Tuan Kcw Malabar Pap

8/2/2019 Tuan Kcw Malabar Pap

1/31

Optimum dietary protein and lipid specifications for juvenile malabar

grouper (Epinephelus malabaricus)

Le Anh Tuan1

and Kevin C. Williams2*

5

10

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: ..............................


2/31

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).

5

10

15

20

25

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.

2


3/31

Key words: nutrition, feeding, requirements, protein to energy, cod, retention

3


4/31

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.

5

10

15

20

25

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

4


5/31

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.

5

10

15

20

25

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

5


6/31

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).

5

10

15

20

25

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

6


7/31

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.

5

10

15

20

25

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

7


8/31

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.

5

10

15

20

2.4 Measurements and statistical analysis

8


9/31

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

31

31

100)/(%

5

10

15

20

25

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).

9


10/31

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.

5

10

15

20

25

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

10


11/31

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%).

5

10

15

20

25

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

11


12/31

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.

5

10

15

20

25

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

12


13/31

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).

5

10

15

20

25

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

13


14/31

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).

5

10

15

20

25

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

14


15/31

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.

5

10

15

20

25

15


16/31

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).

5

10

15

20

25

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

16


17/31

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

15

20

25

AOAC International, 1999. Official Methods of Analysis, 16th

edn. Association of

Official Analytical Chemists International, Maryland, USA.

Baile, C.A., Della-Fera, M.A., Martin, R.J., 2000. Regulation of metabolism and body

fat mass by leptin. Ann. Rev. Nutr. 20, 105-127.

Beglinger, C., Degen, L., 2004. Fat in the intestine as a regulator of appetite role of

CCK. Physiol. Behav. 83, 617-621.

Beveridge, M.C.M., 1996. Cage Aquaculture, 2nd

edn. Fishing News Books, Oxford.

Boonyaratpalin, M., 1997. Nutrient requirements of marine food fish cultured in

Southeast Asia. Aquaculture 151, 283-313.

Boujard, T., Gelineau, A., Coves, D., Corraze, G., Dutto, G., Gasset, E., Kaushik, S.,

2004. Regulation of feed intake, growth, nutrient and energy utilisation in European

sea bass (Dicentrarchus labrax) fed high fat diets. Aquaculture 231, 529-545.

Boujard, T., Medale, F., 1994. Regulation of voluntary feed intake in juvenile rainbow

trout fed by hand or by self feeders with diets containing two different

protein/energy ratios. Aquat. Living Resour. 7, 211-215.

17


18/31

Chen, H-Y., 2001. Nutritional studies and feed development of theEpinephelus

groupers in Taiwan. In: Liao, I.C., Baker, J. (Eds.), Aquaculture and Fisheries

Resources Management. TFRI Conference Proceedings 4, Taiwan Fisheries

Research Institute, Tungkang, Taiwan, pp. 169-172.

5 Chen, H-Y., Tsai, J-C., 1994. Optimal dietary protein level for the growth of juvenile

grouper,Epinephelus malabaricus, fed semipurified diets. Aquaculture 119, 265-

271.

Cho, C.Y., Slinger, S.J., Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy

intake, expenditure and productivity. Comp. Biochem. Physiol. 73B, 25-41.

10

15

20

25

FAO, 2005. Fishstat Plus. http://www.fao.org/fi/statist/FISOFT/FISHPLUS.asp#General

Folch, J., Lees, M., Sloane-Stanley, G.H., 1957. A simple method for the isolation and

purification of total lipid from animal tissues.J. Biol. Chem., 226, 497-509.

Geary, N., 2004. Endocrine controls of eating: CCK, leptin, and ghrelin. Physiol.

Behav. 81, 719-733.

Gelineau, A., Bolliet, V., Corraze, G., Boujard, T., 2002. The combined effects of

feeding time and dietary fat levels on feed intake, growth and body composition in

rainbow trout. Aquat. Living Resour. 15, 225-230.

