tuan kcw malabar pap
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Optimum dietary protein and lipid specifications for juvenile malabar
grouper (Epinephelus malabaricus)
Le Anh Tuan1
and Kevin C. Williams2*
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1Faculty of Aquaculture, University of Fisheries, Nha Trang, Khanh Hoa, Viet Nam.
2CSIRO Marine Research, PO Box 120, Cleveland, Qld. 4163, Australia.
*Corresponding author: Dr Kevin Williams
CSIRO Division of Marine Research
PO Box 120, Cleveland, Qld. 4163, Australia.
Ph +61 7 3826 7284 Fax +61 7 3826 7222
E-mail [email protected]
Submitted to:
Received: ..............................
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Abstract
An 8-week comparative slaughter experiment was carried out to determine the
optimal dietary dry matter (DM) crude protein (CP) and lipid for growth and nutrient
retention of malabar grouperEpinephelus malabaricus. Fingerlings of mean ( SD)
start weight of 17 1.3 g were fed twice daily to satiety one of 16 pelleted dry feeds (~
93% DM) that provided a 4 x 4 factorial comparison of serially incremented CP (from
44 to 60%) and lipid (from 7 to 23%) with three tank replicates (10 fish per tank).
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Tanks (100 L) were situated within an enclosed laboratory and provided with bio-
filtered, constant temperature (29 0.7C) recirculated seawater and supplementary
aeration.
Fish survival averaged 94 5.3% and was unaffected by treatment. Modelling of
the fishs response to dietary CP and lipid showed that feed intake, growth rate, feed
conversion ratio (FCR) and dietary energy retention were all optimized when dietary
CP was 55-56%; N retention was maximized at 47.3% CP. The optimal dietary lipid
level depended on the response criterion: 7.2% for feed intake and energy retention;
12% for N retention; 13% for growth rate; and 16.8% for FCR. Changes in the whole
body (WB) composition of the fish were more direct: protein composition decreased
linearly as dietary protein increased (with increasing dietary lipid tending to have an
opposing effect), while lipid composition increased linearly as dietary protein and lipid
both increased. Thus, WB protein was greatest for fish fed the lowest protein (44%) and
highest lipid (23%) diet while WB lipid was highest for fish fed the lowest protein
(44%) and the lowest lipid (7%) diet. Recommended dietary protein, lipid and protein
to energy ratio specifications for optimal productivity of juvenile malabar grouper are
55%, 12% and 28 g CP:MJ gross energy, respectively.
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Key words: nutrition, feeding, requirements, protein to energy, cod, retention
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1.Introduction
Improved hatchery technology and a more reliable supply of hatchery-reared fry has
resulted in a 10-fold expansion in grouper aquaculture production in eastern Asia since
the mid-1990s, with production now more than 52,000 metric tonnes per annum (FAO,
2005). Trash fish is presently the main food source for rearing grouper in the region but
its decreasing supply, increasing cost and downstream environmental impacts
(Beveridge, 1996; New, 1996; Williams, 2002) are heightening the need for pelleted
feeds and in turn, a greater knowledge of the fishs nutritional requirements.
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The nutritional requirements of groupers have been reviewed most recently by
Boonyaratpalin (1997) and Chen (2001) who concluded that diets need to be high in
crude protein (CP; 45 to 55%) and with up to 14% lipid to ensure good growth rates of
the fish. More recent studies have indicated that dietary protein and lipid requirements
of groupers may differ both between species and with size of the fish. For juvenile
Epinephelus coioides, Luo et al. (2004) found fish growth rate and feed conversion
ratio (FCR) were best when diets contained 48% CP (and 11% lipid) and 53% CP (and
9% lipid), respectively. For the much slower growing humpback grouperCromileptes
altivelis, Williams et al. (2004) found growth rate and FCR of juveniles improved
linearly up to the maximum examined dietary CP level of 63% dry matter (DM) (58%
as-fed basis) and this was independent of dietary lipid over the range of 15 to 24% DM
(14 to 22%, as-fed basis). For humpback grouper of 150-400 g size, the optimal dietary
CP and lipid specification was found to be 53% and 12%, respectively (Usman et al.,
2005). Using a factorial approach to determine nutritional needs of the Mediterranean
white grouperEpinephelus aeneus, Lupatsch and Kissil (2005) advocated for optimal
nutrient efficiency, that dietary CP specification should decrease from 55 to 40% as fish
grew from 2 to 500-700 g; protein to energy ratio correspondingly should decrease
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from 29 to 21 g CP per MJ gross energy, which could be accommodated by increasing
dietary lipid from 10 to 14%.
