Effect of dietary protein and lipid levels on

Effect of Dietary Protein and Lipid Levels on GrowthPerformance, Carcass Composition, and Digestive Enzyme of the Juvenile Spotted Babylon, Babylonia areolata Link 1807 Shu Y.. Zhou Labo

Effect of Dietary Protein and Lipid Levels on Growth

Performance, Carcass Composition, and Digestive Enzyme

of the Juvenile Spotted Babylon, Babylonia areolata Link 1807

Shu Y Chi

Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong

Ocean University, Zhanjiang 524025, China

Qi C Zhou1

Laboratory of Fish Nutrition, College of Life Science and Biotechnology, Ningbo University,

Ningbo 315211, China

Bei P Tan, Xiao H Dong, Qi H Yang, and Jian B Zhou

Laboratory of Aquatic Economic Animal Nutrition and Feed, College of Fisheries, Guangdong

Ocean University, Zhanjiang 524025, China

Abstract

This study was undertaken to evaluate the effects of dietary protein and lipid levels on growth performance, feed utilization, carcass composition, and digestive enzyme activity of the juvenile

spotted babylon, Babylonia areolata Six experimental diets were formulated to contain three protein

levels (25, 35, and 45%) at two lipid levels (8 and 12%) Triplicate groups of 40 animals (average weight 5.05 ± 0.08 g) were stocked in 120-L tanks and fed to apparent satiation twice daily for

8 wk Growth performance and feed utilization were significantly affected by dietary protein and

lipid levels (P < 0.05) Protein efficiency ratio (PER), specific growth rate (SGR), and weight gain (WG) were the best at 45%/8% treatment (P < 0.05) There was no significant interaction between

different levels of dietary protein and lipid on survival rate and the soft body to shell ratio (SB/SR) There was an interaction effect between dietary treatments on PER, SGR, and WG, in which shellfish

fed with 45% protein at 8% lipid had the highest interaction (P < 0.05) There was an interaction

effect between dietary protein and lipid levels on pepsin, tryptase, and lipase activities in soft body Tryptase enzyme activity of 45%/8% treatment was the lowest and the highest was found in 25%/8% treatment which also had the highest activity of lipase Results indicated that the juvenile spotted babylon would obtain better growth performance when fed with diets containing 45% dietary protein

at 8% dietary lipid.

Members of the genus Babylonia are distributed

in the Indo-Pacific region, of which spotted

babylon, Babylonia areolata, a large marine

gastropod (adult size 50–60 mm) extends from

Sri Lanka and the Nicobar Islands through

the Gulf of Siam, along the Vietnamese and

Chinese coast to Taiwan (Altena and

Gitten-berger 1981) Juvenile spotted babylon is one

of the most extensively cultured marine

mol-lusks in the Southeast Asian countries, and

it is the second most economically important marine gastropods for human consumption in Thailand (Kritsanapuntu et al 2009) It is a carnivore inhabiting the muddy/sandy subti-dal zone at depths of 4–20 m (summer) and 40–60 m (winter) (Cai et al 1995) It was previously abundant, but declined in number because of overfishing since the late 1980s In recent years, there has been a rapid increase

in market demand for this species in Thailand and other Asian countries As a result, this species has attracted a great interest of shellfish

© Copyright by the World Aquaculture Society 2010

903

farmers because of its resistance to handling,

rapid growth, delicious meat, and high market

price (Zhou et al 2007a) However, a main

con-straint to spotted babylon culture development

is the limited supply of trash fish or crabs that

are presently the main feed source for

grow-out The use of formulated feeds for growing

to marketable size would be practical and

effi-cient in terms of labor cost compared with the

present practice of using trash fish as the rearing

diet The requirement level for dietary nutrients

is the basis for their inclusion levels in the feed

formula Limited studies have been reported on

the nutrient requirements of spotted babylon

(Ke et al 1997; Xu 2006; Zhou et al 2007a,

2007b; Zhang et al 2009)

Protein is one of the most important nutrient

categories for growth and the most expensive

macrocomponent of fish feed because of its

bulk in the feed formula (National Research

Council 1993) Protein requirements are always

studied in aquaculture species with the aim of

determining the minimum amount requirement

to produce maximum growth The relevant

studies for shellfish mainly focused on scallop,

abalone, and spotted babylon Uriarte and

Farías (1999) reported that the postlarvae

of Chilean scallop, Argopecten purpuratus,

showed significantly better growth and survival

when fed with the higher protein diet For

abalone, some researchers reported that protein

requirements ranged from 20 to 44% (Uki

et al 1985; Mai et al 1995; Coote et al 2000;

