rainbow trout fed the
experimental normal protein (NP) and high protein (HP) diets, in vitro protein
digestibility was expressed asμmol DL-alanine
equivalent liberated reactive amino group of cleaved peptides per mg feed after
standardising trypsin activity in each dialysed pooled crude enzyme extract to
300 μmol p-nitroaniline
produced h−1 mL−1, and
the values with asterisk (*) are significantly different (P<.05).
ParametersNP-diet group (n=56)HP-diet group (n=55)Initial weight in May 2002 [g]140±5148±5September 2003Weight [g]2120±552151±44Length [cm]47.9±0.37*49.0±0.34*Condition factor [g cm−3]1.91±0.02*1.82±0.02*November 2003Final weight [g]2622±732560±60Final length [cm]51.0±0.451.6±0.4Final condition factor [gcm−3]1.96±0.03*1.85±0.03*Specific growth rate (Sept–Nov 2003) [%day−1]0.50±0.02*0.40±0.02*Feed efficiency [daily-gain in g per g feed-intake]Average of all three X-ray sessions1.73±0.13*1.31±0.09*Trypsin specific activity [μmol p-nitroaniline
h−1 mg protein−1]28.7±2.2*41.0±4.5*low growth capacity group30.1±4.5 (n=17)39.3±7.9 (n=17)medium growth capacity group27.7±3.3 (n=17)40.2±7.0 (n=19)high growth capacity group28.3±3.7 (n=22)43.4±8.8 (n=19)Chymotrypsin specific activity [μmol p-nitroaniline
h−1 mg protein−1]102±9*143±12*Low growth capacity group108±19 (n=17)139±20 (n=17)Medium growth capacity group103±17 (n=17)138±15 (n=19)High growth capacity group96±13* (n=22)151±24* (n=19)Protease activity ratio of trypsin to chymotrypsin
(T/C ratio)0.32±0.02*0.27±0.01*Low growth capacity group0.31±0.02 (n=17)0.25±0.02 (n=17)Medium growth capacity group0.33±0.04 (n=17)0.28±0.03 (n=19)High growth capacity group0.32±0.03 (n=22)0.29±0.03 (n=19)In vitro digestibility [μmol DL-alanine
equivalent mg feed−1]Control diet4.25±0.45  (n=16)4.61±0.69 (n=16)NP–diet4.24±0.45 (n=16)4.80±0.75 (n=16)HP–diet4.92±0.48 (n=16)5.49±0.80 (n=16)In vitro digestibility regardless of diet typeLow growth capacity group4.32±0.44 (n=15)3.85±0.48 (n=15)Medium growth capacity group4.17±0.39 (n=15)3.91±0.43 (n=15)High growth capacity group (P<.06)4.85±0.37 (n=18)6.77±0.49 (n=18)Intestinal weight [g]340±12*279±9*Gonad weight [g]60±772±7Carcass weight [g]2191±602196±50Proximate composition [% on wet weight basis]Muscle protein22.9±0.4*24.8±0.4*Muscle lipid8.6±0.3*6.3±0.3*Muscle ratio of protein to lipid (P/L ratio)2.92±0.14*4.44±0.22*Body protein15.7±0.07*16.4±0.07*Body lipid22.1±0.17*20.2±0.19*Body ratio of protein to lipid (P/L ratio)0.71±0.01*0.82±0.01*Figure 1
Weight and condition factor of
rainbow trout fed normal protein (NP) and high protein (HP) diets during the
entire experimental period. The values with asterisk (*) are significantly
different (P<.05) between the dietary groups.There
were relationships between body weight and carcass weight, and between body
weight and intestinal weight (R2=0.49–0.98, P<.0001). The
relationships between SGR with carcass weight and between SGR with intestinal
weight were also observed (R2=0.08–0.29, P<.0001). This
was consistent for both dietary treatments. However, intestinal weight was
lower in the HP-diet fish (P<.0001; Table 2), due to reduction in
lipid deposits around the intestine (observed visually). No differences in
carcass and gonad weights (Table 2) were observed between the two dietary
groups.Maturation
rate seemed to be higher in the HP-diet than the NP-diet fish in all growth
capacity groups, varied from 64.7–84.2% in the HP-diet fish compared
to 52.9–70.6% in the NP-diet fish.