Malabar grouperEpinephelus malabaricus is a highly valued fish in the Asian live
fish markets and is one of the most commonly farmed grouper species in SE Asia
(Boonyaratpalin, 1997; Miao and Tang, 2002). However, there is very little published
information on its protein and lipid (energy) requirements. Chen and Tsai (1994) fed
juvenile malabar grouper casein-based semi-purified diets and found a dietary CP level
of 48% resulted in maximal growth. With fish meal-based semi-purified diets, Shiau
and Lan (1996) reported juvenile malabar grouper did best on a diet containing 50% CP
when the fat content was 7% but increasing the fat to 13-14% enabled the CP content to
be reduced to 45% without a significant adverse effect on growth rate. More recently,
the effect of varying the lipid content of isonitrogenous (50% CP) diets on the growth
and the immune response of malabar grouper was investigated by Lin and Shiau (2003).
They found fish grew well on diets containing from 4 to 12% lipid (optimum being
about 9%) while growth, but not immune competence, was depressed with 16% lipid.
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This paper reports a comparative slaughter experiment in which juvenile malabar
grouper were fed diets that varied factorially in protein and lipid over a wide range.
Productivity, body composition and nutrient retention responses of the fish were
modelled to better understand the independent and interactive effects of the diets and
this information used to derive optimal dietary protein and lipid specifications for
juvenile malabar grouper.
2. Materials and methods
2.1 Experimental design and diets
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An 8-week growth and nutrient retention experiment was carried out with juvenile
malabar grouper to examine the interactive effects of varying dietary protein and lipid
on growth, nutrient retention and body composition. Sixteen diets were formulated to
provide a 4 x 4 factorial of CP (from 44 to 60% DM at equal increments) and lipid
(from 7 to 23% DM at equal increments), with three tank replicates of fish per
treatment. Changes in the dietary concentrations of CP and lipid in a fish meal-based
formulation were achieved by serial adjustment of casein (for protein) or a mixture of
fish and soybean oil (for lipid) at the expense of tapioca starch (Table 1).
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Feed ingredients were finely ground and dry-mixed in a 20 L Chufood planetary
dough mixer/meat mincer (CS 200, Chuseng Food Machinery Works Co. Ltd,
Taichung, Taiwan, R.O.C.) before the oil and sufficient water were added to form a
dough of approximately 40 to 50% moisture. The dough was twice extruded through a 3
mm diameter die plate and the resultant feed strands transferred to a commercial
steaming oven (Stoddart Metal Fabrication P/L, Sunnybank, Queensland, Australia ) for
5 min. After steaming, the feed strands were dried overnight at 40 C in a forced
draught oven, broken into pellets of 3 to 4 mm length and stored at 20 C until just
before use.
2.2 Fish, tanks and experimental management
Fingerlingswere purchasedfrom a local hatchery and transported to the University
of Fisheries seawater laboratory at Nha Trang, Vietnam. After an 1-week period of
acclimatization, fish were sorted by weight and freedom from physical abnormalities
into a uniform group of 500 fish of mean ( SD) weight of 17 1.3 g. Four hundred
and forty eight of these fish were randomly distributed to the experimental tanks at an
equal stocking rate of 10 fish per tank. Ten of the remaining fish were sacrificed in
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groups of five fish to provide an estimate of initial whole body chemical composition.
The experimental system comprised 48 rectangular polyethylene tanks (100 L; 0.24 m2
surface area), which were arranged as three replicate blocks of 16 tanks within an
enclosed seawater laboratory. Each tank was supplied with bio-filtered, constant
temperature (29 0.7C) recirculated seawater ( 33 35 ) at an exchange rate of
500%/d. Each tank was provided with supplementary aeration by means of an airstone
and water temperature and salinity were monitored daily and weekly, respectively.
Photoperiod was held to a constant 12:12 h light-dark cycle.
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During the experiment, fish in each tank were weighed individually at the start and
end of the 8-week experiment and bulk-weighed at intervening fortnightly periods.
Stress at weighing was minimised by mild sedation of the fish using the aquatic
anaesthetic, iso-eugenol (AQUI-S, New Zealand) provided in an aerated water bath at
27 mg/ L. Fish were offered their respective diets to satiety twice daily (nominally at
08:00 and 17:00 h) except on the day of weighing when the morning feed was not fed.
At each feeding, a weighed amount of food was offered to excess on three or four
occasions during a feeding period of about 40 min. All uneaten feed was collected and
dried. Feed intake was calculated as the difference between the amount of feed offered
and the amount of uneaten refusal, after correcting for the DM of the diet and leaching
loss (average of DM retention measurements made after immersion of the diet in water
for 15 and 30 min). At the end of the experiment, a representative sample of two fish
was taken from each tank for determination of whole-body (WB) chemical
composition.
