Aquaculture 434 (2014) 340347

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Aquaculture
journal homepage: www.elsevier.com/locate/aqua-online

Effects of dietary choline supplementation on growth performance

and hepatic lipid transport in blunt snout bream
(Megalobrama amblycephala) fed high-fat diets
Jun-yi Li, Ding-dong Zhang, Wei-na Xu, Guang-zhen Jiang, Chun-nuan Zhang, Xiang-fei Li, Wen-bin Liu
Key Laboratory of Aquatic Animal Nutrition and Feed Science of Jiangsu Province, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, China

a r t i c l e

i n f o

Article history:
Received 7 April 2014
Received in revised form 26 July 2014
Accepted 5 August 2014
Available online 13 August 2014
Keywords:
Choline supplementation
High-fat diets
Growth
Lipid transport
Blunt snout bream

a b s t r a c t
In this study, we elucidated the choline metabolic pathways and its effects on growth and hepatic lipid transport
in blunt snout bream, Megalobrama amblycephala. Fish were fed six experimental diets containing three lipid
levels (5%, 8%, and 11%) and two choline supplementation levels (1200 and 1800 mg kg1) for 8 weeks. After
the feeding trial, viscera/body ratio, hepatosomatic index, plasma aspartate aminotransferase activities, and
plasma triglyceride and cholesterol contents as well as liver lipid content all increased signicantly (P b 0.05)
as dietary lipid levels increased from 5 to 11% in terms of dietary lipid levels, whereas feed conversion ratio,
plasma cholesterol content and liver lipid content decreased signicantly (P b 0.05) with increasing choline supplementations. Weight gain increased signicantly (P b 0.05) with increasing dietary choline supplementations
when dietary lipid levels reached 11%. Additionally, hepatic lipid accumulation was further observed by oil red
O staining. An increase in hepatic S-adenosylmethionine was found in sh fed diets supplemented with
1800 mg kg1 choline, indicating that extra choline supplementation increases very low density lipoprotein
(VLDL) production through methylation of phosphatidylethanolamine to synthesize phosphatidylcholine.
Additionally, increased SAM production resulted in a subsequent increase in glutathione concentration, suggesting
a possible benet in mitigating oxidative stress induced by a high-fat diet. 1800 mg kg1 choline supplementation
in diet signicantly up-regulated the relative mRNA expression levels of apolipoprotein B-100 and microsomal
triglyceride transfer protein, which induced VLDL synthesis and assembly.
2014 Elsevier B.V. All rights reserved.

1. Introduction
Choline is an essential nutrient that is primarily provided by the diet.
It plays a pivotal role in maintaining cell structure and movement of
lipids in and out of the cells (Blusztajn, 1998). It is a precursor to phosphatidylcholine (Mehta et al., 2010), which is an important constituent
of lipoproteins. Moreover, choline (after it has been irreversibly
oxidized to betaine by a two-stage reaction) is a provider of free methyl
groups (Simon, 1999), which act in methylation. Previous studies with
several species of sh showed or indicated that dietary choline is required for optimum growth as well as for preventing lipid deposition
(Hung, 1989; Criag and Gatlin, 1997; Jiang et al., 2013). Hence, the
choline metabolic pathways and the effects on lipid metabolism in the
liver of sh need to be understood.
Under normal physiological conditions, there is a balance between
lipid deposition and transportation to sustain lipid homeostasis of

Corresponding author at: Nanjing Agricultural University, Weigang No. 1, Nanjing

210095, Jiangsu Province, China. Tel./fax: +86 25 84395382.
E-mail address: wbliu@njau.edu.cn (W. Liu).

http://dx.doi.org/10.1016/j.aquaculture.2014.08.006
0044-8486/ 2014 Elsevier B.V. All rights reserved.

the liver. Very low dense lipoprotein (VLDL) delivers endogenous and
dietary lipids from the liver to peripheral tissues for storage or use
(Davis et al., 1979). VLDL is a complex particle consisting of a core of neutral lipids (mostly triglycerides) surrounded by a monolayer of amphipathic lipids, such as phospholipids (Olofsson et al., 2000). Hence, the
process of VLDL production involves the biosynthesis of phosphatidylcholine, which is derived from free choline molecules (Kennedy and Weiss,
1956), as well as a stepwise methylation of phosphatidylethanolamine
(PEMT pathway) (Bremer and Greenberg, 1961; Vance and Ridgway,
1988). The latter synthetic pathway requires S-adenosylmethionine
(SAM), which functions as a direct methyl donor (Blusztajn et al., 1985).
Additionally, apolipoprotein B-100 (apoB-100) is the major apolipoprotein in VLDL secreted exclusively from the liver (Brodsky and Fisher,
2008). Microsomal triglyceride transfer protein (MTTP) is an intracellular
protein that enhances the rate of neutral lipid transfer (Hussain et al.,
2003; Wetterau et al., 1997) located within the endoplasmic reticulum
of apoB-containing lipoprotein-secreting cells, predominantly hepatocytes and intestinal enterocytes (Raabe et al., 1999; Wetterau et al.,
1997), thus plays a vital role in the control of VLDL synthesis, assembly,
and secretion (Shindo et al., 2010) and are closely associated with hepatic
lipid transport. Although choline is regarded as a lipotropic agent in some