Gelineau, A., Boujard, T., 2001. Oral administration of choleocystokinin receptor

antagonists increase feed intake in rainbow trout. J. Fish Biol. 58, 716-724.

Hervey, G.R., 1958. The effects of lesions in the hypothalamus in parabiotic rats. J.

Physiol. 145, 336-352.

Houseknecht, K.L., Portocarrero, C.P., 1998. Leptin and its receptors: Regulators of

whole-body energy homeostasis. Dom. Anim. Endocrin. 15, 457-475.

Jobling, M., Johansen, S.J.S., 1999. The lipostat, hyperphagia and catch-up growth.

Aquac. Res. 30, 473-478.

18


19/31

Jobling, M., Miglavs, I., 1993. The size of lipid depots a factor contributing to the

control of food intake in Arctic charr, Salvelinus alpinus? J. Fish Biol. 43, 487-

489.

Jobling, M., Wandsvik, A., 1983. An investigation of factors controlling food intake in

Arctic charr, Salvelinus alpinus L. J. Fish Biol. 23, 397-404.5

10

15

20

25

Johansen, S.J.S, Ekli, M., Jobling, M., 2002. Is there lipostatic regulation of feed intake

in Atlantic salmon Salmo salarL.? Aquacult. Res. 33, 515-524.

Johansen, S.J.S., Sveier, H., Jobling, M., 2003. Lipostatic regulation of feed intake in

Atlantic salmon Salmo salarL. defending adiposity at the expense of growth?

Aquacult. Res. 34, 317-331.

Kaushik, S.J., Medale, F., 1994. Energy requirements, utilization and dietary supply to

salmonids. Aquaculture 124, 81-97.

Kennedy, G.C., 1953. The role of depot fat in the hypothalamus control of food intake

in the rat. Proc. R. Soc. Lond. B. 140, 578-592.

Lin, X., Volkoff, H., Narnaware, Y., Bernier, N.J., Peyon, P., Peter, R.E., 2000. Brain

regulation of feeding behaviour and food intake in fish. Comp. Biochem. Physiol.

126A, 415-434.

Lin, Y-H., Shiau, S-Y., 2003. Dietary lipid requirement of grouper,Epinephelus

malabaricus, and effects on immune responses. Aquaculture 225, 243-250.

Luo, Z., Liu, Y.J., Mai, K.S., Tian, L.X., Liu, D.H., Tan, X.Y., 2004. Optimal dietary

protein requirement of grouperEpinephelus coioides juveniles fed isoenergetic

diets in floating net cages. Aquacult. Nutr. 10, 247-252.

Luo, Z., Liu, Y-J., Mai, K-S., Tian, L-X., Liu, D-H., Tan, X-Y., Lin, H-Z., 2005. Effect

of dietary lipid level on growth performance, feed utilization and body composition

of grouperEpinephelus coioides juveniles fed isonitrogenous diets in floating

netcages. Aquacult. Intl. 13, 257-269.

19


20/31

Lupatsch, I., Kissil, G. Wm., 2005. Feed formulations based on energy and protein

demands in white grouper (Epinephelus aeneus). Aquaculture 248, 83-95.

Lupatsch, I., Kissil, G. WM., Sklan, D., Pfeffer, E., 2001. Effects of varying dietary

protein and energy supply on growth, body composition and protein utilization in

gilthead seabream (Sparus aurata L.). Aquacult. Nutr. 7, 71-80.5

10

15

20

Miao, S., Tang, H-C., 2002. Bioeconomic analysis of improving management

productivity regarding grouperEpinephelus malabaricus farming in Taiwan.

Aquaculture 211, 151-169.

Morrison, W.R., Smith, L.M., 1964. Preparation of fatty acid methyl esters and

dimethylacetals from lipids with boron fluride-methanol. J. Lipid Res. 5, 600-608.

New, M.B., 1996. Responsible use of aquaculture feeds. Aquaculture Asia, 1: 3-15.