2.3 Chemical analyses
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For determination of WB composition, weighed whole fish were frozen individually
in treatment lots and then minced twice through a 2.5 mm diameter die plate of the
screw mincer attachment of the Chufood mixer/mincer. The minced sample was freeze-
dried and ground with a mortar and pestle to a uniform powder. Samples of finely
ground diets and homogenised fish were analysed in duplicate by standard laboratory
methods essentially in accordance with AOAC International (1999). DM was
determined by drying at 105 C to constant weight and ash by ignition at 600 C for 2
h. Total N was determined by a macro Kjeldadl technique using mercury as the catalyst
in the digestion and titration to an end point pH of 4.6. CP was calculated by using the
conversion factor of 6.25 irrespective of the nature of the N. Total lipid was determined
gravimetrically following chloroform-methanol (2:1) extraction using the method of
Folch et al. (1957). Fatty acids in the total lipid extract were derivatized to their methyl
esters (FAME; Morrison and Smith, 1964) and analysed by capillary gas
chromatography using an Agilent 6890 capillary GC (Agilent Technologies, USA) with
direct on-column injection and flame ionization detection. The FAME were separated
on a 50-m polar capillary column (BP20, 0.33 mm i.d., 0.5 m film thickness) with
hydrogen carrier gas flowing at 2.7 mL/min. Identification and quantification were by
comparison with internal standards (tridecanoic acid (13:0) and heneicosanoic acid
(21:0)) in conjunction with fatty acid mixed standards (Nu-Check-Prep, Elysian, MN,
USA). All composition results, subsequent calculations and discussion of the results are
expressed on a DM basis unless otherwise stated. The determined chemical
composition of the diets is shown in Table 2.
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2.4 Measurements and statistical analysis
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Average daily gain (ADG) was determined as the difference between end (We) and
start (Ws) weights divided by the number of days (d) on the experiment. Daily growth
coefficient (DGC) was calculated as:
=
d
WWdDGC se
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100)/(%
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Nutrient and energy retentions were calculated as the net gain of the nutrient or energy
of the fish over the experimental period divided by the corresponding nutrient or energy
intake of the fish over the same period and expressed on a daily basis. The gross energy
content of the diets and fish was calculated from the determined chemical analysis
using the conversion factors of 17.2, 23.4 and 39.2 kJ/g for carbohydrate, protein and
lipid, respectively (Cho et al., 1982); carbohydrate was determined as the difference
between the total and the sum of moisture, ash, protein and lipid contents.
Fish response data were subjected to an analysis of variance in accordance with the
4 x 4 factorial design of the experiment using prepared statistical programs. Percentage
data were analysed as the natural and arcsine-transformed data but as the F-statistic was
of a similar magnitude for both analyses, only the analyses for the natural data are
reported. The effect of dietary protein and lipid concentration on fish productivity and
the fishs retention of dietary N and energy were subsequently examined using
multivariate regression analysis. Relationships for each of the lipid series were
examined for homogeneity of residual variances (Bartletts test), parallelism of the
regression lines and differences of the regression intercept (Snedecor & Cochran 1989).
Differences between treatment effects were examined a-posteriorly using Fischer's
protected 't' test (Snedecor & Cochran, 1989) wherein differences between means were
examined only where the F-test of the ANOVA was significant (P < 0.05).
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3. Results
3.1 Productivity responses
No water quality problems were experienced during the experiment with water
temperature averaging 29 C (SD 0.7) and the fish remained healthy throughout. Out
of the initial placement of 480 fish, 30 died over the course of the experiment (survival
of 94 5.3%), many apparently due to handling stress at weighing. There were no
significant differences in survival rate between treatments with most tanks experiencing
a single loss except for one tank where two fish died.
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Significant (P < 0.05) interactions between the main effects of protein and lipid
content of the diet were observed for all measured productivity traits (Table 3). Multi-
variate regression analysis (Table 4) showed that these trait responses were well
modelled by quadratic functions of dietary protein and lipid with 78% or more of the
variance being explained. Fish performance improved as the protein content of the diet
increased to an optimum of 55-56% for all traits; the lipid content of the diet that
optimized feed intake, growth rate (both ADG and DGC) and FCR was 7.2, 13 and
16.8%, respectively. Increasing the dietary lipid content beyond these optima caused
feed intake and growth rate to decline while FCR values were less affected (Fig. 1).
3.2 Fish body composition and retention
Table 5 shows the effect on WB composition of varying the concentration of protein
and lipid in the diet. Other than for a very minor interaction between dietary protein and
lipid for lipid composition, varying the level of protein or lipid in the diet had
independent effects on the final composition of the fish. Over the course of the eight
week experiment, the lipid content of the fish on a wet basis increased 2- to 3-fold from
2.2% at the start, to from 4.3 to 6.8% at the end. Increasing the protein content of the
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diet resulted in a corresponding increase in the protein and lipid composition, and a
decrease in the moisture composition, of the fish. Increasing the dietary lipid content
increased and decreased the lipid and protein contents of the fish, respectively. Thus,
the fattest fish (6.8%) were those fed the 60/23 diet while fish with the highest protein
composition (18.9-19.0%) were those fed the two lowest lipid and highest protein diets,
that is, diets 60/7 and 60/12. Conversely, fish fed the lowest protein and lowest lipid
diet (44/7) had the least fat (4.3%).