## 3.2. Digestive Proteases in the Pyloric Caeca

At
maturity in November 2003, rainbow trout fed on HP-diet had higher specific
activities of the protease enzymes trypsin (P<.02) and chymotrypsin (P<.01), and lower T/C ratio (P<.03), than those fed on NP-diet (Table 2).
When the fish were grouped according to family growth capacity, the same trends
were also observed within each growth capacity group, but the difference was
only significant in the chymotrypsin specific activity between the fish fed HP-
and NP-diets in the high growth capacity families (P<.05; Table 2).Trypsin
(P<.002; Figure 2(a)) and chymotrypsin (P<.003; Figure
2(b)) specific activities were inversely correlated with the intestinal weight.
In contrast, the T/C ratio was positively correlated with the intestinal weight
(P<.03; Figure 2(c)). Similarly, the specific activities of trypsin
(R2=0.05, P<.002) and chymotrypsin (R2=0.04, P<.05) were inversely correlated with body weight, and the
T/C ratio showed a trend of positive correlation with fish body weight although
insignificant (data not shown). Among the protease parameters measured, only
the specific activity of trypsin was inversely correlated with SGR (R2=0.04, P<.04) during
maturation, regardless of dietary type (data not shown). Within each dietary
group, these relationships were not significant, except for the negative
correlation in the HP-diet group between intestinal weight and trypsin specific
activity (P<.03; Figure 2(a)) and
between final weight and trypsin specific activity (R2=0.09, P<.03). The observations of similarity in slope T/C ratio
(Figure 3) between the HP-diet (0.222) and the NP-diet (0.212) groups, and the
lack of the relationship between trypsin specific activity and the T/C ratio
(see details in [12, 14]) indicated that the fish were at a similar steady growth
phase at sampling at the end of the experiment.Relationships
between intestinal weight with trypsin specific activity [a],
chymotrypsin specific activity [b], and protease activity ratio of
trypsin to chymotrypsin (T/C ratio) [c]. The protease specific
activities were expressed asμmol p-nitroaniline
produced h−1 mg protein−1.
(a)(b)(c)Figure 3
Relationships
between trypsin (T) and chymotrypsin (C) specific activities showing the slope
T/C ratios (bold values) 	 of rainbow trout fed
normal protein (NP) and high protein (HP) diets at sampling at the end of the
experiment in November 2003.


## 3.3. White Muscle and Body Compositions

SGR
correlated positively with lipid concentration (P<.0001) in the white muscle, and negatively
with moisture (P<.0001) and protein (P<.02) concentrations (Figure 4). This resulted in an inverse
relationship between SGR and the composition ratio of protein to lipid (P/L
ratio) in the white muscle (P<.001; Figure 4). Within each dietary group, the relationships
were significant only in the NP-diet fish (P<.02), except for protein concentration that no correlations were
observed in both dietary groups (Figure 4).Figure 4
Relationships
between specific growth rate (SGR) with white muscle composition and white
muscle ratio of protein to lipid (P/L ratio). Regardless of dietary group, the
regressions arey=−4.0641x+72.166 (R2=0.1579,n=111, P<.0001 for moisture), y=−5.0925x+26.137 (R2=0.0553,n=111, P<.02 for protein), y=6.1719x+4.636 (R2=0.1444,n=111, P<.0001 for lipid) and y=−3.4688x+5.2283 (R2=0.1053,n=111, P<.001 for P/L ratio).At successive
measurements, protein concentration increased significantly in the white muscle
(P<.0001) from November 2002 (20.50±0.08%) to November 2003
(24.34±0.12%). Lipid concentration (7.48±0.19 vs 7.71±0.08%), P/L
ratio (3.53±0.09 vs 3.62±0.05) also increased, albeit
insignificant. Moisture concentration, on the other hand, decreased (P<.0001) from November 2002 (72.12±0.21%) to November 2003 (70.14±0.05%). At the end of the experiment, the moisture concentration in the white
muscle was significantly higher in fish fed on HP-diet than those fed on
NP-diet (71.0±0.2 vs 69.7±0.2%, P<.0001). The concentration
of protein (P<.004) and the P/L ratio (P<.0001) in the white muscle were also higher and the lipid
concentration was lower (P<.0001) in the fish fed the HP-diet than
the NP-diet (Table 2). According to the dietary P/L ratio levels in the HP (2.1–2.7) and the NP (1.2–1.5) diets (Table 1), rainbow trout
at late stage seemed to double the P/L ratios in the white muscle (HP-diet
fish: 4.44±0.22; NP-diet fish: 2.92±0.14) (Table 2) with respect to the P/L
ratio of the diet consumed.For
whole body composition, SGR also showed positive correlation with lipid
concentration (R2=0.09, P<.002), and negative
correlation with protein concentration (R2=0.10, P<.001), resulting in an inverse relationship with the P/L ratio (R2=0.11, P<.001). Similar to white muscle composition, whole body
concentrations of protein (November 2002: 9.94±0.14%; November 2003: 16.04±0.06%), lipid (17.98±0.08 vs 21.14±0.16%), and P/L ratio (0.56±0.01 vs
0.77±0.01) increased between successive measurement periods (P<.0001). At the end of the experiment, fish fed on HP-diet had higher
concentrations of whole body protein (16.44±0.07 vs 15.65±0.07%) and P/L
ratio (0.82±0.01 vs 0.71±0.01), and lower concentration of whole body lipid
(20.15±0.19 vs 22.10±0.17%) than the NP-diet fish (P<.0001;
Table 2). Moreover, there was a significant inverse relationship between body
P/L ratio and the condition factor, regardless of dietary group (P<.0003; Figure 5). Within the dietary groups, the relationship was significant
only in the NP-diet fish (P<.004; Figure 5).Figure 5
Relationship
between body P/L ratio and condition factor.Among different
composition parameters studied, white muscle protein concentration was the only
factor that significantly correlated with feed efficiency, with a negative
relationship (R2=0.11, P<.05), regardless of
dietary type.