G´omez-Montes et al 2003; Thongrod et al

2003) As for spotted babylon, the protein

requirements ranged from 25 to 48% (Ke et al

1997; Xu 2006; Zhou et al 2007a)

Lipid is one of the important nutrients to

provide energy for mollusk, especially at larval

and juvenile stages Lipid provides a source

of energy, essential fatty acids and other lipid

classes such as phospholipids, sterols, and

fat-soluble vitamins (Watanabe 1982) The optimal

dietary lipid level had been demonstrated

for mollusk species, such as Haliotis discus

hannai (Uki et al 1985; Mai et al 1995)

and Haliotis tuberculata (L.) (Mai et al 1995).

Zhou et al (2007b) reported that the optimal

dietary lipid requirement for maximum mean

protein gain of juvenile spotted babylon was about 6.54% of dry diet with 43% crude protein Britz and Hecht (1997) reported that the combination of 34% protein and 2–6% lipid

was optimum for the growth of abalone Haliotis

midae Therefore, this study was undertaken to

determine the optimal levels of dietary protein and lipid to support optimum growth response, body composition, and digestive enzyme of the juvenile spotted babylon

Materials and Methods

Diet Preparation

Six diets were formulated to contain three protein levels (25, 35, and 45%) at two lipid levels (8 and 12%) for each protein (Table 1) Fish meal and soybean meal were used as protein sources Fish oil/soybean oil (1:1) and wheat meal were used as lipid and carbohydrate sources, respectively Diet ingredients were ground through an 80-mesh sieve Lipid and distilled water (40% w/w) were added to the premixed dry ingredients and thoroughly mixed until homogenous in a Hobart-type mixer The 1-mm diameter pellets were wet extruded using

a pelletizer (Institute of Chemical Engineer-ing, South China University of Technology, Guangzhou, China), air-dried, and then sealed

in plastic bags and stored at−20 C before use

Animal Rearing

Juvenile spotted babylon was obtained from

a local shellfish farm (Dongding breeding farm, Zhanjiang, China) Management was as described in our previous study (Zhou et al 2007a) Prior to the start of the trial, juvenile spotted babylon was acclimatized to a com-mercial diet (containing 42% crude protein and 6% crude lipid) and was fed twice daily to apparent satiation for 2 wk A 2 × 3 factorial experiment in a completely randomized design was used Each experimental diet was randomly assigned to three tanks The acclimated spotted babylon (initial mean weight, 5.05 ± 0.08 g) was sorted into 18 120-L cylindrical fiberglass tanks at a stocking density of 40 spotted baby-lon per tank Juvenile spotted babybaby-lon was fed

to visual satiety twice daily at a rate of 4% wet

Table 1 Ingredients and composition of experimental diets (g/kg dry matter).

Diets (protein, %/lipid, %)

Ingredients

Composition

d-calcium pantothenate, 30; folic acid, 2.4; biotin, 0.2; and inositol, 60.

body weight for 8 wk, 30% of the ration was

fed at 0900 h, and 70% at 1800 h, which was

the start of the dark phase during which most

feeding activity occurs (Liu and Xiao 1998)

Feed consumption was recorded for each tank

and animals were bulk weighed and counted

every 2 wk to adjust the quantity of feed

Uneaten feed were removed daily before the

next feeding, dried, and weighed to calculate

the feed intake They were provided with

sand-filtered seawater (2 L/min) with continuous

aer-ation The bottom of each tank was covered

with about 4 cm clean sea sand, which

simu-lated the natural environment that they normally

inhabit Tanks were thoroughly cleaned and the

sea sand was changed biweekly Water quality

parameters were monitored daily between 0900 and 1800 h During the feeding trial, water tem-perature ranged from 28.5 to 30.5 C, salinity from 27 to 32 ppt, and pH from 7.8 to 8.0 Ammonia nitrogen was maintained from 0.02

to 0.03 mg/L and dissolved oxygen was from 6.0 to 6.5 mg/L

Sample Collection and Chemical Analyses

At the end of the growth trial, spotted baby-lon was starved 24 h and weighed A sample of