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The proportion of dietary N and GE retained by the fish is presented in Table 6.
Multi-variate regression analysis showed that 87 to 89% of the variation in these
retentions could be explained as linear or quadratic functions of dietary protein and
lipid (Table 4). Increasing the lipid content of the diet from 7 to 12% increased N
retention but it was depressed at higher dietary lipid and at high protein levels (Fig.
2A). N retention was highest for dietary protein and lipid contents of 47.3 and 12.0%,
respectively. Retention of dietary energy increased with increasing dietary protein and
decreased with increasing dietary lipid, and was maximized at dietary protein and lipid
contents of 56 and 7%, respectively (Fig. 2B).
4. Discussion
Modelling of the fishs response to dietary protein and lipid (Table 4, Fig. 1 and 2)
showed that growth rate, FCR, feed intake and dietary energy retention were all
optimized when the protein content of the diet was 55-56%. The only benefit of
feeding a lower dietary protein content was to improve dietary N retention, which was
maximized at a dietary protein level of 47.3%. The optimal dietary lipid level depended
on the response criterion: 7.2% for feed intake and energy retention; 12% for N
retention; 13% for growth rate; and 16.8% for FCR (Fig. 1 and 2). Changes in the
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whole body composition of the fish resulting from dietary manipulation were more
direct: protein composition decreased linearly as dietary protein increased (with
increasing dietary lipid tending to have an opposing effect), while lipid composition
increased linearly as dietary protein and lipid both increased (Table 4). Thus, WB
protein composition was greatest for fish fed the lowest protein (44%) and highest lipid
(23%) diet while WB lipid composition was highest for fish fed the lowest protein
(44%) and the lowest lipid (7%) diet.
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Our findings that growth rate and efficiency of feed utilization were best when the
fish were fed a diet that contained 55-56% protein and 12% lipid differs somewhat from
other studies with juvenile malabar grouper. Using semi-synthetic diets based on
casein and dextrin and without fish meal, Chen and Tsai (1994) found growth and feed
efficiency utilization of malabar fry of 4 g initial weight were optimized in a 50-day
experiment when an 8% lipid diet contained 48% protein. Moreover, they found body
lipid composition decreased as dietary protein content was increased from 24 to 42%
but further increases up to 54% protein resulted in a slight but significant increase in
fish adiposity. However, the growth of these fish was exceedingly poor, only 0.18 g/day
on the best diet, probably because of the unpalatability of the casein/dextrin diets. Shiau
and Lan (1996) examined the protein requirement of malabar juveniles of 9 g initial
weight when fed for 8-weeks semi-synthetic diets that were based on fish meal and
starch. They found growth rate and FCR of the fish improved linearly as dietary protein
increased from 0 to 51%, but no further improvement was seen when dietary protein
increased to 57%. Maximum growth rate achieved by the fish in their experiment was
0.93 g/day, slightly less than that of the slightly larger fish (17 g) used in our study (viz.
1.13 g/day; Table 3). The lipid composition of the fish in the study of Shiau and Lan
(1996) increased more or less linearly with increasing dietary protein, an effect that we
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also observed in our study. In a second experiment examining protein to energy
requirements of juvenile malabar of 10 g initial weight, Shiau and Lan (1996) used
diets that contained either 45 or 50% protein in combination with four lipid levels that
varied serially between 7 and 18%. Survival of fish in that experiment was 67 to 88%
and the best growth rate was poor, only 0.68 g/day, which occurred on the diet that
contained 50% protein and 13% lipid. For both the 45 and 50% protein series,
increasing the lipid content above 13% reduced the growth rate of the fish; the
efficiency of feed utilization was not impaired with the 50% protein series but it
progressively got worse with the 45% protein series. The lipid composition of the fish
tended to increase with increasing dietary lipid but with no clear difference between the
50 and 45% dietary protein series. While the differences in the observed fish
performance between our study and the two aforementioned studies could be attributed
to the different experimental methods employed, particularly the type of feed
ingredients used, the underlying factor governing the nature of the fishs response is
surely feed intake and the absolute supply of ingested nutrients (and energy).
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In our study, feed intake was inversely related to dietary lipid content and to WB
lipid composition, but not dietary energy contentper se. This suggests that feed intake
regulation in this species may operate through a lipostatic mechanism similar to that of
mammals. The existence of a negative feedback mechanism between body adiposity
and feed intake was first postulated by Kennedy (1953) and evidence of a circulatory
lipostatic agent provided a few years later by Hervey (1958). However, it was not until
36 years later that Zhang et al. (1994) confirmed the existence of the circulatory factor,
now know to be leptin, as an 146 amino acid cytokine peptide. Leptin is synthetized
and secreted by adipocytes in proportion to the amount of lipid stored in the body and,
through feedback inhibition of appetite, assists the animals regulation of energy
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balance (Houseknecht and Portocarrero, 1998; Woods and Seeley, 2000; Baile et al.,
2000). Lipostatic control of feed intake and energy regulation has been postulated for
Arctic charrSalvelinus alpinus (Jobling and Miglavs, 1993; Jobling and Johansen,
1999), Atlantic salmon Salmo salar(Johansen et al., 2002, 2003) and barramundi,Lates
calcarifer(Tian and Qin, 2003, 2004). Direct evidence for lipostatic regulation of feed
intake in fish, however, is scant (Lin et al., 2000). Perhaps the most convincing
evidence for a lipostatic mechanism in fish is provided by the studies of Volkoff et al.