J. Li et al. / Aquaculture 434 (2014) 340347

species of animals, our knowledge about the link between dietary choline
and lipid metabolism in sh is limited.
Blunt snout bream (Megalobrama amblycephala) is a major herbivorous freshwater sh native to China where it is widely favored due to its
tender esh, high survival rate, fast growth, use of natural food, and high
disease resistance (Zhou et al., 2008). Like other herbivorous freshwater
sh, blunt snout bream cannot tolerate high dietary lipids, and excess
lipids may cause lipid deposition in the liver (Arzel et al., 1994; Lee
et al., 2002; Stephan et al., 1996; Stowell and Gatlin, 1992), which
leads to impaired liver function and metabolic and physiological
abnormities (Lu et al., 2013a,b) and reduced resistance to disease (Li
et al., 2012). Bearing this in mind, more information on the effects of
dietary choline supplementation in blunt snout bream fed a diet with
different lipid levels could be important for commercial production.
The principal goal of this study is to elucidate the metabolic pathway
of choline and the effects of choline on growth and hepatic lipid transport in blunt snout bream in order to further understand how choline
supplementation attenuates the harmful effects induced by high-fat
diets in this sh species.
2. Materials and methods
2.1. Experimental diets
A 3 2 factorial design with four replicates was used in this study.
Six isonitrogenous (320 g kg 1 crude protein) semi-puried diets
were formulated to contain three lipid levels (5%, 8%, and 11%) and
two choline supplementation levels (1200 and 1800 mg kg1). Dietary
protein was supplied by casein, gelatin, and sh meal. Soybean oil was
used as the lipid source. The diets were formulated to meet the optimal
dietary protein, lipid and choline requirements of juvenile blunt snout
bream (Li et al., 2010; Jiang et al., 2013). The experimental diets were
formulated with a choline-free vitamin premix. Choline chloride
(Sigma, St. Louis, MO, USA) was supplemented in the basal diet to formulate experimental diets containing 1195 and 1782 mg kg1 choline
(Venugopal, 1985). Formulation and proximate composition of the
experimental diets are presented in Table 1. All diets were prepared in
the laboratory. Ingredients were nely ground, well mixed, and pelletized through a pellet machine. After drying, all diets were stored at
20 C in plastic bags until use.
2.2. Experimental sh and feeding trial
Juvenile blunt snout bream (M. amblycephala) were obtained from
the sh hatchery of Yangzhou (Jiangsu, China). Prior to the experiment,
sh were kept in an indoor circulatory system to acclimate to the experiment conditions by feeding a diet which contains 5% lipid for one
month. After a one-month acclimation, sh of similar sizes (average initial weight: 9.80 0.35 g) were randomly distributed into 24 tanks
(300 L each) at a rate of 30 sh per tank. Fish in each aquarium were
randomly assigned one of six experimental diets. Each diet was tested
in four replicates. Fish were hand-fed to apparent satiation three times
daily (08:00, 12:00 and 17:00 h) for 8 weeks. A 12:12 h light:dark
regime (07:3019:30 h, light period) was maintained by timed uorescent lighting. Water temperature varied from 25 to 28 C, and pH
uctuated between 7.2 and 7.6. Ammonia nitrogen and nitrite nitrogen
were maintained below 0.4 mg/L and 0.064 mg/L, respectively.
Dissolved oxygen was maintained above 6.00 mg L1 during the feeding trial.
2.3. Sample collection
At the end of the feeding trial, sh were starved for 24 h to evacuate
alimentary tract contents prior to harvest. Then, sh were euthanized
by MS-222 (tricaine methanesulfonate, Sigma, USA) at a concentration
of 100 mg L1. No mortalities were observed during the experiment.

341

Table 1
Ingredient and proximate composition of experimental diets.
Choline supplementation mg kg1
1200

1800

Dietary lipid level %

5

11

11

Ingredient composition %
Caseina
Gelatinb
Fish mealb
Soybean oilc
Corn starchb
Cellulosed
Vitamin and mineral premixe
Calcium biphosphated
Sodium chloride
Carboxymethyl cellulosed
Choline chloride

26.0
6.50
5.00
4.70
39.0
13.0
1.20
1.80
0.50
2.00
0.29

26.0
6.50
5.00
7.80
39.0
10.0
1.20
1.80
0.50
2.00
0.29

26.0
6.50
5.00
10.9
39.0
7.00
1.20
1.80
0.50
2.00
0.29

26.0
6.50
5.00
4.70
39.0
13.0
1.20
1.80
0.50
2.00
0.45

26.0
6.50
5.00
7.80
39.0
10.0
1.20
1.80
0.50
2.00
0.45

26.0
6.50
5.00
10.9
39.0
7.00
1.20
1.80
0.50
2.00
0.45

Proximate composition %
Moisture
Crude protein
Crude lipid
Ash
Crude ber
Nitrogen-free extractf
Methionine

