1 1 2 3 2 2 1

1 1 1 1 1

1 Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, East China Normal University,

Shanghai, China; 2 Department of Aquaculture, Hainan University, Haikou, China; 3 School of Biological Sciences, Flin-

ders University, Adelaide, SA, Australia

Juvenile Litopenaeus vannamei were fed for 8 weeks with

diets containing four ratios of protein to carbohydrate

(CBH) at P26 : C30, P30 : C25, P34 : C19 and P38 : C14,

respectively, at 3.0 g L�1 salinity. Shrimp weight gain of

P34 : C19 group was the highest and differed from the

shrimp fed the P26 : C30 or P30 : C25 diet. Shrimp fed

the P26 : C30 diet obtained higher survival than those

fed other diets. Shrimp fed the P34 : C19 diet contained

the highest body protein and lipid, which were signifi-

cantly higher than those fed the P38 : C14 diet. Shrimp

fed the P30 : C25 diet had the highest haemolymph glu-

cose content, which was significantly higher than those

fed the P26 : C30 or P38 : C14 diet. Shrimp muscle gly-

cogen of the P26 : C30 group was the highest. Hepato-

pancreas B-cell number of shrimp fed the P26 : C30 diet

was lower than those fed other diets, and the R cell

number was the highest in the shrimp fed the P30 : C25

diet. This study indicates that the protein-sparing effect

by CBH occurred in the P30 : C25 and P34 : C19 groups

because these proteins to CBH ratios can support normal

growth. Within the range of basic energy demand, the

high dietary CBH to protein ratio can improve L. vanna-

mei survival at low salinity.

KEY WORDS: ammonia tolerance, carbohydrate, Litopenaeus

vannamei, low salinity, protein-sparing effect

Received 11 January 2014; accepted 15 May 2014

Correspondence: E. Li and L. Chen, School of Life Sciences, East China

Normal University, Shanghai 200062, China.

E-mails: ecli@bio.ecnu.edu.cn and lqchen@bio.ecnu.edu.cn

The white shrimp Litopenaeus vannamei is the major

shrimp species in aquaculture, representing over 70% pro-

duction of the farmed shrimp in the world (CFSY 2011).

With the development of inland shrimp farming at low

salinity, L. vannamei has become one of the most popular

shrimp species for aquaculture in the United States, Thai-

land, Ecuador and China (Saoud et al. 2003; Cheng et al.

2006). Although L. vannamei is euryhaline and able to tol-

erate a wide range of salinity from 1 to 50 g L�1 (Pante

1990), the low salinity in estuary can affect its growth

performance and physiological responses (Fry 1971; Kinne

1971). At low salinity (<5 g L�1), slow growth, low survival

(Diaz & Farfan 2001; Li et al. 2007) and poor stress toler-

ance (Lin & Chen 2001, 2003; Li et al. 2007, 2008; Wang

et al. 2013) have been found in L. vannamei. The culture of

L. vannamei at low salinity is a trend that will continue to

grow throughout the world in future (Roy et al. 2010).

Therefore, there is a need to further explore the physiologi-

cal adaptation of shrimp hypo-osmoregulation and to

identify a practical way to improve growth performance

and antistress ability of L. vannamei at low salinity.

Protein is an indispensable nutrient required to repair tis-

sue damage and maintain routine body functions. High-

protein diet can improve the growth of L. vannamei at low

salinity because dietary amino acids can contribute to

energy requirement and therefore promote somatic growth

(Cuzon et al. 2004; Li et al. 2011). But high dietary protein

means high cost of feed and environmental pollution from

waste, which will not support sustainable development of

shrimp aquaculture. Identification of optimal dietary pro-

tein is essential because it is not only important for animal

growth but also for reduction of organic load in an ecosys-

tem and environmental pollution (Singh et al. 2006). In

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ª 2014 John Wiley & Sons Ltd

2014 doi: 10.1111/anu.12221. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition

animal nutrition research, the increase of non-protein

ingredients (e.g. lipid and CBH) has been used as a strategy

to minimize the use of dietary protein (Cho & Kaushik

1990). Although shrimp have limited ability to utilize CBH

and cannot adapt to a high level of dietary CBH (Shiau

et al. 1991), CBHs are often included in artificial diets for

crustacean as an energy source to spare protein (Cruz-

Suarez et al. 1994; Cuzon et al. 2004).

In addition, CBH can meet the high-energy requirement

of aquatic animals in a stress condition (Welcomme &

Devos 1991; Arasta et al. 1996; Tseng & Hwang 2008;

Wang et al. 2012) as CBH is the primary and immediate

source of energy for most crustacean species (Lehninger

1978). Under the stress of low salinity, blood glucose

increases in Chasmagnathus granulate (Santos & Nery 1987)

and Crangon Crangon Linnaeus (Spaargaren & Mors 1985).

The protein-sparing effect of CBH has been observed in

many aquatic species, such as rainbow trout (Pieper &

Pfeffer 1980), channel catfish (Garling & Wilson 1976),

European eel (Hidalgo et al. 1993) and Penaeus monodon

(Cruz-Suarez et al. 1994). However, the supplementation of

CBH as a non-protein energy source has not been tested in

an environment where energy requirement is escalated due

to the osmoregulation of invertebrates at low salinity.

As L. vannamei is often cultured at low salinity especially

in inland saline waters, there is a need to understand the

use of nutritional manipulation to improve its physiological

adaptation and growth rate at a hyposmotic condition.