(2003) with goldfish Carassiys auratus: central or peripheral injection of murine leptin
into the fish brought about a significant decrease in feed intake, which could be
reversed if the lectin was co-injected with the neuropeptide Y (Volkoff et al., 2003). In
mammals, the neuropeptide Y exerts a strong stimulatory effect on appetite
(Houseknecht and Portocarrero, 1998; Woods and Seeley, 2000).
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Since feed intake was reduced in line with increasing adiposity of the fish in the
present study, this is consistent with an hypothesis of lipostatic control of appetite.
However, it might equally be explained as a more acute satiation response caused by
ingesting high lipid diets, rather than a true lipostatic effect emanating from increasing
body adiposity. In mammals, ingestion of lipid brings about a cascade of dose-
dependent events, preeminent of which is the secretion of choleocystokinin (CCK),
bombesin-like peptides, gastric leptin and enterostatin from the gut, that culminate in
meal termination and appetite suppression (Ritter, 2004; Beglinger and Degen, 2004;
Geary, 2004). Similar mechanisms appear to operate in fish (Shearer et al., 1997;
Gelineau and Boujard, 2001; Volkoff and Peter, 2004; Volkoff et al., 2005) and this
could account for the intake depression observed in the present experiment with
malabar grouper. Other grouper species such as polka dot grouperCromileptes altivelis
(Williams et al., 2004; Usman et al., 2005; Williams et al., 2006) and gold-spot grouper
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Epinephelus coioides (Luo et al., 2005) show a similar suppressed appetite response to
high lipid diets.
While we can only speculate as to the physiological mechanisms controlling the
fishs appetite in our experiment, what is clear is that feed intake was not being closely
regulated to meet some predetermined energy requirement of the fish. Energy intake of
the fish can be calculated from data in Tables 2 and 4. At each dietary protein level,
increasing the lipid content of the diet resulted in energy intake increasing by an
average of 13.1% whereas feed intake fell, on average, by about 6.0%. Conversely, at
each dietary lipid level, increasing the protein content of the diet resulted in an
increased energy intake of 12.8% and an increase in feed intake of 8.0%. Thus, feed
intake, and energy intake, both increased as the protein content of the diet increased
whereas feed intake, but not energy intake, decreased with increasing dietary lipid. A
similar finding wherein feed intake appeared to be controlled more by protein intake
than energy intake was observed with European sea bass Dicentrarchus labrax by
Peres and Oliva-Teles (1999) and gold spot grouper (Luo et al., 2004). However, these
findings should not be interpreted to indicate that energy density of the diet is not
involved in feed intake regulation as this has been convincingly shown to occur in
numerous species including Arctic charr (Jobling and Wandsvik, 1983), salmonids
(Boujard and Medale, 1994; Kaushik and Medale, 1994; Rasmussen et al., 2000;
Gelineau et al., 2002), gilthead seabream Sparus aurata (Lupatsch et al., 2001), turbot
Scophthalmus maximus (Saether and Jobling, 2001) and European sea bass (Boujard et
al., 2004). Rather it emphasizes the preference of malabar grouper to meet cellular
energy requirements by oxidizing protein instead of lipid.
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That malabar grouper preferred protein to lipid as an energy source is clearly
illustrated by the energy and protein retention responses of the fish to dietary protein
and lipid manipulation. For each lipid series of diets, increasing dietary protein resulted
in an 11 to 27 percentage unit improvement in energy retention whereas an opposite
effect, but of a lower magnitude, was seen when dietary lipid was increased (Fig. 2B).
Increasing the lipid content of the diet from 7 to 12% resulted in a significant
improvement in protein retention indicative of a protein-sparing effect but higher
levels of dietary lipid resulted in an equally significant decrease in protein retention
(Fig. 2A). As expected, increasing the amount of protein in the diet resulted in a fall in
protein retention, with the effect being greatest for the diet with the highest
concentrations of lipid and protein (Table 6). Thus, under the conditions of the
experiment, feed intake appeared to be more responsive to dietary protein content than
to either dietary lipid or energy. However, lipid at levels up to about 12% had a
protein-sparing effect, which is similar to that observed for this and other species of
grouper (Shiau and Lan, 1996; Williams et al., 2004; Usman et al., 2005; Luo et al.,
2005; Williams et al., 2006).