13.0
32.5
5.22
4.71
12.2
32.4
0.76

13.2
32.9
8.07
4.80
9.41
31.6
0.75

13.0
33.0
11.2
5.09
7.36
30.4
0.76

13.2
32.9
5.21
4.98
12.8
31.1
0.76

12.9
32.7
8.11
5.14
9.82
31.3
0.76

13.1
32.5
11.0
4.67
6.88
31.9
0.76

Provided by Hulunbeier Sanyuan Milk Co., Ltd (Inner Mongolia, China).

Provided by Zhengchang Feed Industry Co., Ltd (Huaian, China).
c
Purchased from Coland Feed Industry Co., Ltd (Wuhan, China).
d
Provided by Lanping Industry Co., Ltd (Shanghai, China).
e
Premix supplied the following minerals (g/kg) and vitamins (IU or mg/kg):
CuSO45H2O 2.0 g, FeSO47H2O 25 g, ZnSO47H2O 22 g, MnSO44H2O 7 g, Na2SeO3
0.04 g, KI 0.026 g, CoCl26H2O 0.1 g, VA 900 000 IU, VD 200 000 IU, VE 4500 mg, VK3
220 mg, VB1 320 mg, VB2 1090 mg, VB6 500 mg, VB12 1.6 mg, VC 10000 mg, pantothenate
1000 mg, folic acid 165 mg, niacin 2500 mg, biotin 100 mg, myoinositol 150 mg.
f
Calculated by difference (100 moisture crude protein crude lipid ash
crude ber).
b

Blood sample was rapidly taken from caudal vessel into heparinized
Eppendorf tubes and centrifuged at 3000 rpm (850 g) for 10 min at
4 C. The supernatant was stored at 70 C until analysis. After blood
collection, the total number and weight of sh in each cage were determined. Six sh were randomly sampled from each tank for analysis of
body weight and length, viscera/body ratio and hepatosomatic index.
Also, individual liver from fteen sh in each tank was quickly removed
and stored at 70 C for subsequent analysis.
2.4. Analysis
2.4.1. Proximate composition analysis
The proximate composition of diets was analyzed following the standard methods (A.O.A.C., 1995). Moisture was determined by oven drying
until constant weight (105 C); crude protein (nitrogen 6.25) was
determined by the Kjeldahl method using an Auto Kjeldahl System
(FOSS KT260, Switzerland); crude lipid by ether-extraction using Soxtec
System HT (Soxtec System HT6, Tecator, Sweden); ash by combustion at
550 C for 4 h; crude ber by fritted glass crucible method using an automatic analyzer (ANKOM A2000i, USA) and gross energy by an adiabatic
bomb calorimeter (PARR 1281, USA). Liver lipid was extracted with a
chloroform: methanol (2:1 V:V) mixture according to Folch et al. (1957).
2.4.2. Measures of plasma biochemical parameters
Activities of plasma aspartate aminotransferase (AST) and alanine
aminotransferase (ALT) were both measured following the methods
detailed by Rietman and Frankel (1957). Concentrations of triglycerides
(TG) and cholesterol (T-CHO) in plasma were determined by colorimetric enzymatic methods using commercial kits (Beijing BHKT Clinical
Reagent Co., 101 Ltd, China). Plasma glucose level was measured by
the glucose oxidase method as described by Asadi et al. (2009).

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J. Li et al. / Aquaculture 434 (2014) 340347