This study aimed to investigate the protein-sparing effect

of CBH on this economically important shrimp species in

aquaculture by evaluating growth performance, body com-

position, key enzyme activities in glycometabolism, hepato-

pancreatic histology and ammonia tolerance of

L. vannamei at 3 g L�1 salinity when shrimp were fed on

diets with different protein and CBH ratios. Results of this

study will provide fundamental bases to adjust the protein

and energy ratio in shrimp feed with a potential to reduce

feed costs in shrimp farming.

Four iso-energetic diets, using fish meal and soybean meal

as protein, fish oil and soybean oil as lipid and wheat

starch as CBH, were formulated to contain four different

protein (P) : CBH (C) ratios at P26 : C30; P30 : C25;

P34 : C19 and P38 : C14, respectively (Table 1). Dietary

ingredients were ground with 80-lm mesh and weighed to

the nearest 0.1 mg. All dry ingredients were mixed before

lipids containing 150 mg kg�1 ethoxyquin and water were

added for further mixing. Diets were processed into 3-mm

diameter pellets, air-dried at room temperature to a moisture

content of < 10 g 100 g-1, ground and sieved to appropriate

size, and stored at �20 °C until use (Peres et al. 2003).

Juvenile L. vannamei were obtained from a local company

in Hainan, China. Shrimp were cultured in fibreglass tanks

(80 9 60 9 50 cm) at a salinity of 32.0 g L�1 for 1 week

and then were acclimated to the target salinity (3.0 g L�1)

by changing 2.0 g L�1 per day. During acclimation, shrimp

were fed with a commercial L. vannamei feed. Seawater

was pumped from the deep sea of Baisha Coast, Haikou,

China. Tap water was thoroughly aerated before being

Table 1 Compositions of experimental diets

Ingredients

Diets g kg�1

1 2 3 4

Ratio of

protein

and CBH

P26 : C30 P30 : C25 P34 : C19 P38 : C14

Fish meal 280 320 360 400

Soybean meal 200 240 280 300

Wheat starch 300 250 190 140

Fish oil 27.2 25 22.8 20.5

Soybean oil 25 25 25 25

Lecithin 10 10 10 10

Cholesterol 5 5 5 5

Vitamin premix1 20 20 20 20

Vitamin C 1 1 1 1

Mineral premix2 5 5 5 5

Carboxymethyl

cellulose

30 30 30 30

Cellulose 76.8 49 31.2 23.5

Calcium

carbonate

20 20 20 20

Crude protein 268 306 347 382

Crude lipid 86.0 87.5 88.7 85.8

1 Vitamin premix, diluted in cellulose, provided the following

vitamins (g kg�1 premix): thiamine HCl 0.5, riboflavin 3.0, pyro-

doxine HCl 1.0, DL Ca-Pantothenate 5.0, nicotinic acid 5.0, biotin

0.05, folic acid 0.18, vitamin B12 0.002, choline chloride 100.0,

inositol 5.0, menadione 2.0, vitamin A acetate (20 000 IU g�1)

5.0, vitamin D3 (400 000 IU g�1) 0.002, dl-alphatocopherol ace-

tate (250 IU g�1) 8.0, alpha-cellulose 865.266.2 Trace mineral premix provided the following minerals

(g 100 g�1 premix): cobalt chloride 0.004, cupric sulphate penta-

hydrate 0.250, ferrous sulphate 4.0, magnesium sulphate hepta-

hydrate 28.398, manganous sulphate monohydrate 0.650,

potassium iodide 0.067, sodium selenite 0.010, zinc sulphate hep-

tahydrate 13.193, sodium dihydrogen phosphate 15, filler 38.428.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

added to the tanks to adjust the salinity level. Daily water

exchange was 30% of the tank volume. Water quality

parameters including pH, salinity, temperature, dissolved

oxygen and ammonia were monitored 2–3 times a week

throughout the feeding trial. The water quality parameters

across all feeding treatments were maintained at 7–8 pH,

3 g L�1 salinity, 27.0–30.0 °C, 6.25–7.47 mg dissolved oxy-

gen L�1, and <0.06 mg total ammonia nitrogen L�1.

After salinity acclimation from 32 to 3.0 g L�1, 40 juve-

niles (0.270 � 0.013 g) were randomly assigned to one of

the four experimental diets in each tank

(47 9 58 9 60 cm) in triplicate and were fed twice daily

(0800 and 1700 h) to apparent satiation for 8 weeks. Based

on the amount of feed left in the previous day, daily

rations were adjusted to slight over satiation. The uneaten

feed was daily removed with a siphon tube. At the end of

the experiment, all shrimp in each tank were counted and

deprived of feed for 24 h to evacuate the gut content

before body weight determination. The indexes for the

assessment of growth performance and morphometric

indexes were calculated as follows:

Weight gain ð%Þ ¼ 100� ðWt �W0Þ=W0;

whereW0 is the initial weight andWt is the final weight;

Survival rate ð%Þ ¼ 100� ðfinal shrimp numberÞ=ðinitial shrimp numberÞ;

Condition factor ðCF;%Þ ¼ 100�W=L3;

whereW is weight (g) andL is length (cm);

Hepatosomatic index ðHSI;%Þ¼ 100� ðhepatopancreas weight/body weightÞ:

All experimental diets and shrimp samples were analysed in

triplicate for proximate composition following the standard

methods (AOAC, 1990). Moisture was determined by oven

dry at 105 °C to a constant weight. Samples used for dry

matter determination were digested with nitric acid and

incinerated in a muffle furnace (XL-3; TIANYOULI, Tian-

jin, China) at 600 °C overnight for ash determination. Pro-

tein was measured with the combustion method using an

FP-528 nitrogen analyser (Leco Corporation, St. Joseph,

MI, USA). Lipid was determined by the ether extraction

method using the Soxtec system (2055 Soxtec Avanti; Foss

Tecator, Hoganas, Sweden).