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A number of conclusions can be drawn from this study. Firstly, juvenile malabar
grouper grow best when given diets that contain high levels of protein (at least 55%)
and lipid levels that do not exceed about 12-13%. While slightly higher lipid diets may
improve FCR, this will be at the expense of some growth because of a concomitant
depression of feed intake. Secondly, increasing the amount of lipid in the diet above
7% results in a progressive decrease in feed intake, with this effect intensifying as the
protein content of the diet reduces. Thirdly, whole body fat content of the fish increases
with increasing dietary lipid but this increase in adiposity is attenuated when low
protein diets are fed because the feed intake depression of lipid is amplified with low
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protein diets. Finally, on the basis that the optimal protein and lipid specification of the
diet for juvenile malabar grouper is 55 and 12%, respectively, the optimal protein to
energy ratio is calculated to be 28 g crude protein per MJ of gross energy.
5. Acknowledgements5
The research was carried out as part of an Australian AusAID project with Vietnam
(CARD Project 15) and this financial support is acknowledged. We thank ????? for
technical assistance in the conduct of the experiment and ???? for chemical analyses.
6. References10
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Table 1
Formulation (% of air-dry ingredients) of diets fed to juvenile malabar grouper
Feed ingredient Diet label (protein/lipid)
44/7 50/7 55/7 60/7 44/12 50/12 55/12 60/12 44/16 50/18 55/18 6
Fishmeal (Chile 65%) 35 35 35 35 35 35 35 35 35 35 35 3
Krill hydrolysate 5 5 5 5 5 5 5 5 5 5 5
Wheat gluten 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5 10.5
Casein 6 12 18 24 6 12 18 24 6 12 18 2
Tapioca flour 33 27 21 15 28 22 16 10 23 17 11
Fish oil 1.5 1.5 1.5 1.5 5.5 5.5 5.5 5.5 9.5 9.5 9.5
Soybean oil 1 1 1 1 2 2 2 2 3 3 3Diatomaceous earth 6.45 6.45 6.45 6.45 6.45 6.45 6.45 6.45 6.45 6.45 6.45
Vitamin mix1
1 1 1 1 1 1 1 1 1 1 1
Mineral mix2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Carophyll pink3
0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05
1Custom premix made by Rabar Nutrition, Beaudesert, Australia to provide in final diet (mg/kg): Retinol (A), 1.8; ascorbic acidmenadione (K3), 10.0; d/l-tocopherol (E), 200; choline, 500; inositol, 100; thiamine (B1), 15; riboflavin (B2), 20; pyridoxine (nicotinic acid, 75; biotin, 0.5; cyanocobalamin (B
512), 0.05; folic acid, 5; and ethoxyquin, 150.
2Custom premix made by Rabar Nutrition, Beaudesert, Australia to provide in final diet (mg/kg): Co (as CoCl2.6H2O), 0.5; Cu (40; I (as KI), 4; Cr (as KCr.2SO4), 0.5; Mg (as MsSO4.7H2O), 150; Mn (as MnSO4.H2O), 25; Se (as NaSeO3), 0.1; and Zn (as Z
3 Product of F. Hoffmann-La Roche Ltd, Basel, Switzerland, containing 8% astaxanthin.
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Table 2
The dry matter (DM), crude protein (CP), ash, lipid, fatty acid and calculated gross energy (GE) composition
Feed ingredient Diet label (protein/lipid)
44/7 50/7 55/7 60/7 44/12 50/12 55/12 60/12 44/17 50/17 55/17 6
DM (% as fed) 93.3 92.6 91.9 91.9 93.5 93.9 93.6 93.9 94.5 93.9 93.5 9
DM basis
CP (%) 43.7 49.5 55.5 60.9 43.6 48.8 54.4 59.6 43.1 48.8 54.4 5
Ash (%) 14.7 14.9 15.1 15.1 14.6 14.6 14.7 14.7 14.3 14.5 14.6
Lipid (%) 6.9 7.0 7.0 7.0 12.2 12.2 12.2 12.2 17.4 17.5 17.6
EPA + DHA (%)1 2.0 2.0 2.0 2.0 2.9 2.9 2.9 2.9 3.8 3.8 3.8
n-3:n-6 (%)2 2.1 2.1 2.2 2.2 2.1 2.1 2.1 2.1 2.1 2.1 2.1
GE (kJ/g)3 18.9 19.2 19.6 19.9 20.1 20.4 20.7 21.1 21.2 21.6 21.9 2
CP:GE (mg/kJ) 23.1 25.7 28.3 30.6 21.7 23.9 26.2 28.3 20.3 22.6 24.8 2
1Sum of eicosapentaenoic (EPA; C20:5n3) and docosahexaenoic (DHA; C22:6n3) fatty acids.5
2Ratio of the sum of n3 and n6 fatty acids.