150 animals at the initiation of the feeding trial and 20–25 animals per tank at termination were used for carcass proximate analysis, and then shell and soft body tissues were individually weighed for the calculation of soft body to

shell ratio (SB/SR) (Mai et al 1995) Soft

body of spotted babylon was sampled, sealed

in plastic bags, and stored frozen (−20 C)

for the analysis of nutrient composition Crude

protein, crude lipid, moisture, and ash in diets

and soft body were determined by standard

methods (AOAC 1995) Moisture was

deter-mined by oven-drying at 105 C for 24 h Crude

protein (N × 6.25) was determined by the

Kjel-dahl method after acid digestion using an Auto

Kjeldahl System (1030-Auto-analyzer, Tecator,

H¨ogan¨as, Sweden) Crude lipid was determined

by ether extraction using a Soxtec System HT6

(Tecator) Ash was determined by muffle

fur-nace at 550 C for 24 h

Digestible Enzyme Analyses

The soft body of 15 spotted babylon from

each tank was homogenized in 10 volumes

(w/v) of ice-cold double distilled water by

an electric homogenizer (IKA, T-25, Staufen,

Germany) Homogenates were centrifuged at

10,000 g for 30 min at 4 C to analyze

pro-tease activity and 1660 g for 20 min at 4 C

to analyze lipase activity, respectively After

centrifugation, the supernatants were collected

and stored frozen at−70 C until analyzed The

assays for pepsin and tryptase activity were

measured using the casein hydrolysis method

of Liu et al (1991) and Pan et al (2005)

The substrate was 0.5% casein (Sigma, St

Louis, MO, USA) in citric acid (China National

Medicines Corporation Ltd., Shanghai, China)

buffer (pH 3.0) for pepsin and in borax–sodium

hydroxide (China National Medicines

Corpo-ration Ltd.) buffer (pH 9.8) for tryptase The

reaction proceeded at 37 C for 15 min and

was stopped with trichloroacetic acid (China

National Medicines Corporation Ltd.)

Perco-late was filtered and mixed with 0.5 mol/L

Na2CO3 Color was allowed to develop for

20 min after adding forint-hydroxybenzene At

20 min, the enzyme activity was calculated

from the light absorption at 680 nm One unit

of protease activity was defined as 1-μg

tyro-sine liberated by hydrolyzing casein in 1 min

at 37 C Lipase activity was determined by the

method of Pan and Wang (1997) Homogenates

were incubated with 2% polyvinyl alcohol (Sigma, N81384) in 25-mM phosphate buffer,

pH 7.5, containing 25% olive oil (China National Medicines Corporation Ltd.) as an emulsifying agent at 40 C for 15 min, and then 15-mL 95% ethanol was added to terminate the reaction Two to three drops of phenolphthalein were added to the solution and a titration action with 0.05 mol/L sodium hydroxide was per-formed Consumed volume of sodium hydrox-ide was measured when the solution showed light red One unit of lipase activity was defined

as the amount of enzyme that catalyzed the release of 1μmol of fatty acids in 1 min at pH 7.5 and 40 C Specific activities were expressed

as enzyme activity per milligram protein The protein concentration in homogenates was determined by Bradford (1976) and bovine serum albumin (China National Medicines Corporation Ltd.) as the standard

Calculations and Statistical Analysis

The parameters were calculated as follows: Specific growth rate (SGR, %) = (ln Wt−

ln Wi) × 100/t.

Percent weight gain (WG, %)= 100 × (Wt−

Wi) /Wi Protein efficiency ratio (PER) = (Wt− Wi)/ protein intake (g)

Soft body to shell ratio (SB/SR) = Ws/shell weight (g)

where Wt (g) is final body weight, Wi (g) the

initial body weight, Ws (g) the soft body

weight, and t the experimental duration in day.

Results are presented as mean ± SE of the three replicates All data were analyzed using two-way ANOVA and Tukey’s multiple range test (Puri and Mullen 1980) All statistical analyses were performed by SPSS version 13.0 (SPSS, Chicago, IL, USA)

Results

Growth performance and feed utilization of the juvenile spotted babylon fed with different dietary protein and lipid levels are shown in Table 2 There was no significant interaction

between different levels of dietary protein and

lipid on survival rate and SB/SR Regardless of

protein levels, the SB/SR of shellfish fed with

8% lipid (0.41± 0.02) was significantly higher

than 12% lipid (0.39 ± 0.04) (P < 0.05).

However, there was an interaction on PER,

SGR, and WG among the dietary treatments,

in which shellfish fed with 45% protein at 8%

lipid had the highest interaction (P < 0.05).

Regardless of lipid levels, PER, SGR, and WG

of shellfish fed with 45% protein were the

highest (P < 0.05) (Figs 1, 2).