2.4.3. Analysis of hepatic glutathione (GSH) and glutathione disulde (GSSG)

concentrations, S-adenosylmethionine (SAM) and S-adenosylhomocysteine
(SAH) levels
GSH and GSSG concentrations were all measured according to the
methods described by Lygren et al. (1999). Liver tissue was homogenized
in 1.15% KCl, and then added to one volume of 10% trichloroacetic acid.
The homogenate was incubated on ice for 15 min, vortexed and centrifuged at 10000 rpm (9500 g) for 20 min. The supernatant was passed
through a 0.45 m lter and then applied to a high-performance liquid
chromatography (HPLC) column. The contents of SAM and SAH were
measured as described previously (She et al., 1994).
2.4.4. Oil red O staining
After in vivo xed by 4% paraformaldehyde and removed from body,
liver tissue was embedded in OCT, snap-frozen in liquid nitrogen, and
stored at 80 C. Frozen liver tissue was sectioned (5 m) and committed to neutral lipid staining with oil red O (Direct Red 80; Aldrich,
Milwaukee, WI, USA) following standard procedure (Gao et al., 2010).
2.4.5. Total RNA extraction, reverse transcription and real-time PCR
Total RNA was extracted from the liver tissue using Trizol
(Invitrogen, CA, USA). RNA samples were treated by RQ1 RNase-Free
DNase prior to RT-PCR (Takara Co. Ltd, Japan) to avoid genomic DNA
amplication. cDNA was generated from 500 ng DNase-treated RNA
using an ExScript RT-PCR kit (Takara Co. Ltd, Japan), and the mixture
consisted of 500 ng RNA, 2 l buffer (5), 0.5 l dNTP mixture (10 mM
each), 0.25 l RNase inhibitor (40 U/l), 0.5 l dT-AP primer (50 mM),
0.25 l ExScript RTase (200 U/l), and DEPC H2O, with a total volume
up to 10 l. The reaction conditions were as follows: 42 C for 40 min,
90 C for 2 min, and 4 C thereafter. Real-time PCR was employed to
determine mRNA levels based on the SYBR Green I Fluorescence Kit.
Primers (Table 2) for the real-time PCR analysis were designed using
the Primer5 Software, based on the cDNA sequences available in
published papers. Real-time PCR was performed in a Mini Option realtime detector (BIO-RAD, USA). The uorescent quantitative PCR reaction solution consisted of 12.5 l SYBR premix Ex Taq (2), 0.5 l
PCR forward primer (10 M), 0.5 l PCR reverse primer (10 M), 2.0 l
RT reaction (cDNA solution), and 9.5 l dH2O. The reaction conditions
were as follows: 95 C for 3 min followed by 45 cycles consisting of
95 C for 10 s and 60 C for 20 s. The orescent ux was then recorded,
and the reaction continued at 72 C for 3 min. The dissolution rate was
measured between 65 and 90 C. Each increase of 0.2 C was maintained for 1 s, and the orescent ux was recorded. All amplicons
were initially separated by agarose gel electrophoresis to ensure that
they were of correct size. A dissociation curve was determined during
the PCR program to make sure that specic products were obtained in
each run. To calculate relative expression levels, the -actin gene of
blunt snout bream was used as internal control to normalize the Ct
value in each sample, and the relative expression levels under different
experimental diets were calculated by 2 Ct method.

variances were normally distributed, Tukey's test for multiple comparisons of means was applied. Differences were considered signicant at a
level of 95% (P b 0.05). All data are presented as mean of 4 tank replicates.
3. Results
3.1. Growth performance
Growth, feed utilization, and biometric parameters are presented in
Table 3. Both WG and condition factor were not affected by either lipid
levels or choline supplementation levels. VBR and HSI both increased
signicantly (P b 0.05) as dietary lipid levels increased from 5 to 11%,
whereas FCR decreased signicantly (P b 0.05) with increasing choline
supplementations. In addition, a signicant interaction between dietary
lipid and choline was observed in WG and FCR (P b 0.05). They both
showed little difference (P N 0.05) with increasing dietary choline
supplementations when dietary lipid was 5 and 8%. However, weight
gain increased signicantly (P b 0.05) with increasing choline supplementations from 1200 to 1800 mg kg 1 when dietary lipid level
reached 11%, whereas the opposite was true for FCR.
3.2. Plasma biochemistry parameters
Plasma parameters, indicating liver damage, and lipid metabolites
are presented in Table 4. Both ALT and glucose were not affected by either lipid levels or choline supplementation levels. AST activity, TG and
T-CHO content all increased signicantly (P b 0.01) as dietary lipid
levels increased from 5 to 11%, whereas T-CHO decreased signicantly
(P b 0.01) with increasing choline supplementations. Additionally,
they all showed no signicant (P N 0.05) interaction between dietary
lipid and choline levels.
3.3. Hepatic lipid content and GSH and GSSG concentrations
Hepatic lipid content and GSH and GSSG concentrations are presented in Table 5. Both GSH concentration and GSH/GSSG were not affected
by either lipid levels or choline supplementation levels. Liver lipid content increased signicantly (P b 0.01) as dietary lipid levels increased
from 5 to 11%, whereas liver lipid content decreased signicantly
(P b 0.01) with increasing choline supplementations. In addition, a
signicant interaction between dietary lipid and choline was observed
in liver lipid content and GSH concentration (P b 0.01). They both
showed little difference (P N 0.05) with increasing dietary choline
supplementations when dietary lipid was 5 and 8%. However, liver
lipid content decreased signicantly (P b 0.05) with increasing choline
supplementation levels from 1200 to 1800 mg kg 1 when dietary
lipid level reached 11%, whereas the opposite was true for GSH
concentration.
3.4. Hepatic SAM and SAH levels

2.5. Statistical analysis

All data were analyzed using the statistical software SPSS (version
16.0, Michigan Avenue, Chicago, IL, USA). Normality of the data and homogeneity of the variance were tested to ensure that the assumptions of
analysis of variance (ANOVA) were satised using Levene's tests. If the

Hepatic SAM and SAH levels are shown in Table 6. Both SAM levels
and SAM/SAH decreased signicantly (P b 0.05) as dietary lipid levels
increased from 5 to 11%. Additionally, hepatic SAM level signicantly
(P b 0.05) elevated with increasing choline supplementations with
the highest value in sh fed 11% lipid and 1800 mg kg 1 choline