3 Calculated using energy conversion factors of 23.4, 39.2 and 17.2 kJ/g for protein, lipid and carbohydrate,Carbohydrate was determined as the total less the sum of moisture, protein, ash and lipid.
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Table 3
Interaction effects of dietary protein and lipid on productivity traits of Malabar grouper
Diet protein labelDiet lipidlabel 44 50 55 60 Mean
Final weight (g)1
7 67.3h 78.4ef 80.1d 78.8e 76.1B
12 70.0g
82.0b
83.7a
81.0c
79.2A
17 67.2h
78.0f
79.1e
78.7e
75.7C
23 66.8h 77.9f 78.9e 77.8f 75.3D
Mean 67.8Z
79.1Y
80.5X
79.1Y
0.232
Feed intake (g as-fed/d)1
7 1.81f 1.88a 1.89a 1.88ab 1.86A
12 1.73h 1.87bc 1.88ab 1.87c 1.84B
17 1.68i
1.83e
1.84d
1.84de
1.80C
23 1.63j 1.79g 1.80fg 1.80fg 1.76D
Mean 1.71Z 1.84Y 1.85X 1.85XY 0.0052
Gain (g/d)1
7 0.90f 1.10cd 1.13b 1.11c 1.06B
12 0.95e 1.16a 1.19a 1.15a 1.11A
17 0.90f
1.09d
1.11c
1.10cd
1.05C
23 0.89f
1.09d
1.11c
1.09d
1.04C
Mean 0.91Z 1.11Y 1.13X 1.11Y 0.0042
Daily growth coefficient (%)1
7 2.69
h3.05
ef3.10
c3.08
cd2.98
B
12 2.77g 3.18b 3.23a 3.15b 3.08A
17 2.68h 3.05ef 3.07de 3.05ef 2.96C
23 2.67h
3.04ef
3.08cd
3.04f
2.96C
Mean 2.70Z 3.08Y 3.12X 3.08Y 0.0102
Feed conversion ratio (g as fed feed/ g fish gain)1
7 2.01
i1.72
f1.68
e1.70
f1.78
D
12 1.82g 1.61b 1.58a 1.63c 1.66A
17 1.87h
1.67e
1.66de
1.67e
1.72C
23 1.84g
1.64cd
1.63bc
1.66de
1.69B
Mean 1.88Z 1.66Y 1.64X 1.67Y 0.0072
1 For each productivity criterion and within main effect or interaction comparisons,
means with a common superscript letter do not differ (P > 0.05).52
Standard error of the mean for the protein x lipid interaction term.
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Table 4
Regression statistics for relationships describing the effects of dietary dry matter (DM) concentrations of prot
DM feed intake (DFI), gain, daily growth coefficient (DGC), DM feed conversion ratio (FCR), dietary retent
whole wet body protein (WBP) and lipid (WBL) composition responses (Y) of malabar grouper
5Response Statistics for the derived relationship (Y = a + bP + cP
2+ dL + eL
2) and regression coefficient (R
2)
trait (Y) a bP SE bP cP2
SE cP2
dL SE dL eL2
SE eL2
DFI (g/d) -1.408 0.115 0.0117 -0.0011 0.00011 0.005 0.0037 -0.0003 0.00012Gain (g/d) -4.454 0.202 0.0134 -0.0018 0.00013 0.015 0.0043 -0.0006 0.00014DGC (%/d) -7.333 0.378 0.0473 -0.0034 0.00046 2.788 0.0150 -0.0010 0.00050FCR (g :g) 7.420 -0.211 0.0214 0.0019 0.00021 -0.019 0.0068 0.0006 0.00023
N retn (%) -24.08 1.908 0.3086 -0.0202 0.00298 0.491 0.0980 -0.0204 0.00324E retn (%) -41.65 2.262 0.2787 -0.0200 0.00269
0.120 0.0885 -0.0085 0.00293
WBP (%) 21.77 -0.0911 0.0253 0.0537 0.0264WBL (%) 0.205 0.0949 0.0111 0.0392 0.0116
1
Significance of the regression terms: * = P < 0.05; ** = P < 0.001.