Before sample collections, 50–200 lL haemolymph was

extracted through a needle at the base of the fifth pereopod

after the shrimp had been dried on a paper towel. After

collecting 200 lL haemolymph, a subsample of 100 lL was

obtained from each animal with a syringe containing 20%

anticoagulation to prevent clotting. The glucose concentra-

tion in haemolymph was measured with a commercial kit

for clinical diagnosis. The glycogen concentrations of hepa-

topancreas and muscle homogenates were measured with

the method of Dubois et al. (1965). Homogenate

(100 mg mL�1, w/v) was prepared in the 5% trichloroace-

tic acid buffer for 2 min at 8000 g. After collection, the

glycogen was dissolved by adding 0.5 mL of distilled water

and then 5 mL of concentrated sulphuric acid and phenol

(5%) were added and mixed. The glycogen content was

read at 490 nm on a spectrophotometer (Spectrumlab 22pc;

LINGGUANG, Shanghai, China), and the glycogen con-

centration was expressed as milligrams glycogen per gram

wet weight (mg g�1).

Pyruvate kinase (PK) activity was determined according to

the method of Feska et al. (2003). Samples of hepatopan-

creas from different treatments were homogenized (1 : 10

w/v) in an ice-cold buffer with a homogenizer. PK activity

was assayed in an incubation medium containing 0.1 M

Tris–HCl buffer, 10 mM MgCl2, 0.16 mM NADH, 75 mM

KCl, 5.0 mM ADP, 1.0 U of L-lactate dehydrogenase, and

10 lL mitochondria-free supernatant at pH 7.5 in a final

volume. The reaction was started after adding 1.0 mM

phosphoenolpyruvate. All assays were performed in dupli-

cate at 25 °C. Results were expressed as U mg�1 protein.

Samples of hepatopancreas from different feeding groups

were separately homogenized (1 : 3, w/v) in an ice-cold

buffer. Next, the homogenate was centrifuged at 18 000 g

for 20 min at 4 °C. The supernatant was used to determine

enzyme activity. Hexokinase (HK) activity was assayed

according to the method of Brito et al. (2001). In addition

to 0.1 mL supernatant, the assay medium also contained

75 mM Tris–HCl, 7.5 mM MgCl2, 0.8 mM EDTA, 1.5 mM

KCl, 4.0 mM mercaptoethanol, 0.4 mM nicotinamide-

adenine dinucleotide phosphate NADP+, 2.5 mM ATP,

10 mM creatine phosphate, 1 mM glucose, creatine phos-

phokinase (100 g, 1.8 units) and glucose-6-phosphate dehy-

drogenase (10 g, 1.4 units). The reaction was started after

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

adding 0.4 mM NADP+. All assays were performed at

25 °C. Results were expressed as U mg�1 protein.

After shrimp were sampled for assay, the remaining nine

shrimp in each tank were challenged with 9.33 mg L�1

ammonium nitrogen (Li et al. 2007) at 3 g L�1 for 96-h

without feeding. The testing medium was refreshed every

24 h at 26.0–27.5 °C and pH 7.8 � 0.2. Shrimp mortalities

were recorded at 6, 12, 24, 48, 72 and 96 h.

Upon termination of the experiment, three shrimp in the

intermoult stage (C) were taken from each tank and fixed

in Bouin’s solution for 24 h. The fixed tissues were dehy-

drated in ascending concentrations of alcohol, cleared in

toluene, embedded in paraffin and sectioned with a rotary

microtome at 5 lm thick. Sectioned tissues were stained

with haematoxylin and eosin (H&E).

To compare the effects of dietary treatments on all the

parameters tested and the survival rate at each time point

in the challenge trial for ammonia tolerance, data were

subjected to one-way analysis of variance (SPSS 19.0; IBM

SPSS, Chicago, IL, USA). Some data were transformed

with arcsine function to comply with the assumptions for

normality and homogeneity of variance. If a significant dif-

ference was identified for the diet treatment, the differences

between all diet levels were compared by Duncan’s multi-

ple range tests. The significant difference was set at

P < 0.05 for all statistical tests.

Weight gain of shrimp fed the P34 : C19 diet was highest

and differed from those fed P26 : C30 and P30 : C25 diets

(P < 0.05, Table 2). Shrimp fed P26 : C30 had the lowest

weight gain in all feeding groups (P < 0.05). The survival

rate of the shrimp tended to increase with the increased

dietary CBH levels. Shrimp fed P26 : C30 had the highest

survival rate and was significantly higher than those fed

other three diets (P < 0.05). There were no significant dif-

ferences in survival between P34 : C19 and P38 : C14

groups. No differences were found in the hepatosomatic

index and the condition factor among all the treatments

(Table 2).