Peres, H., Oliva-Teles, A., 1999. Effect of dietary lipid level on growth performance

and feed utilization by European sea bass juveniles (Dicentrarchus labrax).

Aquaculture 179, 325-334.

Rasmussen, R.S., Ostenfeld, T.H., McLean, E., 2000. Growth and feed utilization of

rainbow trout subjected to changes in feed lipid concentrations. Aquacult. Intl. 8,

531-542.

Ritter, R.C., 2004. Gastrointestinal mechanisms of satiation for food. Physiol. Behav.

81, 249-273.

Saether, B-S., Jobling, M., 2001. Fat content in turbot feed: influence on feed intake,

growth and body composition. Aquacult. Res. 32, 451-458.

Shearer, K.D., Silverstein, J.T., Plisetskaya, E.M., 1997. Role of adiposity in food

intake control of juvenile Chinook salmon (Oncorhynchus tshawytscha). Comp.

Biochem. Physiol. 118A, 1209-1215.

20


21/31

Shiau, S-Y., Lan, C-W., 1996. Optimum dietary protein level and protein to energy

ratio for growth of grouper (Epinephelus malabaricus). Aquaculture 145, 259-

266.

Snedecor, G.W. & Cochran, W.G. (1989) Statistical Methods, 8th

edn. Iowa State

University Press, Ames, Iowa, USA, 503 pp.5

10

15

20

25

Tian, X., Qin, J.G., 2003. A single phase of food deprivation provoked compensatory

growth in barramundiLates calcarifer. Aquaculture 224, 169-179.

Tian, X., Qin, J.G., 2004. Effects of previous ration restriction on compensatory growth

in barramundiLates calcarifer. Aquaculture 235, 273-283.

Usman, Rachmansyah, Laining, A., Ahmad, T., Williams, K.C., 2005. Optimum dietary

protein and lipid specifications for grow-out of humpback grouperCromileptes

altivelis (Valenciennes). Aquacult. Res. 36, 1285-1292.

Volkoff, H., Canosa, L.F., Unniappan, S., Cerda-Reverter, J.M., Bernier, N.J., Kelly,

S.P., Peter, R.E., 2005. Neuropeptides and the control of food intake in fish. Gen.

Comp. Endocrin. 142, 3-19.

Volkoff, H., Eykelbosh, A.J., Peter, R.E., 2003. Role of leptin in the control of feeding

of goldfish Carassius auratus: interactions with cholecystokinin, neuropeptide Y

and orexin A, and modulation of fasting. Brain Res. 972, 90-109.

Volkoff, H., Peter, R.E., 2004. Effects of lipopolysaccharide treatment on feeding of

goldfish: role of appetite-regulating peptides. Brain Res. 998, 139-147.

Williams, I., Williams, K.C., Smith, D.M., Jones, M., 2006. Polka dot grouper,

Cromileptes altivelis, can utilize dietary fat efficiently. Aquacult. Nutr. 12, In press.

Williams, K.C., Irvin, S., Barclay, M., 2004. Polka dot grouperCromileptes altivelis

fingerlings require high protein and moderate lipid diets for optimal growth and

nutrient retention. Aquacult. Nutr. 10, 125-134.

21


22/31

Williams, M.J., 2002. Asian fisheries in the 21st

century: Which way to prosperity?

(keynote address to the 6th Asian Fisheries Forum, Kaohsiung, Taiwan November

25-29 2001) http://www.compass.com.ph/~afs/mjwkeynote.html

Woods, S.C., Seeley, R.J., 2000. Adiposity signals and the control of energy

homeostasis. Nutrition 16, 894-902.5

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., Friedman, J.M., 1994.

Positional cloning of the mouse obese gene and its human homologue. Nature

372, 425-432.

22


23/31

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.


24/31

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.


25/31

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.

25


26/31

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.


27/31

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


28/31

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).

28


29/31

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.

29


30/31

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


31/31

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

Documents

Tuan Kcw Malabar Pap