## 3.4. In Vitro Digestibility and Comparisons between Different Growth Capacity Groups

Low growth
capacity families had lower final weight (2253±63 g) than those from medium
(2684±63 g) and high (2791±85 g) growth capacities (P<.0001). Within each growth capacity group,
no differences in weight (Figure 6(a)) and the T/C ratio (Figure 6(b)) were
observed between the fish fed NP-diet and HP-diet. Feed efficiency was
significantly different between the two dietary treatments only in the medium
growth capacity families (P<.01; Figure 6(c)). The P/L ratio in the white muscle was higher in
the HP-diet than the NP-diet fish in all growth groups (P<.02; Figure 6(d)).
Interestingly in the high growth families, the white muscle P/L ratio of the
HP-diet fish increased 80% more than the NP-diet fish, whereas the increase was
only 40% in the low and medium growth families (P<.03; Figure 6(d)).Final body
weight [a], protease activity ratio of trypsin to chymotrypsin (T/C
ratio) [b], feed efficiency [c], and white muscle composition
ratio of protein to lipid (P/L ratio) [d], in groups of rainbow trout
with low, medium, and high growth capacity, subdivided according to feeding
normal protein (NP) or high protein (HP) diet. The values with different superscripts are significantly different (P<.05).
(a)(b)(c)(d)The digestive ability
was different among different growth capacity groups (Figure7).  The enzyme extracts from families with high
growth capacity resulted in relatively higher in vitro protein digestibility
values for C-diet (P<.05), NP-diet (P<.05), and HP-diet (P>.05) than those from low and medium
growth capacity families (Figure 7). Paired comparison indicated significantly
higher in vitro protein digestibility of the HP-diet than the C-diet and
the NP-diet, regardless of the enzyme extracts used (P<.0001).
Within each growth capacity family, a tendency of higher in vitro protein
digestibility was observed in the enzyme extracts from fish fed HP-diet, albeit
insignificant (Table 2). A significant inverse relationship between in vitro protein digestibility values and SGR (R2=0.18, P<.02) was observed at maturity in November 2003, regardless of dietary
type.Figure 7
In vitro protein digestibility of control
(C), normal protein (NP), and high protein (HP) diets, among rainbow trout with
low, medium, and high growth capacities. The values with different superscripts
are significantly different (P<.05).