Table 2 Growth performance and morphological parameter of different treatments (n = 3)

Ratio of protein and CBH Weight gain (%) Survival rate (%) Hepatosomatic index (%) Condition factor (%)

P26 : C30 630.21 � 14.49a 96.67 � 0.83a 4.79 � 0.63 0.94 � 0.04

P30 : C25 845.34 � 37.26b 86.67 � 1.67b 5.42 � 0.33 1.11 � 0.05

P34 : C19 1021.00 � 66.19c 69.17 � 3.00c 4.65 � 0.23 1.01 � 0.10

P38 : C14 904.37 � 80.11bc 68.33 � 3.00c 5.26 � 0.57 1.08 � 0.11

The different superscripts of the same column indicate significant difference (P < 0.05).

Table 3 Whole body proximate composition (g kg�1 wet weight basis) of Litopenaeus vannamei fed different diets (n = 3)

Ratio of protein and CBH Moisture Crude protein Crude lipid Ash

P26 : C30 784.62 � 3.52 161.45 � 2.06b 9.48 � 0.21ab 37.36 � 0.19

P30 : C25 783.87 � 4.01 158.85 � 0.42ab 8.16 � 0.54ab 35.98 � 0.70

P34 : C19 776.14 � 2.97 169.91 � 1.72c 11.12 � 1.57a 37.14 � 0.97

P38 : C14 784.79 � 3.30 156.60 � 0.55a 7.69 � 0.57b 37.23 � 0.03

The different superscripts in the same column indicate significant difference (P < 0.05).

Table 4 The haemolymph glucose, glycogen in hepatopancreas and muscle and hexokinasse (HK) and pyruvate kinase (PK) activities of

Litopenaeus vannamei fed different diets (n = 3)

Ratio of protein

and CBH

Haemolymph

glucose (mM)

Glycogen in

hepatopancreas (mg g�1)

Glycogen in

muscle (mg g�1)

HK activities

(U mg�1 protein)

PK activities

(U mg�1 protein)

P26 : C30 11.00 � 1.62a 5.68 � 0.84 5.59 � 0.14a 1.34 � 0.09 11.71 � 1.39

P30 : C25 22.93 � 1.95b 4.94 � 0.43 1.87 � 0.52b 1.61 � 0.13 11.11 � 1.16

P34 : C19 20.35 � 1.93b 4.62 � 0.33 2.12 � 0.14b 1.81 � 0.10 9.08 � 0.84

P38 : C14 8.68 � 0.23a 4.34 � 0.24 2.08 � 0.05b 1.57 � 0.20 9.19 � 0.63

The different letters in the same column indicate significant difference (P < 0.05).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

Shrimp fed P34 : C19 had the highest crude body pro-

tein and was significantly higher than those fed other three

diets (P < 0.05, Table 3). The crude body lipid of shrimp

fed P34 : C19 was also the highest and significantly higher

than that in the P38 : C14 group (P < 0.05), but there were

no differences between other two groups. No significant

differences were found in moisture and ash content among

all the treatments.

Shrimp fed P26 : C25 had the highest level of haemol-

ymph glucose, which was significantly higher than the

shrimp fed P26 : C30 or P38 : C14 (P < 0.05, Table 4). No

differences in haemolymph glucose between P30 : C25 and

(a1) (a2)

(b1) (b2)

(c1)

(d1)

(c2)

(d2)

Figure 1 Changes of hepatopancreas histology in juvenile white shrimp fed different diets at 2009 (left) and 4009 (right) magnifications.

The dietary protein to carbohydrate ratios were (a) P26 : C30, (b) P30 : C25, (c) fed P34 : C19 and (d) fed P38 : C14. Shrimp hepatopan-

creas is composed of many hepatopancreas tubules with four dominant types, namely E (embryonalzellen or embryonic) cells, R (restzellen)

cells, F (fibrillenzellen or fibrous) cells and B (blasenzellen) cells.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

P26 : C30 were found. Shrimp fed P26 : C30 had the high-

est level of glycogen in muscle, which were significantly

higher than those fed other diets (P < 0.05), although no

significant differences were found between other groups.

Shrimp fed P26 : C30 had the highest level of hepatopan-

creas glycogen, but no differences were found in hepato-

pancreas glycogen between all feeding groups. Pyruvate

kinase activities and hexokinasse activities were not signifi-

cantly affected by the CBH to protein ratio.

The mean number of B cells in hepatopancreas tubules

in shrimp fed P26 : C30 was 5–6 and was significantly

lower than that in shrimp fed other three diets. Shrimp fed

P30 : C25 had the most abundant R cells nearly 6–7 in the

hepatopancreas tubules, which was significantly higher than

shrimp fed other diets (P < 0.05, Fig. 1).

After the 96-h ammonia nitrogen challenge, the survival

rate of the shrimp fed P34 : C19 was highest, followed by

that of shrimp fed P30 : C25 and P38 : C14. The lowest

survival rate was found in shrimp fed P26 : C30, but no

significant differences in shrimp survival rate were observed

between treatments (Fig. 2).