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Table 5
The pre- and post-experiment whole body moisture, protein, lipid and ash composition1 of
malabar grouper fed diets varying in protein and lipid content
5
Diet lipid Diet protein label
Label 44 50 55 60 Mean
Moisture content (%, wet fish)2
7 72.4 72.0 71.3 70.5 71.5
12 72.3 71.9 71.1 70.4 71.4
17 72.3 71.9 71.2 70.5 71.5
23 72.4 72.0 71.3 71.0 71.7
Mean 72.3W 71.9W 71.2X 70.6Y 0.293
Ash content (%, wet fish)2
7 4.3 4.3 4.3 4.3 4.3
12 4.3 4.3 4.3 4.3 4.3
17 4.3 4.3 4.3 4.3 4.3
23 4.3 4.3 4.2 4.2 4.3
Mean 4.3 4.3 4.3 4.3 0.073
Protein content (%. wet fish)2
7 18.0 18.1 18.5 18.9 18.4A
12 17.9 18.2 18.6 19.0 18.4A
17 17.3 17.5 17.9 18.3 17.7B
23 16.6 16.8 17.2 17.2 16.9C
Mean 17.4X
17.6X
18.0WX
18.3W
0.413
Lipid content (%, wet fish)2
7 4.3h 5.0fg 5.2fg 5.3ef 5.4D
12 5.4ef
5.4ef
5.3efg
5.4ef
5.6C
17 5.7de 5.9cd 6.1c 6.3bc 5.8AB
23 6.3
bc
6.2
bc
6.6
ab
6.8
a
6.0
A
Mean 4.9W
5.4X
6.0Y
6.5Z
0.133
1The chemical composition (% wet fish) of representative fish at the start of the experiment was (mean
SD): moisture, 74.3 1.06; ash, 5.0 0.08; crude protein, 17.4 1.10; and lipid, 2.2 0.38.
2 For each composition attribute and within main effect or interaction comparisons, means without superscript
letters, or with a common superscript letter, do not differ (P > 0.05).
3Standard error of the mean for the protein x lipid interaction term.10
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Table 6
Interaction effects of dietary protein and lipid on protein and energy retentions of malabar
grouper
Diet protein labelDiet lipidLabel 44 50 55 60 Mean
Protein retention (%)7 22.2f 23.2d 22.1f 20.2k 21.9B
12 24.3b
25.0a
23.6c
21.2i
23.5A
17 22.7e 22.8e 21.3i 19.8l 21.7C
23 21.8h 22.1fg 20.7j 18.5m 20.8D
Mean 22.8X
23.3W
21.9Y
19.9Z
0.101
Energy retention (%)7 18.0j 21.6d 22.7a 22.8a 21.3B
12 19.6i 21.9c 22.6a 22.2b 21.5A
17 18.0jk
20.2g
20.8f
21.1e
20.0C
23 17.8k 19.6i 20.4g 19.9h 19.4D
Mean 18.3Z
20.8Y
21.6W
21.5X
0.081
5 1 Standard error of the mean for the protein x lipid interaction term.
2 For each productivity criterion and within main effect or interaction comparisons, means
without a common superscript letter differ (P < 0.05).
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Figure captions
Fig. 1. Effect of varying the dry matter (DM) protein and lipid content of the diet on the feed
intake (A), daily growth coefficient (DGC) (B) and feed conversion ratio (FCR) (C)responses of malabar grouper. Regression statistics for these modeled responses are given in
Table 4.
5
10
15
Fig. 2. Effect of varying the dry matter (DM) protein and lipid content of the diet on the
retention of dietary N (A) and gross energy (B) by malabar grouper. Regression statistics for
these modeled responses are given in Table 4.
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5
10
15
20
25
30
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
Feedintake
(g DM/d)
Dietprotein
(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
2.5
2.7
2.9
3.1
3.3
DGC(g/d)
Dietprotein(% DM)
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
1.9
FCR(g DM:g fish)
Diet lipid (% DM)
Dietprotein
(% DM)
A
B
C
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
Feedintake
(g DM/d)
Dietprotein
(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
2.5
2.7
2.9
3.1
3.3
DGC(g/d)
Dietprotein(% DM)
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
1.9
FCR(g DM:g fish)
Diet lipid (% DM)
Dietprotein
(% DM)
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
Feedintake
(g DM/d)
Feedintake
(g DM/d)
Dietprotein
(% DM)
Dietprotein
(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
2.5
2.7
2.9
3.1
3.3
DGC(g/d)DGC(g/d)
Dietprotein(% DM)
Dietprotein(% DM)
7 12.2 17.522.9
43.5
49.0
54.6
60.0
1.4
1.5
1.6
1.7
1.8
1.9
FCR(g DM:g fish)
FCR(g DM:g fish)
Diet lipid (% DM)
Dietprotein
(% DM)
Dietprotein
(% DM)
A
B
C
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5
10
712.2
17.522.9
43.5
49.0
54.6
60.0
16
18
20
22
24
E retn(%)
Diet lipid (% DM)
Dietprotein(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
17
19
21
23
25
N retn(%)
Dietprotein(% DM)
A
B
712.2
17.522.9
43.5
49.0
54.6
60.0
16
18
20
22
24
E retn(%)
Diet lipid (% DM)
Dietprotein(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
17
19
21
23
25
N retn(%)
Dietprotein(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
16
18
20
22
24
E retn(%)E retn(%)
Diet lipid (% DM)
Dietprotein(% DM)
Dietprotein(% DM)
712.2
17.522.9
43.5
49.0
54.6
60.0
17
19
21
23
25
N retn(%)N retn(%)
Dietprotein(% DM)
Dietprotein(% DM)
A
B