Table 2
Primers used in the experiment.
Name

Target

Forward (53)

Reverse (53)

Fragment length (bp)

apoB-100
MTTP
-Actin

qPCR for apoB-100

qPCR for MTTP
qPCR for -actin

ATTCCAACAACACGGCATTT
TACAAGGCTACCAAACA
CGGACAGGTCATCACCATTG

GAGCAGTGGCAGTTTCATTT
AGACTTCCCACTGACG
CGCAAGACTCCATACCCAAGA

426
111
196

ApoB-100: apolipoprotein B-100; MTTP: microsomal triglyceride transfer protein.

J. Li et al. / Aquaculture 434 (2014) 340347

343

Table 3
Growth performance in blunt snout bream fed diets containing three levels of lipid with two choline supplementation levels.
Diets
Choline mg kg1

Lipid %

1200

5
8
11
5
8
11

1800

Pooled SEM

Weight gain (WG)1

Feed conversion ratio (FCR)2

Condition factor

Viscera/body ratio (VBR)3

Hepatosomatic index (HSI)4

115.30ab
139.18b
95.62a
118.47ab
122.83ab
145.44b
5.31

2.30ab
2.13a
2.73c
2.05a
2.38ab
2.01a
0.08

1.93
1.97
1.98
1.86
1.89
1.93
0.02

7.39
8.45
9.48
7.46
8.77
8.79
0.17

1.21
1.23
1.47
1.11
1.18
1.29
0.04

Two-way ANOVA
Dietary lipid
Choline supplementation
Interaction

0.427
0.192
0.022

0.588
0.036
0.014

0.335
0.052
0.915

0.000
0.726
0.342

0.049
0.144
0.700

Means and pooled SEM are presented for each parameter. Means in the same column with different superscripts are signicantly different (P b 0.05).
1
Weight gain (WG, %) = (Wt W0) 100/W0.
2
Feed conversion ratio (FCR, %) = feed consumption (g) / sh weight gain (g).
3
Viscera/body ratio (VBR, %) = (viscera weight 100) / wet body weight.
4
Hepatosomatic index (HSI, %) = liver weight (g) 100 / body weight (g).

supplementation. Hepatic SAH level was little signicantly (P N 0.05) affected by either lipid levels or choline supplementations.
3.5. Morphology of liver tissue
As shown in Fig. 1, frozen tissue sections of sh liver were stained
with oil red O. Signicant differences were observed between sh fed
experimental diets containing various levels of lipids and choline
supplementations. Small individual lipid droplets within the liver cells
appeared in sh with 5% and 8% dietary lipids (Fig. 1A, B, D, and E),
while a large number of lipid droplet clusters were seen in sh fed
diet with 11% lipid and 1200 mg kg1 choline supplementation
(Fig. 1C). In contrast, decreased clusters of lipid droplets were observed
in liver of sh fed with 11% lipid and 1800 mg kg1 choline supplementation (Fig. 1F).
3.6. Expressions of apolipoprotein B-100 (apoB-100) and microsomal
triglyceride transfer protein (MTTP) mRNA
ApoB-100 and MTTP mRNA expressions in the liver are presented in
Figs. 2 and 3, respectively. Both apoB-100 and MTTP mRNA expression
levels signicantly (P b 0.05) increased as dietary lipid levels increased
from 5 to 11%. Additionally, apoB-100 and MTTP mRNA expression
levels signicantly (P b 0.05) increased with increasing choline supplementations from 1200 to 1800 mg kg1. There is also a signicant
Table 4
Plasma biochemistry parameters in blunt snout bream fed diets containing three levels of
lipid with two choline supplementation levels.
Diets
Choline
mg kg1

Lipid
%

1200

5
8
11
5
8
11

1800

Pooled SEM

ALT
TG
T-CHO
Glucose
AST
(U L1) (U L1) (mmol L1) (mmol L1) (mmol L1)

2.99
3.05
4.61
3.13
2.90
3.99
0.17

0.77
0.51
1.18
0.65
0.78
0.82
0.06

1.36
1.17
2.07
1.38
1.01
1.67
0.11

1.29
1.58
2.21
0.98
1.01
1.65
0.09

3.36
3.98
4.90
3.50
3.86
3.88
0.17

Two-way ANOVA
Dietary lipid
Choline supplementation
Interaction

0.003
0.135
0.372

0.098
0.796
0.175

0.009
0.390
0.710

0.000
0.006
0.828

0.081
0.316
0.340

Means and pooled SEM are presented for each parameter. Means in the same column with
different superscripts are signicantly different (P b 0.05).