Liu et al. (2005) and Li et al. (2001) reported the protein

requirement for optimum growth of L. vannamei at low

salinity (3 g L�1) is 400 to 441 g kg�1. The inclusion of

high levels of dietary protein can improve the osmoregula-

tory capacity of L. vannamei with more energy intake at

low salinity (Li et al. 2011). However, in the present study,

when shrimp were fed on a lower protein diet (P34 : C19),

the weight gain seemed higher than those fed on a higher

protein diet (P38 : C14), although no significant difference

was found between these two groups. There were also no

differences between P30 : C25 and P38 : C14 group in

weight gain, indicating that protein spare effect by CBH

has occurred in shrimp fed the P30 : C25 diet. Similarly,

Cruz-Suarez et al. (1994) found that soft wheat meal in the

diet of Penaeus monodon also provoked protein-sparing

effect. Shiau & Peng (1993) found that the decrease of die-

tary protein from 280 g kg�1 to 240 g kg�1 by increasing

the dietary starch or dextrin from 370 g kg�1 to 410 g kg�1

did not reduce weight gain and feed efficiency ratio (FER).

The diet containing 400 g kg�1 dextrin with 300 g kg�1

protein achieved the highest growth by sparing protein in

Cirrhinus mrigala fry (Singh et al. 2006).

Although the growth of shrimp fed P26 : C30 or

P30 : C25 was inferior to those fed P34 : C19, the survival

rate of the shrimp fed the diet over 250 g kg�1 CBH was

higher than that of shrimp fed <190 g kg�1 dietary CBH.

Shrimp fed P26 : C30 showed the highest survival rate and

was significantly higher than shrimp in other three groups.

This result differed from a previous study on crustacean

that the optimum CBH level for the best survival rate of

L. vannamei at low salinity was 20% (Guo et al. 2011). Li

et al. (2011) recommended that the increase of dietary pro-

tein could improve the osmoregulatory capacity of L. van-

namei by providing more energy and amino acids. It is

possible that the protein content in the P26 : C30 diet can-

not meet the protein requirement of L. vannamei. Although

shrimp can utilize dietary CBH for growth, the lowest

weight gain was found in shrimp fed a low-protein diet.

The survival rate showed an increasing trend with the

increase of dietary CBH, indicating the importance of die-

tary CBH to improve L. vannamei survival at low salinity.

As CBH is the primary source of energy for shrimp in nat-

ure (Lehninger 1978), the high dietary CBH may meet the

high-energy demand of aquatic animals in a stress condi-

tion (Tseng & Hwang 2008; Wang et al. 2012). Therefore,

when the basic level of protein has met the demand of

shrimp, the addition of high CBH can improve L. vanna-

mei survival at low salinity. However, the relationship

between protein and CBH on growth of shrimp is not fully

understood and needs further investigation.

Although a high-protein diet can improve the growth of

L. vannamei at low salinity (Li et al. 2011), this study

revealed the best protein–CBH ratio for L. vannamei

growth. The shrimp fed the P34 : C19 diet showed the

highest contents of crude body protein and lipid, suggesting

that the dietary protein to CBH ratio of 34 : 19 can meet

Figure 2 The survival rate of shrimp fed different diets containing

glucose, sucrose, wheat starch, corn starch and potato starch dur-

ing a 96-h ammonia challenge. Vertical bars represent SE.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

the requirements of both energy and protein for L. vanna-

mei at low salinity. The shrimp fed P26 : C30 had the high-

est muscle glycogen and were significantly higher than

those fed other diets. The excess dietary CBH that is not

used for energy may contribute to lipid and glycogen depo-

sition in shrimp after biochemical conversion (Hutchins

et al. 1998; Gumus & Ikiz 2009). Shrimp fed P30 : C25

and P34 : C19 contained higher haemolymph glucose con-

tents than those fed P30 : C30 or P38 : C14, implying that

glucose mobilization in these two groups exceeds the

amount of glucose required in the glycolytic pathway,

which can lead to the accumulation of glucose in haemol-

ymph of shrimp as reported in other studies (Anderson

et al. 1994; Zou et al. 1996; Hervant et al. 1997).

Although pyruvate kinase and hexokinase activities are

the key enzymes in the glycolysis process, no differences of

these two enzymes were found between all treatments, indi-

cating that the glycolysis pathway is not affected by the

dietary ratio of CBH and protein at low salinity. Similarly,

the hepatic hexokinase activities in rainbow trout, gilthead

seabream, gibel carp and common carp are not affected by

the level of starch in diet (Panserat et al. 2000; Tan et al.

2009). But in European sea bass and gibel carp, the hepatic

pyruvate kinase activity increased when fish fed on diets

with high starch (Enes et al. 2006; Tan et al. 2009). In pre-

vious studies, the CBH metabolism is involved in osmoreg-

ulation of aquatic animals (Charmantier-Daures et al.

1988; Spanings-Pierrot et al. 2000; Morris 2001), but the

mechanism of CBH for osmoregulation in crustacean war-

rants further study.

In this study, the number of B cells in hepatopancreas

tubules of shrimp fed P26 : C30 was significantly lower

than those fed other three diets. This may be related to the

B-cell function as the B cell is the main site for nutrient

absorption and digestion (Al-Mohanna & Nott 1989; Li

et al. 2008). The low rate of synthesis and the release of

digestive and antioxidant enzymes may be indicative of the

relatively simple metabolic process. Because CBH is an

important source of dietary energy for omnivorous aquatic

animals (Welcomme & Devos 1991; Arasta et al. 1996;

Tseng & Hwang 2008; Wang et al. 2012), it can possibly

supply the energy demand for shrimp, especially when os-

morelation requires more energy at low salinity. Further-

more, shrimp fed P30 : C25 had the most abundant R cells

in the hepatopancreas tubules. As the R cell is the main

site for nutrient reserve in hepatopancreas (Al-Mohanna &

Nott 1987, 1989), the increase of R cell number might be

due to the excess energy supply in the P30 : C25 diet,

which can lead to the protein-sparing effect in shrimp.