## 3.5. Analysis of Covariance

The continuous
variable white muscle P/L ratio was included as covariate in the models in
order to study its effect on growth together with fish growth capacity group or
family fed on the different diets. Due to class-by-covariate interaction being
significant (P<.05), the
statistical analysis was performed separately per class. The effects on the
growth parameters (final weight, condition factor, and SGR) by growth capacity
group and muscle P/L-ratio, and by family and muscle P/L-ratio, were studied
separately for each dietary group (Table 3). Differences in fish growth
capacity significantly affected final body weight for both diets but did not
affect condition factor and SGR. Family influenced fish SGR for both diets, and
the growth of the fish fed on NP-diet was highly affected by family and muscle
P/L ratio compared to the HP-diet fish. The results clearly indicated that the
differences in fish growth capacity among different families and the level of
P/L ratio in the white muscle affected fish growth, and their effects varied
according to the dietary P/L ratio.Table 3
Analysis of covariance showing the effects of fish growth capacity
(Group) or family and white muscle ratio of protein to lipid (P/L ratio) as
covariate, on final weight, condition factor, and specific growth rate (SGR)
during September–November 2003. Due to class-by-covariate interaction was
significant (P<.05); the
statistical analysis was performed separately for the experimental normal
protein (NP) and high protein (HP) diets. Probabilities with significant
effects are shown by the P values <.05 (bold values).
(a)Covariance effectdfFinal weightCondition factorSGRNPHPNPHPNPHPGroup20.00030.00220.41510.84530.97030.7316Muscle P/L ratio10.00120.17760.00330.71620.01250.1689Family150.00380.0961<0.00010.16800.00790.0465Muscle P/L ratio10.00310.10330.00890.45140.00670.2924(b)NPHPGroupFinal weightR2=0.389(P<.0001)R2=0.224(P<.005)Condition factorR2=0.186(P<.015)R2=0.013(P=.883)SGRR2=0.117(P=.089)R2=0.066(P=.321)FamilyFinal weightR2=0.606(P<.0005)R2=0.407(P=.107)Condition factorR2=0.725(P<.0001)R2=0.370(P=.195)SGRR2=0.560(P<.0025)R2=0.467(P<.035)In order to study the association
between SGR, dietary quality, and digestive abilities of different growth
capacity groups, the continuous variable SGR was included as covariate in the
models. Similarly, the class-by-covariate interaction was significant (P<.05); thus, the statistical analysis was also performed separately per
class. The effects on specific activities of trypsin and chymotrypsin, T/C
ratio, and in vitro protein digestibility of diet, by dietary P/L ratio
and SGR were studied for each growth capacity group (Table 4). The results
indicated an effect of the diet on the T/C ratio of the low growth capacity
group. There was an indication of the associations between SGR and the specific
activities of trypsin and chymotrypsin. Trypsin specific activity was affected
in the high growth capacity group, whereas chymotrypsin specific activity was
affected in the low growth capacity group. There was an association between SGR
and in vitro protein digestibility of the experimental diets in the high
growth capacity group.Table 4
Analysis
of covariance showing the effects of dietary P/L ratio level (Diet) and
specific growth rate (SGR) during September–November 2003 as covariate, on
the expression of trypsin and chymotrypsin (expressed asμmol p-nitroaniline h−1 mg protein−1), the activity ratio of trypsin to
chymotrypsin (T/C ratio), and in vitro protein digestibility (expressed as μmol DL-alanine equivalent per
mg feed after standardising trypsin activity to 300 μmol p-nitroaniline
produced  h−1mL−1). Due to class-by-covariate interaction was
significant (P < .05); the statistical analysis was performed
separately for low (L), medium (M), and high (H) growth capacity groups.
Probabilities with significant effects are shown by the P values <.05 (bold values).
(a)Covariance effectdfTrypsinChymotrypsinT/C ratioIn vitro digestibilityLMHLMHLMHLMHDiet10.17960.32760.49050.10330.36470.20350.04580.48640.44710.32860.69380.4804SGR10.10590.10550.03200.01740.08540.09050.97050.18260.74240.44030.48070.0403(b)LMHTrypsinR2=0.111(P=.163)R2=0.139(P=.085)R2=0.173(P<.03)ChymotrypsinR2=0.205(P<.035)R2=0.147(P=.078)R2=0.169(P<.03)T/C ratioR2=0.131(P=.123)R2=0.092(P=.213)R2=0.015(P=.746)In vitro digestibilityR2=0.041(P=.523)R2=0.016(P=.764)R2=0.163(P<.035)The
analyses of covariance (Tables3 and 4) indicated the associations between
dietary P/L ratio, fish digestive ability (trypsin and chymotrypsin specific
activity, T/C ratio, in vitro protein digestibility), white muscle P/L
ratio, growth parameters (fish weight, condition factor, SGR), and variations
in growth capacity of different family memberships.