(P b 0.05) interaction between dietary lipid and choline in apoB-100

and MTTP mRNA expression. They both increased signicantly
(P b 0.05) with increasing choline supplementations from 1200 to
1800 mg kg1 when dietary lipid levels reached 8 and 11%.
4. Discussions
Lipids, an effective energy source for sh, play a prominent role in
providing essential fatty acids and phospholipids, which are needed
for maintaining biological structure and normal functions of tissues
and organelles (Sargent et al., 1999). However, in many sh species
an increase in dietary lipids may lead to increased lipid deposition in
tissues (Du et al., 2005), thus possibly being disadvantageous to
the growth and health of sh (Lu et al., 2013a). In the present study,
weight gain tended to increase with increasing lipid levels, whereas
the lowest weight gain was observed in sh fed diet with 11% lipid and
1200 mg kg1 choline supplementation. However, 1800 mg kg1 choline
supplementation gave the best growth performance in high-fat (11%)
diet. The interactive effect of dietary lipid levels and choline supplementations on weight gain and FCR indicates that the appropriate increase in
dietary lipid could improve growth performance and feed utilization
(Johnsen et al., 1993; Peres and Oliva-Teles, 1999; Watanabe et al.,
1978). Furthermore, extra choline supplementation may be benecial to
mitigate the harmful effects of a high-fat diet on growth.
ALT and AST are quantitatively the most important aminotransferases in sh and are commonly recognized as a valuable tool for indications of tissue damage (Wroblewski, 1959; Wroblewski and Ladue,
1956a,b). The elevated plasma AST and ALT levels in experimental sh
fed the diet consisting of 11% lipid and 1200 mg kg1 choline supplementation suggest that there was a release of intracellular enzymes
into the blood (Cheng and Kong, 2011), possibly indicating damage to
membranes of hepatocytes from oxidative stress and further lipid
peroxidation. However, after 1800 mg kg1 choline was added to the
high-fat diet, plasma AST and ALT activities as well as the hepatic lipid
content returned to normal values. This might be attributable to the resolution of hepatic steatosis and injury (Takeuchi-Yorimoto et al., 2013).
The levels of energetic metabolites (triglycerides, cholesterol) of sh
are also considered important indices of health status (Coz-Rakovac
et al., 2005, 2008; Mensinger et al., 2005; Wagner and Congleton,
2004). Fish fed experimental diets with increased lipids had elevated
plasma triglycerides and cholesterol levels, with the highest values in
sh fed the 11% lipid and 1200 mg kg 1 choline supplementation.
This may indicate metabolic disorders of lipids and lipoproteins as
well as liver dysfunction (Mensinger et al., 2005; Takeuchi-Yorimoto
et al., 2013). Likewise, the higher glucose level in sh fed the diet of

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J. Li et al. / Aquaculture 434 (2014) 340347

Table 5
Hepatic lipid content and GSH and GSSG concentrations in blunt snout bream fed diets
containing three levels of lipid with two choline supplementation levels.
Diets
Choline
mg kg1

Lipid %

1200

5
8
11
5
8
11

1800

Pooled SEM

Liver lipid
content
(mg kg1)

GSH
(mg g prot1)

GSSG
(mg g prot1)

GSH/GSSG

117bc
123b
157a
108c
114bc
125b
4.80

10.7bc
11.9c
7.01a
10.3b
10.7bc
9.45b
0.23

1.07
1.29
1.31
1.03
1.05
1.24
0.04

10.1
10.2
5.60
9.68
8.89
7.04
0.43

Two-way ANOVA
Dietary lipid
Choline supplementation
Interaction

0.001
0.000
0.005

0.630
0.000
0.003

0.472
0.589
0.844

0.745
0.066
0.267

Means and pooled SEM are presented for each parameter. Means in the same column with
different superscripts are signicantly different (P b 0.05).

11% lipid with 1200 mg kg1 choline supplementation compared to the

other experimental groups suggests a stress response to high-fat diets in
this sh species since glucose production in sh is mostly mediated by
the action of cortisol, which stimulates liver glucogenesis and gluconeogenesis when sh are subjected to suboptimal or stressful conditions
(Iwama et al., 1999). In contrast, plasma triglycerides, cholesterol,
and glucose concentrations showed a reversed trend with a diet of
1800 mg kg1 choline supplementation and 11% lipid, suggesting a
role of choline in alleviating liver damage caused by a high-fat intake.
However, supplementing 1800 mg kg 1 choline in the diet may not
have a signicant inuence on blunt snout bream fed with low (5%) or
optimum (8%) lipid levels.
Oil red O staining is commonly used for the detection of triacylglycerols and cholesteryl esters and for quantitative evaluation of steatosis
(Fowler and Greenspan, 1985; Takeuchi-Yorimoto et al., 2013) and is
a valuable tool in the diagnostic histopathologic process (Dekker et al.,
1979). The appearance of lipid droplets in the liver cells stained with
oil red O further conrmed the content of the liver lipids in the present
work, which are stored in the primary form of triglycerides (Gaylord
and Gatlin, 2000). Few lipid droplets were found in sh fed 5% and 8%
lipids. Conversely, a large number of lipid droplets in sh fed the diet
containing 11% lipid and 1200 mg kg1 choline supplementation indicated excessive triglyceride accumulation in the cytosol of hepatocytes;
this condition did not occur when supplemented with 1800 mg kg1
choline, suggesting that hepatic lipidosis is a result of defective VLDL
production and the subsequent reduced excretion of lipids or triglycerides (Chan, 1990; Corbin and Zeisel, 2012; de Wit et al., 2012; Walkey
Table 6
Hepatic SAM and SAH levels in blunt snout bream fed diets containing three levels of lipid
with two choline supplementation levels.
Diets
Choline mg kg1