As the optimal salinity for the growth of L. vannamei is

around 20 g L�1 (Huang 1983; Li et al. 2007), the white

shrimp at a lower salinity is more susceptible to ambient

toxicants, such as ammonia (Lin & Chen 2001; Li et al.

2007), boron (Li et al. 2008), nickel (Leonard & Barcaolli

2011), beta-cypermethrin and acephate (Wang et al. 2013).

Extremely low salinity (<5 g L�1) can exert severe stress on

L. vannamei, and more energy is needed for osmoregula-

tion. After shrimp were challenged with ammonia nitrogen

for 96-h, shrimp fed the C19 : P34 had the highest survival,

suggesting that under hypo-osmotic stress, the dietary pro-

tein to CBH ratio can affect the efficiency of energy utiliza-

tion and shrimp survival.

Overall, the protein-sparing effect of CBH in shrimp was

pronouncedly shown by feeding on the P30 : C25 and

P34 : C19 diets. Under the basic demand for protein, the

addition of appropriate CBH can improve L. vannamei sur-

vival at low salinity. The best ratio of protein to CBH for

the optimum growth is recommended at P34 : C19. This

ratio can meet both energy and protein requirements of

L. vannamei at low salinity and result in the best growth

and alleviation of ammonia stress on shrimp at low salin-

ity.

This research was supported by grants from the Special

Fund for Agro-scientific Research in the Public Interest

(No. 201003020, 201203065), National ‘Twelfth Five-Year’

Plan for Science & Technology Support (2012BAD25B03),

the National Basic Research Program (973 Program, No.

2009CB118702), Shanghai Committee of Science and Tech-

nology, China (10JC1404100), National Natural Science

Foundation of China (No. 31172422, 31001098), Special-

ized Research Fund for the Doctoral Program of Higher

Education of China (No. 20100076120006), Shanghai Uni-

versity Knowledge Service Platform Shanghai Ocean Uni-

versity Aquatic Animal Breeding Centre (ZF1206), and

partly by the E-Institute of Shanghai Municipal Education

Commission (No. E03009) and ECNU innovation fund.

Al-Mohanna, S.Y. & Nott, J.A. (1987) R-cells and the digestive

cycle in Penaeus semisulcatus (Crustacea:Decapoda). Mar. Biol.,

95, 129–137.Al-Mohanna, S.Y. & Nott, J.A. (1989) Functional cytology of the

hepatopancreas of Penaeus semisulcatus (Crustacea: Decapoda)

during molt cycle. Mar. Biol., 102, 535–544.Anderson, S.J., Taylor, A.C. & Atkinson, J.A. (1994) Anaerobic

metabolism during anoxia in the burrowing shrimp Calocaris

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

macandreae Bell (Crustacea: Thalassinidea). Comp. Biochem.

Physiol., 108, 515–522.AOAC (Association of Official Analytical Chemists) (1990) Asso-

ciation of Official Analytical Chemists. Arlington, VA.

Arasta, T., Bais, V.S. & Thakur, P. (1996) Effect of Nuvan on

some biological parameters of Indian catfish, Mystus vittatus. J.

Environ. Biol., 17, 167–169.Brito, S.R.C., Moura, M.A.F., Kawashita, N.H., Brito, M.N.,

Kettelhut, I.C. & Migliorini, R.H. (2001) Glucose uptake and

glycolytic flux in adipose tissue from rats adapted to a high pro-

tein, carbohydrate-free diet. Metabolism, 50, 1208–1212.CFSY (2011) China Fishery Statistical Yearbook. China Agricul-

ture Press, Beijing, China.

Charmantier-Daures, M.N., Bouaricha, P. & Thuet, D.E. (1988)

Ontogeny of osmoregulation and salinity tolerance in two deca-

pod crustaceans: Homarus americanus and Penaeus japonicus.

Biol. Bull., 175, 103–110.Cheng, K.M., Hu, C.Q., Liu, Y.N., Zheng, S.X. & Qi, X.J.

(2006) Effects of dietary calcium, phosphorus and calcium/

phosphorus ratio on the growth and tissue mineralization of

Litopenaeus vannamei reared in low-salinity water. Aquaculture,

251, 472–483.Cho, C.Y. & Kaushik, S.J. (1990) Nutrition energetics in fish:

energy and protein utilization in rainbow trout (Salmo gairdneri).

World Rev. Nutr. Diet., 61, 132–172.Cruz-Suarez, L.E., Ricque-Marie, D., Pinal-Mansilla, J.D. &

Wesche-Ebelling, P. (1994) Effect of different carbohydrate

sources on the growth of P. vannamei: economic impact. Aqua-

culture, 123, 349–360.Cuzon, G., Lawrence, A., Gaxiola, G., Rosas, C. & Guillaume, J.

(2004) Nutrition of Litopenaeus vannamei reared in tanks or in

ponds. Aquaculture, 235, 513–551.Diaz, F. & Farfan, C. (2001) Effects of temperature and salinity

fluctuation on the ammonium excretion and osmoregulation of

juveniles of Penaeus vannamei, Boone. Mar. Freshw. Behav.