## 4. Discussion

Fish growth was affected by dietary
P/L ratio and by the inherent growth capacity of the fish as well as by white
muscle P/L ratio (Table3) and by genetic differences in protease digestive
ability (Table 4). At late stage, rainbow trout seemed to double their white
muscle P/L ratio with respect to the dietary P/L ratio (Tables 1 and 2).
Increased dietary protein concentration resulted in elevated protein and
reduced lipid concentrations in fish body ([1, 6, 35], current work). In the
white muscle, increased dietary protein resulted in elevation of both protein
and moisture contents [current work]. Dietary protein concentration is also
inversely correlated with protein efficiency ratio and productive lipid value [3].
Increasing dietary protein concentration has been associated with increased
muscle protein in salmonids ([35], current work), channel catfish Ictalurus
punctatus [2] and grass carp Ctenopharyngodon idella [4], and
increased fillet mass and decreased liver and adipose tissue mass in the
abdominal cavity in African catfish Clarias gariepinus [7]. In addition,
fish fed high protein diets tend to have lower total lipids, triglycerides, and
cholesterol in fillets, with higher percentage of polyunsaturated fatty acids
(PUFAs) and lower saturated fatty acids [7]. Thus, increasing protein in the
diet could result in improved fillet quality. Moreover, HP-diet appeared to
promote an increase in length rather than weight during growth (Figures 1 and
5). Our work illustrates the importance of dietary protein level not only on
fillet quality but also on skeletal growth. Deformities in juvenile tench (Tinca
tinca L.) with significantly elevated whole body lipid have been reported
[36]. Increased growth in Atlantic salmon (Salmo salar L.) stimulated by
continuous light, which is not caused by increased protein deposition [10] but
by increased lipid deposition [13], results in reduced mechanical strength and
growth of the vertebrae in Atlantic salmon postsmolts [37]. Thus, stimulating
fish growth without concerning with appropriate dietary P/L ratio and P/L ratio
deposition in muscle or body may affect the strength of skeletal growth and
cause deformities. Fish growth could be explained not only by body weight but
also by body length illustrated as condition factor ([26], current work).
Therefore, the conventional study for dietary protein requirement through
optimal weight gain in fish (recently, i.e., [1, 3, 5, 6, 8]) should probably
instead indicate the effect of optimal dietary protein level on the change of
fish growth performance to greater increase in length than weight.It is interesting to
note that fish with different growth capacities [current work] and trypsin
phenotypes [14, 38] have different protease digestive abilities. Fish from high
growth group had a relatively higher digestive ability for all feed types,
compared to those from low and medium growth groups (Figure 7). There were
associations between growth and protein digestibility (Table 4) as well as
between growth and muscle P/L ratio (Table 3). The high growth capacity
families had higher capacity to increase their growth through increase in white
muscle protein deposition (Figure 6(d)). In addition, white muscle fibre size was
also significantly larger in the high growth capacity fish compared to the
medium and low growth capacity groups (pooled data, P<.01),
irrespective of the diet (unpublished data). Furthermore, genetic correlations
between percent white muscle area and protein percent in rainbow trout cutlets
were very high on both diets, 0.76 in NP-diet and 0.92 in HP-diet groups [39].
These results indicate the advantage of high growth capacity in protein growth
performance quality. For more understanding on the effect of dietary P/L ratio
on muscle composition and growth, if possible, other parameters such as
cellular growth should be investigated in the future in order to study the insight
effect of genetics and nutrition on fish growth. Growth rate was correlated
with feed intake but was not correlated with feed efficiency (FE), as FE
reduces when consumption increases beyond a threshold level [11, 21]. The inverse relationship observed between FE and white muscle protein
concentration (not with body protein concentration) was probably due to a
limited capacity to deposit muscle protein. Differences in FE have been
associated with variations in the levels of total free amino acids in the
plasma and white muscle as well as in the ratio of essential to nonessential
free amino acids, and with variations in free hydroxyproline and RNA
concentrations in the white muscle [18]. In addition, relationships have been
observed between T/C ratio in the pyloric caeca and concentration ratios of
essential to nonessential free amino acids in plasma and white muscle as well
as between T/C ratio with free hydroxyproline and RNA concentrations in the
white muscle [10]. Moreover, a significant positive relationship between FE and
T/C ratio has been observed in fish fed diets varying in dietary quality [11, 16]. The current work further emphasises these associations between FE, T/C
ratio, and levels of protein and lipid utilisation for deposition in the white
muscle. High protein concentration in the white muscle of maturing rainbow
trout, as high as 30% with 24±0.1% on total average
observed in the current study (Table 2) compared to normal values of 15–20% reported [40], was probably due
to complete solubilisation of protein by using the newly developed method, as
higher protein solubility resulted in higher percentage protein measurements.
The high white muscle protein concentration might as well be a characteristic
of maturing salmonids, as it has also been observed in maturing Atlantic salmon
[11], which is probably due to mobilisation of muscle lipid as energy for