Lipid %

1200

5
8
11
5
8
11

1800

Pooled SEM

SAM
(nmol g1)

SAH
(nmol g1)

SAM/SAH

4.27
4.31
4.16
4.51
4.67
4.22
0.06

0.439
0.429
0.455
0.401
0.419
0.430
0.009

9.36
10.1
8.98
10.8
10.9
10.0
0.25

Two-way ANOVA
Dietary lipid
Choline supplementation
Interaction

0.025
0.046
0.357

0.189
0.686
0.840

0.009
0.492
0.858

Means and pooled SEM are presented for each parameter. Means in the same column with
different superscripts are signicantly different (P b 0.05).

et al., 1998). Additionally, aberrant VLDL-mediated secretion of triglycerides is a central mechanism in hepatic steatosis, which is an early
manifestation of liver dysfunction (Corbin and Zeisel, 2012; Vance,
2008). The results were coincided with those reported by Jiang et al.
(2013), who demonstrated a negative correlation between dietary
choline levels and liver lipid contents. Moreover, the increased lipid
concentration of dressed carcass with increasing dietary choline supplementations observed in that study, may indicate that extra choline
supplemented in high-fat diet enhanced the transport of lipid from
liver to other tissues, especially muscle for storage and use, as might
lead to the diminished hepatic lipid deposition, which was observed
in the present study
SAM occupies a central position in the metabolism of all cells as a precursor molecule to three main pathways: methylation, transulfuration,
and aminopropylation (Bottiglieri, 2002). A sufcient amount of SAM is
essential for sustaining the normal function of these pathways. SAH, a
potent competitive inhibitor of transmethylation reactions, is formed
after the transfer of the methyl-group of SAM to a methyl acceptor and
is hydrolyzed to homocysteine (Chiang et al., 1996; Espe et al., 2010).
As we know, there are several potential interactions of choline, methionine and SAM. The excessive methionine may promote the synthesis of
choline and provide sufcient metabolic precursors for SAM production,
thereby masking any effect from dietary choline supplementation
(Kasper et al., 2000). In the present study, the dietary methionine level
of the experimental diet was 7.6 g kg1, which is slightly less than the
optimal requirement of blunt snout bream (7.9 g kg1), so endogenous
synthesis of choline from methionine would be limited. In the liver,
oxidation of choline to betaine is the only known alternative source of
methyl groups for the conversion of homocysteine to methionine
(Abdelmalek et al., 2009). Choline depletion induced by a high-fat intake
might diminish phosphatidylcholine synthesis through the PEMT
methylation pathway (Corbin and Zeisel, 2012; Wortham et al., 2008).
Additionally, the activity of PEMT should be limited by the availability
of SAM (Sha et al., 2010). The present study showed elevated SAM levels
in sh fed diets supplemented with 1800 mg kg1 choline, suggesting
that choline may regulate hepatic lipid transport by involving SAM synthesis (Abdelmalek et al., 2009; Lomba et al., 2010) and subsequently
participating in the phosphatidylcholine synthetic pathway (Cheng and
Blumenthal, 1999; Chiang et al., 1996; Espe et al., 2010).
GSH (gamma-glutamylcysteinylglycine) serves crucial functions
in animals (Wu et al., 2004). Endogenous GSH plays an important role
in scavenging reactive oxygen species (ROS) in the liver. While scavenging ROS, GSH is oxidized to GSSG. In order to maintain the cellular redox
equilibrium, GSSG is reduced to GSH by glutathione reductase (GR)
(Husain and Somani, 1997), forming a redox cycle. Hence, a change in
the GSH/GSSG ratio can be an indication for oxidative stress (Husain
and Somani, 1997). The diet containing 11% lipid and 1200 mg kg 1
choline supplementation resulted in a signicant reduction in hepatic
GSH content as well as an increase in the hepatic GSSG concentration
indicating increased oxidative utilization of GSH induced by high fat
intake (Grifth, 1999; Lu et al., 2000; Speisky et al., 1985). In addition,
severe oxidative stress may suppress the ability of liver cells to reduce
GSSG to GSH, thus leading to GSSG accumulation in hepatocytes (Lu,
1999).
As the ultimate product of SAM transsulfuration reactions, GSH also
functions as a modulator for the feedback regulation of SAM synthesis
(Bottiglieri, 2002). Inactivation of methionine adenosyltransferase
(MAT), which is the enzyme that makes SAM from methionine (Ross,
2003), by ROS can be reversed by physiological concentrations of GSH.
Therefore, liver MAT activity seems to be regulated on a minute-tominute basis by ROS (which maintains the enzyme in an inactive conformation) and GSH (which reactivates the enzyme) (Mato et al.,
2002). This may be the mechanism by which excess lipid intake induces
increased ROS production, which in turn inactivates MAT. This might result in a subsequent reduction in hepatic SAM together with a depletion
of GSH, nally predisposing the liver to oxidative stress and injury.