Physiol., 34, 93–104.Dubois, M.K., Lilles, L.A., Hamilton, J.C., Rebers, P.A. & Smith,

F. (1965) Colorimetric method for determination of sugars and

related substances. Anal. Chem., 28, 350–356.Enes, P., Panserat, S., Kaushik, S. & Oliva-Teles, A. (2006) Effect

of normal and waxy maize starch on growth, food utilization

and hepatic glucose metabolism in European sea bass (Dicentrar-

chus labrax) juveniles. Comp. Biochem. Physiol., 143, 89–96.Feska, L.R., Corn�elio, A.R., Dutra-Filho, C.S., Wyse, A.T.S.,

Wajner, M. & Wannmacher, C.M.D. (2003) Characterization of

inhibition of pyruvate kinase caused by phenylalanine and phe-

nylpyruvate in rat brain cortex. Brain Res., 968, 199–205.Fry, F.E.J. (1971) The effect of environmental factors on the phys-

iology of fish. In: Fish Physiology Vol. VII: Environmental Rela-

tions and Behaviour (Hoar, W.S. & Randall, D.J. eds), pp. 1–98. Academic Press, New York, NY.

Garling, D.L. & Wilson, R.P. (1976) Optimum dietary protein to

energy ratio for channel catfish fingerlings, Ictalurus punctatus. J.

Nutr., 106, 1368–1375.Gumus, E. & Ikiz, R. (2009) Effect of dietary levels of lipid and

carbohydrate on growth performance, chemical contents and

digestibility in rainbow trout. Pak. Vet. J., 29, 59–63.Guo, R., Liu, Y.J., Tian, L.X., Xia, H. & Wang, J.Q. (2011)

Effects of dietary cornstarch levels on fat metabolism of hepato-

pancrease in Litopenaeus vannamei (in Chinese with English

Abstract). Acta Scientiarum Naturalium Un., 50, 105–109.Hervant, F., Mathieu, J. & Messana, G. (1997) Locomotory, venti-

latory and metabolic responses of the subterranean Stenasellus

virei (Crustacea, Isopoda) to severe hypoxia and subsequent

recovery. C. R. Acad. Sci. III, Sci. Vie, 320, 139–148.Hidalgo, M.C., Sanz, A., Garcia Gallego, M., Saurez, M.D. & de

la Higuera, M. (1993) Feeding of the European eel Anguilla

anguilla. I. Influence of dietary carbohydrate level. Comp. Bio-

chem. Physiol., 105A, 165–169.Huang, H.J. (1983) Factors affecting the successful culture of

Penaeus stylirostris and Penaeus vannamei at an estuarine power

plant site: temperature, salinity, inherent growth variability,

damselfly nymph predation, population density and distribution,

and polyculture. PhD dissertation. Texas A&M University, Col-

lege Station, TX, USA, 221 pp.

Hutchins, C.G., Rawles, S.D. & Gatlin, D.M. III (1998) Effects of

dietary carbohydrate kind and level on growth, body composi-

tion and glycemic response of juvenile sunshine bass (Morone

chrysops ♀ 9 M. saxatilis ♂). Aquaculture, 161, 187–199.Kinne, O. (1971) Salinity: animal invertebrates. In: Marine Ecol-

ogy Vol. 1: Environmental Factors. (Kinne, O. ed.), pp. 821–995. Wiley Interscience, London.

(Lehninger, A.L. ed.) (1978) Biochemistry. Kalyani, Ludhiana,

New Delhi.

Leonard, E.M. & Barcaolli, I. (2011) The effects of salinity on

acute and chronic nickel toxicity and bioaccumulation in two

euryhaline crustaceans: Litopenaeus vannamei and Excirolana ar-

mata. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 154, 409–419.

Li, G.L., Zhu, C.H. & Zhou, Q.C. (2001) Effects of dietary protein

level on the growth of Peanaeus vannamei. Mar. Sci., 25, 1–4.Li, E.C., Chen, L.Q., Zeng, C., Chen, X.M., Yu, N., Lai, Q.M. &

Qin, J.G. (2007) Growth, body composition, respiration and

ambient ammonia nitrogen tolerance of the juvenile white

shrimp, Litopenaeus vannamei, at different salinities. Aquaculture,

265, 385–390.Li, E.C., Chen, L.Q., Zeng, C., Yu, N., Xiong, Z.Q., Chen, X.F.

& Qin, J.G. (2008) Comparison of digestive and antioxidant

enzymes activities, haemolymph oxyhemocyanin contents and

hepatopancreas histology of white shrimp, Litopenaeus vannamei,

at various salinities. Aquaculture, 274, 80–86.Li, E.C., Arena, L., Lizama, G., Gzxiola, G., Cuzon, G., Rosas,

Carlos., Chen, L.Q. & Van Wormhoudt, A. (2011) Glutamate

dehydrogenase and Na+-K+ ATPase expression and growth

response of Litopenaesus vannamei to different salinities and die-

tary protein levels. Chin. J. Oceanol. Limnol., 29, 343–349.Lin, Y.C. & Chen, J.C. (2001) Acute toxicity of ammonia on

Litopenaeus vannamei Boone juveniles at different salinity levels.