Moisture, crude protein, and ash in soft body

were not significantly affected by the dietary

protein and lipid levels (P > 0.05) (Table 3).

Juvenile spotted babylon fed with 45% protein

at 12% lipid diet had a significantly higher ether

0

1

2

3

4

5

6

a a

b c

c

b

Protein levels%

PER SGR

Figure 1 Effect of dietary protein levels on the protein

efficiency ratio (PER) and specific growth rate (SGR) of

juvenile spotted babylon Regardless of lipid levels, Fig 1

showed significant differences among protein levels on

PER and SGR The highest PER and SGR were found

in 45% protein treatments (P < 0 05 ).

0

50

100

150

a

b

WG c

200

250

300

350

Protein levels%

Figure 2 Effect of dietary protein levels on the weight

gain (WG) of juvenile spotted babylon Regardless of

lipid levels, Fig 2 showed significant differences among

protein levels on WG, which was found the highest in 45%

protein treatments (P < 0 05 ).

extract content than those fed with the other

diets (P < 0.05) (Table 3).

Pepsin, tryptase, and lipase activities in soft body were significantly affected by the dietary

protein and lipid levels (P < 0.05) (Figs 3, 4

and Table 4) The highest pepsin activity was found in animals fed with the 35%/8% diet

(P < 0.05) The tryptase activity was lowest

in spotted babylon fed with the 45%/8% diet; however, animals fed with the 25%/8% diet had a significantly higher tryptase activity than

those fed with the other diets (P < 0.05) The

lowest lipase activities were found in animals fed with the 35% protein at 8 and 12% lipid diets

0 2 4 6 8 10 12 14 16 18 20

b

a a

a

a

c

c

b b

Protein levels%

Pepsin Tryptase Lipase

Figure 3 Effect of dietary protein levels on the digestive enzyme activities of juvenile spotted babylon Regardless

of lipid levels, significant differences of pepsin activi-ties were found in three protein treatments (P < 0 05 ) Tryptase and lipase activities of 25% protein treat-ments were higher than those of 45% protein treattreat-ments (P < 0 05 ).

0 2 4 6 8 10 12 14 16 18

12 8

Lipid levels%

Pepsin Tryptase Lipase

a

a a

b

b b

Figure 4 Effect of dietary lipid levels on the digestive enzyme activities of juvenile spotted babylon Regardless

of protein levels, significantly higher digestible enzyme activities were found at 8% lipid treatments (P < 0 05 ).

Table 2 Effect of dietary protein/lipid ratio on growth performance, survival, and feed utilization of juvenile spotted babylon.

Diets

(protein, %/

different (P < 0.05).

Table 3 Effect of dietary protein/lipid ratio on soft body composition of juvenile spotted babylon.

Diets

(protein, %/

different (P < 0.05).

Table 4 Effect of dietary protein/lipid ratio on the digestive enzyme activities of juvenile spotted babylon.

Diets

(protein, %/

lipid, %)

Pepsin (U/mg protein)

Tryptase (U/mg protein)

Lipase (U/mg protein)

different (P < 0.05).

Discussion

In this study, PER, SGR, and WG of the

shellfish were significantly affected by dietary

protein and lipid levels Similar results were

observed in abalone fed with diets containing

three protein levels at 24, 34, and 44%, each

with three lipids levels at 2, 6, and 10%,

respec-tively (Britz and Hecht 1997) Juvenile green

abalone, Haliotis rufescens, fed 40.5 and 44.1%

protein diets showed significantly better growth performance than those fed the other diets (26,

31 and 35% protein with the same energy con-tent at about 4.1 kcal/g) (G´omez-Montes et al 2003) Xu (2006) reported that the optimal pro-tein and lipid requirement of juvenile spotted babylon (initial weight 2.16 ± 0.05 g) should

be 36.5–43.1% and 7.8–10.7%, respectively; growth performance would be restrained when the dietary lipid level was under 7.8% In this

study, the maximum growth performance of

spotted babylon was observed in diet

contain-ing 45% protein at 8% lipid However, Liu

et al (2006) indicated that Babylonia formosae

(initial weight 1.60 ± 0.11 g) fed with

differ-ent dietary protein levels (crude protein from

25 to 48%) have no significant differences in

growth performance, which was different from

our results Dietary protein is not enough to

meet the growth requirement; lower growth

rates would be observed (Smith 1989) If the

dietary energy level is insufficient in the diets,

protein will be used as energy for maintenance

(National Research Council 1983) The

esti-mation of protein requirements is affected by

some factors such as rearing conditions, stage of

growth, and sources of protein, but a more

sig-nificant factor may be the dietary energy content

in quantitative determination (Wilson 1989)