J. Li et al. / Aquaculture 434 (2014) 340347

345

Fig. 1. Oil red O staining for liver tissue section to assess lipid accumulation in blunt snout bream fed diets containing three levels of lipid with two choline supplementation levels.
Photomicrographs (400) and scale bar (50 m). (A) 5% lipid and 1200 mg kg1 choline supplementation; (B) 8% lipid and 1200 mg kg1 choline supplementation; (C) 11% lipid and
1200 mg kg1 choline supplementation; (D) 5% lipid and 1800 mg kg1 choline supplementation; (E) 8% lipid and 1800 mg kg1 choline supplementation; (F) 11% lipid and
1800 mg kg1 choline supplementation.

Conversely, 1800 mg kg 1 choline supplementation elevated SAM

levels in liver, which ultimately promote the synthesis of GSH, which
is known to be of major importance in preventing a shift from the cellular redox equilibrium and protecting hepatocytes against oxidative
stress (Mato et al., 2002). Through this conversion to GSH, MAT is
reactivated and subsequently leads to an increase in SAM synthesis. In
this way, the metabolic pathways of choline, SAM, and GSH may further
explain the effects of choline on mitigating abnormal physiological status in blunt snout bream fed high-fat diets.
Furthermore, apoB-100 and MTTP mRNA expression levels can be
markers to indicate the association of VLDL forming and trafcking with
hepatic lipid transport. VLDL synthesis and assembly is a substratedependent process highly regulated by the availability of hepatic triglycerides (Fisher and Ginsberg, 2002; Fisher et al., 2001; Olofsson et al.,
2000). This might explain the up-regulation of apoB-100 and MTTP

mRNA expressions in sh with increasing the lipid levels from 5% to 8%

in this work. However, the MTTP expression of sh fed diet with 11%
lipid and 1200 mg kg1 choline supplementation was signicantly
lower than that of the other groups. The results of hepatic lipid content,
plasma triglycerides, cholesterol, and glucose all showed the highest
values in the same experimental group, suggesting that a high-fat diet
may cause liver damage and dysfunction (Choi and Ginsberg, 2011) and
induce the blockage of VLDL assembly and export. Consequently, VLDL
production was not sufcient to meet the demand for excessive hepatic
lipid transport (Sparks et al., 2006; Tietge et al., 1999). The results further
indicate that the complexity of VLDL synthesis, assembly, and secretion is
generally regulated by the metabolic and physiological status of the liver
(Choi and Ginsberg, 2011). However, 1800 mg kg1 choline supplementation increased the abundance of apoB-100 and MTTP mRNA, especially
in the high-fat diets, suggesting that the mechanism of extra choline

346

J. Li et al. / Aquaculture 434 (2014) 340347

choline metabolism and some related metabolites and genes impact

hepatic lipid transport and liver physiology of blunt snout bream.
Acknowledgments
This work was funded by the National Nature Science Foundation of
China (31172418) and the National Nature Science Youth Foundation of
China (31202005).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.aquaculture.2014.08.006.
References

Fig. 2. Relative mRNA expression analysis of apolipoprotein B-100 (apoB-100) in the

liver of blunt snout bream fed diets containing three levels of lipid with two choline
supplementation levels.

supplementation stimulating excessive hepatic lipid transport might

be through increasing VLDL production, which was ascribed to the upregulation of apoB-100 and MTTP gene expressions.
5. Conclusions
The study showed an overload of lipid deposition in the liver of sh
fed a high-fat diet with 1200 mg kg1 choline supplementation. This
predisposes metabolic and physiological disorders as well as inhibits
the growth of blunt snout bream. However, adequate dietary choline
could resolve hepatic steatosis and enhance lipid transport from
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apoB-100 and MTTP mRNA expressions. Furthermore, extra supplemental choline stimulated hepatic SAM production of blunt snout
bream and subsequently increased GSH concentration, which could
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has contributed to an improved understanding of how the pathway of

Fig. 3. Relative mRNA expression analysis of microsomal triglyceride transfer protein

(MTTP) in the liver of blunt snout bream fed diets containing three levels of lipid with
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Group 11/1200: 11% lipid and 1200 mg kg1 choline supplementation; Group 5/1800:
5% lipid and 1800 mg kg1 choline supplementation; Group 8/1800: 8% lipid and
1800 mg kg1 choline supplementation; Group 11/1800: 11% lipid and 1800 mg kg1
choline supplementation.

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