reproduction. The highest muscle protein concentration observed in maturing
Atlantic mackerel (Scomber scombrus L.) was only 24% with 14±1% on average [12]. Whilst it was 20% with 17±0.1% on average in Atlantic salmon of about 250 g [11], and 25% with 21±0.1% on
average in rainbow trout of about 1 kg [current work]. During the final year of
the experiment, the composition of body and white muscle successively changed
with concentration of protein increasing and moisture concentration decreasing.Higher
dietary protein induced increases in protease specific activities of trypsin
and chymotrypsin in the pyloric caeca (Table2). In a previous experiment using
the same feed but varying rearing conditions, both trypsin specific activity
and T/C ratio were related to growth rate [10], whereas in the current work
varying in feed quality, only the T/C ratio was positively associated with
growth rate (Table 2). Thus, the T/C ratio has an effect on growth efficiency
independent of the specific activity level of the proteases. This has also been
observed in Atlantic salmon [32] and gene manipulated fish [10, 41]. A
reduction in T/C ratio in the HP-diet fish, relative to NP-diet fish, caused by
a greater increase in the activity of chymotrypsin over trypsin confirmed a
reduction in growth rate (Table 2). This has been reported in several works
[11, 12, 14, 18, 32]. The confirmation is also strengthened by the observed
associations between trypsin specific activity and high growth and between
chymotrypsin specific activity and low growth (Table 4). In vitro protein
digestibility values of different diets (Figure 7 and Table 4) were influenced
by fish growth capacity and the levels of protease expression in fish enzyme
extracts. The in vitro protein digestibility
of diets has been affected by genetic differences in trypsin phenotypes [33, 38], and different trypsin phenotypes affected variations in the
protease activity expression levels [14] and FE [42]. At sampling in November
2003, the fish were at a similar steady growth phase showing by similar slope
T/C ratio (Figure 3) (details in [32]) and no correlation between trypsin
specific activity and the T/C ratio (data not shown) (details in [12, 14]). At
steady growth phase, utilization of protein for deposition and growth would be
reduced to a maintenance level that should be steady irrespective of diet. This
is confirmed by the observed higher rate of nitrogen excretion in the HP-diet
fish than the NP-diet fish with similar absolute nitrogen retention between the
two dietary groups at the end of the experiment (D. Tobin, personal
communication). If the fish had been at a growing phase, lower nitrogen excretion
per kg of fish growth in rainbow trout fed diet with higher protein content [43]
and increased nitrogen retention with increased protein deposition [35] would
have been observed in the HP-diet fish. Growth status of the fish is very
important for interpreting biological parameters. At present, the slope T/C
ratio and the relationship between trypsin specific activity and the T/C ratio
in the pyloric caeca seem to be the only indicators available for predicting
relative growth level and growth status of the fish at sampling, respectively,
without knowing fish history see [12, 14, 32]. Our results suggest the
association between the diet dependent in vitro protein digestibility
value and protein growth in fish, whereas the T/C ratio is associated with fish
growth regardless of protein or lipid growth. In vitro digestibility by
protein digestion is the key factor for diet quality, not only in carnivores
but also in herbivores where carbohydrate digestion (the second important
digestion factor in herbivores after protein digestion) is also related to dietary
protein levels [19, 20] while lipid digestion is not related to any diet
quality [19]. A high protein diet may limit feed efficiency, weight, and growth
rate of the fish, as they become mature. Analyses of covariance also indicate
the associations among genetic differences in protease digestive efficiency,
the dietary P/L ratio, and fish growth (Table 4), and showing the influence of
white muscle P/L ratio on the SGR (Table 3).The T/C ratio (directly
calculated from the enzyme activities) and the slope T/C ratio (obtained as
shown in Figure3) are the suitable factors for comparison of growth efficiency
between fish groups over a period of 1-2 months and at sampling,
respectively, regardless of either protein or lipid growth or the protease
specific activity levels ([10–12, 14, 18, 32], current work). It is
important to calculate the T/C ratio and slope T/C ratio in the pyloric caeca
for comparison instead of using trypsin specific activity alone. If fish are
needed alive, biopsy of pyloric caeca could be performed [44], or the T/C ratio
in faeces could also be used for comparison of digestive and growth efficiency
but not for predicting growth status [11]. Most observed relationships between
key factors have very low correlation coefficients [10, 14, 45], current work).
This is due to their indirect interaction in a series of biological mechanisms.
The phenomenon is common, as many factors being involved in biological
processes.The dietary protein
concentration of≥50% used in this work is not actually practical in the
production of large fish due to high cost. It was purely intended to act as a
challenge environment to optimising the genetic response of traits such as
protein and growth efficiency in salmonids [21, 22, 27]. However, as a long-term study, it has enabled us to investigate
the effect of dietary protein on skeletal growth (length), which is important
knowledge for future production of fish with high growth performance quality [32]
as well as for future growth study in natural ecosystems where protein
deposition is the main cause [12].