J. Exp. Mar. Biol. Ecol., 259, 109–119.Lin, Y.C. & Chen, J.C. (2003) Acute toxicity of nitrite on Litope-

naeus vannamei (Boone) juveniles at different salinity levels.

Aquaculture, 224, 193–201.Liu, D.H., He, J.G. & Liu, Y.J. (2005) Effects of dietary protein

levels on growth performance and immune condition of pacific

white shrimp Litopenaeus vannamei juveniles at very low salinity.

Acta Scien. SYSU., 44, 217–223.Morris, S. (2001) Neuroendocrine regulation of osmoregulation

and the evolution of air-breathing in decapod crustaceans.

J. Exp. Biol., 204, 979–989.Panserat, S., Medale, F., Blin, C., Breque, J., Vachot, C., Plagnes-

Juan, E., Gomes, E., Krishnamoorthy, R. & Kaushik, S. (2000)

Hepatic glucokinase is induced by dietary carbohydrates in rain-

bow trout, gilthead seabream, and common carp. Am. J. Phys-

iol., 278, 1164–1170.Pante, M.J.R. (1990) Influence of environmental stress on the heri-

tability of molting frequency and growth rate of the penaeid

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd

shrimp, Penaeus vannamei. University of Houston-Clear Lake,

Houston, TX, USA, M.Sc. Thesis.

Peres, H., Lim, C. & Klesius, P.H. (2003) Nutritional value of

heat-treated soybean meal for channel catfish (Ictalurus puncta-

tus). Aquaculture, 225, 67–82.Pieper, A. & Pfeffer, E. (1980) Studies on the effects of increasing

proportion of sucrose or gelatinized maize starch in diet for rain-

bow trout (Salmo gairdneri, R) on the utilization of dietary

energy and protein. Aquaculture, 20, 333–342.Roy, L.A., Davis, D.A., Saoud, I.P., Boyd, C.A., Pine, H.J. &

Boyd, C.E. (2010) Shrimp culture in inland low salinity waters.

Rev. Aquacult., 2, 191–208.Santos, E.A. & Nery, L.E.M. (1987) Blood glucose regulation in an

estuarine crab, Chasmagnathus granulata (Dana, 1851) exposed

to different salinities. Comp. Biochem. Physiol., 87, 1033–1035.Saoud, I.P., Davis, D.A. & Rouse, D.B. (2003) Suitability studies

of inland well waters for Litopenaeus vannamei culture. Aquacul-

ture, 217, 373–383.Shiau, S.Y. & Peng, C.Y. (1993) Protein sparing effect of carbohy-

drates in diets for tilapia, Oreochromis niloticus 9 O. aureus.

Aquaculture, 117, 327–334.Shiau, S.Y., Lin, S.F. & Lu, L.J. (1991) Effects of different types

of wheat flour in feeds for grass prawn Penaeus monodon. Nip-

pon Suisan Gakkai Shi, 57, 705–710.Singh, R.K., Balange, A.K. & Ghughuskar, M.M. (2006) Protein

sparing effect of carbohydrates in the diet of Cirrhinus mrigala

(Hamilton, 1822) fry. Aquaculture, 258, 680–684.Spaargaren, D.H. & Mors, R. (1985) The effect of environmental

osmotic conditions on blood lipid levels of the shore crab, Carci-

nus maenas. Comp. Biochem. Physiol. A Physiol., 80, 579–585.

Spanings-Pierrot, C., Soyez, D. & Van, H.F. (2000) Involvement

of crustacean hyperglycemic hormone in the control of gill of

the green crab Pachygrapsus marraoratus. Gen. Comp. Endocri-

nol., 119, 340–350.Tan, Q.S., Wang, F., Xie, S.Q., Zhu, X.M., Lei, W. & Shen, J.Z.

(2009) Effect of high dietary starch levels on the growth

performance, blood chemistry and body composition of gibel

carp (Carassius auratus var. gibelio). Aquacult. Res., 40, 1011–1018.

Tseng, Y.C. & Hwang, P.P. (2008) Some insights into energy

metabolism for osmoregulation in fish. Comp. Biochem. Physiol.

C Toxicol. Pharmacol., 148, 419–429.Wang, Y.R., Li, E.C., Yu, N., Wang, X.D., Cai, C.F., Tang, B.,

Chen, L.Q. & Wormhoudt, A.V. (2012) Characterization and

expression of glutamate dehydrogenase in response to acute

salinity stress in the Chinese mitten crab, Eriocheir sinensis.

PLoS ONE, 7, e37316.

Wang, X.D., Li, E.C., Xiong, Z.Q., Chen, K., Yu, N., Du, Z.Y. &

Chen, L.Q. (2013) Low salinity decreases the tolerance to two

pesticides, beta-cypermethrin and acephate, of white-leg shrimp,

Litopenaeus vannamei. J. Aquacult. Res. Dev., 4, 190.

Welcomme, L. & Devos, P. (1991) Energy consumption in the

perfused gills of the euryhaline crab Eriocheir sinensis [H. Man.

Edw.] adapted to freshwater. J. Exp. Zool., 25, 150–159.Zou, E., Du, N. & Lai, W. (1996) The effects of severe hypoxia on

lactate and glucose concentration in the blood of the Chinese

freshwater crab Erioccheir sinensis (Crustacea, Decapoda).

Comp. Biochem. Physiol., 114, 105–109.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Aquaculture Nutrition ª 2014 John Wiley & Sons Ltd