Lee et al (2002) reported that a positive

corre-lation was found between the levels of dietary

digestible protein/digestible energy ratio and

growth performance at the same lipid levels

The results of juvenile spotted babylon fed with

25 and 35% protein indicated a sparing effect

of the lipid for protein on growth performance

Juvenile spotted babylon fed with diet

con-taining 12% lipid had higher PER than that fed

with 8% lipid diet The trend indicated that

spotted babylon can effectively utilize dietary

lipid as an energy source and dietary protein

will be used for growth The theory behind

a protein sparing effect is that, when protein

provides essential amino acids to meet growth

requirements, extra dietary protein will be used

for energy purposes Increases in the

non-protein energy component of diets (at a specific

protein concentration) have been reported to

improve growth and reduce the protein

require-ment through protein sparing in the

Amer-ican lobster (Capuzzo and Lancaster 1979)

When spotted babylon was fed with a diet

con-taining excess energy, WG may be decreased

because of the reduced feed consumption

How-ever, when spotted babylon was fed with a

diet deficient in energy, dietary protein will be

used as an energy source and this elevates the

production cost In this study, there was no sig-nificant sparing effect when the dietary protein increased to 45%

Mai et al (1995) found that SB/SR of abalone did not differ significantly when fed with diets containing 20–50% protein In this study, although the protein and lipid had sig-nificant influences on SB/SR, the interaction between protein and lipid was not significant The main difference in protein and lipid utiliza-tion may be because of the carnivorous feeding activity of spotted babylon, whereas abalone is

a herbivorous mollusk

Protein level in diet would affect the body protein and lipid contents of scallop spat, but there were no effects on protein deposition with the growth change (Uriarte and Farías 1999) However, the increase of dietary lipid levels should be carefully considered as it may affect carcass quality, mainly because of an increase

of lipid deposition (Cowey 1993; Hillestad and Johnsen 1994) Zhou et al (2007a) reported that lipid content in soft body (initial weight=

93.50 ± 1.70 mg) decreased with increasing

dietary protein levels from 27 to 49% with lipid levels from 15 to 3% In this study,

by comparison with the spotted babylon fed with the different protein and lipid levels, ether extracts of soft body of juvenile spotted babylon were significantly affected by the dietary protein and lipid levels; however, there was no significant difference in the protein content of soft body Increasing dietary protein level did not influence the protein content

in soft body However, our previous studies reported that crude protein, moisture, and ash content in soft body significantly decreased when the dietary lipid level increased from 1.83

to 11.73% at 43% dietary protein, but the lipid content was reversed (Zhou et al 2007b) It is speculated that excretive nitrogen level would increase with increasing dietary protein level (Hawkins and Bayne 1991)

In this study, the digestive enzyme activities

in soft body were significantly affected by the dietary protein and lipid levels Protease activity

in the digestive gland is a key determinant enzyme of the digestibility and assimilation efficiency of ingested proteins The results

showed that spotted babylon fed with a diet

with 35% protein and 8% lipid had the highest

pepsin activities of those fed the diets With

the dietary protein level increasing at the same

lipid level, pepsin activity showed a downtrend,

except 35%/8% On the contrary, Pan et al

(2005) and Zhou et al (2007a) reported that the

activities of pepsin and tryptase in soft body

were elevated with an increase in the dietary

protein The main reason may be because of

different species or different dietary lipid level

and/or development stage At the low lipid

level, tryptase activity significantly declined

with the protein increasing However, at the

high lipid level, the trend was adverse precisely

without difference Lipase activities of lipid

level at 12% were lower than those at 8% with

dietary protein level at 25 and 35% The lipase

activities were improved at 45% protein level

To the juvenile spotted babylon, lower protein

level would limit the utilization of higher lipid

The juvenile spotted babylon could digest lipid

and utilize the dietary lipid as an energy source

at higher protein levels

Conclusion

In summary, this study provides some insight

into the nutrition of juvenile spotted babylon

The levels of protein and lipid at 45 and 8%

were recommended for the best growth of

juve-nile spotted babylon (initial mean weight=

5.05 ± 0.08 g).

Acknowledgments

This work was supported by Zhanjiang

Science and Technology Research Program

(grant number 200401) The authors are grateful

for J C Zhang and S L Zeng for their skilled

technical assistance

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