## 5. Conclusions

Dietary P/L ratio affected the level
of digestion and utilisation of protein and lipid for deposition in the white
muscle that influenced feed efficiency and growth of the fish. Muscle protein
concentration increased between successive measurement periods, and older
larger fish had higher concentration than younger smaller fish. Fish with
higher growth capacity had better chance to deposit higher protein. Protein
deposition was associated more with body length than body weight, resulting in
lower condition factor. Fish with higher protein growth had lower feed
efficiency and SGR during maturation. Genetic effect of protease digestive
efficiency on fish growth is evident ([14, 38], current work). In vitro protein
digestibility of experimental diets using fish crude enzyme extract and
standardised by trypsin activity is useful not only for comparison and
prediction of biological quality of different diets [11, 16], but also for
comparison of genetic differences in fish digestive efficiency ([33], current work) and fish growth capacity [current work]. Specific
activity levels of trypsin and chymotrypsin were related to dietary protein
concentrations. The T/C ratio and slope T/C ratio were related to growth efficiency
over a period of 1-2 months and at sampling,
respectively, regardless of protein or lipid growth or the specific activity
levels of the two enzymes ([10, 32], current work). A relatively higher increase
in chymotrypsin over trypsin activity, resulting in lower T/C ratio, affected a
reduction in growth rate and indicated the associations between trypsin
specific activity and high growth and between chymotrypsin specific activity
and low growth ([12, 14, 32], current work). The associations of all parameters
studied by the analyses of covariance (Tables 3 and 4) indicate the influences
of both external (dietary P/L ratio) and internal (variations in inherent
growth capacity of different family memberships) factors on fish growth,
through biological changes in digestive ability (trypsin and chymotrypsin
specific activity, T/C ratio, in vitro protein digestibility) and white
muscle P/L ratio. High dietary protein results in similar weight, but higher
growth in length (skeletal growth) and better fillet quality that would
nutritionally benefit to consumers, compared to high dietary lipid. The work
shows the advantage of using the unique combination of the practical methods
for growth performance quality study that could be performed in any
uncontrolled condition, such as in the natural ecosystem where protein
deposition is important and study on biological responses to environmental
changes for survival has been ignored. It also provides more knowledge on
dietary protein affecting skeletal growth as well as the interaction between
genetics and nutrition affecting digestive ability and growth performance
quality of the animal.



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*Source: 709529-2009-06-07.xml*