Aquaculture nutrition, tập 18, số 6, 2012

Aquaculture Nutrition
2012 18; 581–588

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

1
1

doi: 10.1111/j.1365-2095.2012.00936.x

1,2

CINVESTAV IPN Unidad Me´rida, Laboratorio de Biologı´a y Cultivo de Moluscos, Me´rida, Yucata´n, Me´xico;
des Sciences, Ancien logement des maıˆtres, La Rosie`re, Lamentin, Guadeloupe, FWI, France

One of the bottlenecks for the queen conch, Strombus
gigas, aquaculture is the lack of well-adapted formulated
food for optimal growth. The goals of this study were to
analyse the digestive gland structure of conchs fed with different diets using histochemical techniques and to evaluate
the growth and survival of S. gigas juveniles with nine formulated diets (protein from 190 to 380 g kgÀ1 and lipids
from 26 to 82 g kgÀ1). Proteoglycan granules and acidophilic granules were detected in the digestive cells. The
abundance of both granule types was variable, according
to the nutritional state of the animals. The granular content of the digestive cells of conchs fed with artificial diets
was scarce when compared with conchs fed on natural
food. Of the nine formulated feeds, the diet with 365 and
45 g kgÀ1 of protein and lipids, respectively, gave the best
growth in weight (0.20 g dayÀ1) and was also associated
with digestive cells in the best condition as determined histologically. Histochemical analysis of the digestive gland
differentiated with Alcian blue staining determines the
nutritional status much better than a simple growth index
and is therefore more useful in assessing adjustments to the

feed formulation to meet the real needs of conchs.
KEY WORDS:

aquaculture, Caribbean, diets, feeding, molluscs, physiology

Received 29 January 2011; accepted 14 October 2011
Correspondence: D.A. Aranda, CINVESTAV IPN Unidad Me´rida,
Laboratorio de Biologı´a y Cultivo de Moluscos, Km. 6 antigua carretera
a Progreso. CP 97310 Me´rida, Yucata´n, Me´xico. E-mail: daldana@
mda.cinvestav.mx

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

ª 2012 Blackwell Publishing Ltd

2

Archipel

The queen conch, Strombus gigas, (Linnaeus, 1754) is a
marine resource of ecological and economic importance in
the Caribbean (FAO 2007). Since pre-Columbian time,
queen conchs have been an important source of food for
the inhabitants of Caribbean coasts and islands (Wing
2001). However, queen conch meat that was a popular staple food is now mostly consumed as a tourist delicacy. It is
an important source of income in several exporting countries and is an overexploited fishery (Theile 2001). As populations have been declining for several decades, much of the
current research focuses on aquaculture, restocking and
transplanting techniques to help replenish wild conch populations. Queen conch aquaculture has been developed in the
Turks and Caicos (to expand conch production farm and to
license grow-out farms throughout the Caribbean) and in

Florida Harbor Branch Oceanographic Institution (Davis
2000; Shawl et al. 2008). Even though queen conch aquaculture is a success in terms of hatchery spat production,
growth still depends on the use of large areas of wild environment (Davis 2000). One of the bottlenecks for intensive
conch farming is the lack of formulated food for optimal
growth in hatchery at a reasonable price (Shawl & Davis
2006; Shawl et al. 2008). There is a need to improve husbandry techniques for the grow-out of juveniles with diets
that allow a growth rate equal to or higher than that of wild
juveniles. Moreover, the use of prepared feeds can be very
practical as formulations can be manipulated to obtain an
optimum nutritional value. Furthermore, they are available
on demand and if properly prepared may be stored for a
longer time period (Bautista-Theurel & Millamena 1999).
Aldana Aranda et al. (1996) reported a growth rate of
0.31 mm dayÀ1 with Frippak No. 2 (480 g kgÀ1 of protein).
Glazer et al. (1997) fed conchs with Koi fish food and macroalgae, obtaining a growth rate of 0.34–0.35 mm dayÀ1,
respectively. Rathier (1987), Iversen & Jory (1997) and


Aldana Aranda et al. (2005) obtained growth rates of 0.16,
0.23 and 0.31 mm dayÀ1, respectively in wild juvenile queen
conch fed on natural food. Most studies on the digestive
tract and digestive gland of microphagous prosobranch
Gastropods have been performed on species living in intertidal environments in order to investigate the influence of
tidal variations upon the digestive gland cycle (Nelson &
Morton 1979). Queen conch natural feed is a complex mixture of macroalgae, microbenthos and biofilm involving
ingestion of sediment (Stoner & Waite 1991; Shawl et al.
2008; Serviere et al. 2009). However, real nutrient requirements for queen conch, in terms of energy level, protein and
micronutrients, are unknown. (Amber et al. 2011). Furthermore, shell growth and weight are not precise indices of
optimal growth and assimilation (Lucas & Beninger 1985).
The goals of this study were to compare the status of the

digestive gland of juvenile queen conch fed with different
formulated diets with increasing levels of proteins and lipids
using histochemical techniques and to evaluate the growth
and survival of conchs fed with these diets.

An experimental aquaculture facility was set up at Xcaret
Marine Park, south of Cancun (Mexico), to raise juvenile
queen conch received from Ocean Reef Aquarium society
(ORA) in Fort Pierce, Florida. Experimental cultures were
set up in 45-L raceways with a density of 1.5 conchs LÀ1.
Aquaria were filled with sand-filtered sea water (5 lm)
which was continuously oxygenated using an air pump. Sea
water was renewed (4 times dayÀ1), and raceways were
cleaned twice a week to avoid the development of microalgae and biofilm which could be used as a food source. Juvenile queen conchs were fed 150 mg of diet per conch each
day for 3 months, and the uneaten food was removed daily.
Two experimental treatments and a control were set up.
The experimental treatments were as follows: (i) conchs
were given formulated feed (diets 1, 3, 4, 5, 6 and 9, see
Table 1) for 84 days and (ii) conchs were given formulated
feed (diets 2, 7 and 8) for 84 days and then returned to a
natural diet (a biofilm of 50% of red and green algae and
50% of seagrass and sand) for 21 days. The main ingredients used in these formulated diets were fish meal, soy
flour, wheat flour, spiruline, corn starch, fish oil, vegetable
oil and soy lecithin. Each of the nine diets was tested using
three replicates of 30 conchs each, giving a total of 810
conchs on experimental diets. The control comprised two
replicates of 30 conchs each (n = 60 conchs), which were
kept on a natural food diet (a biofilm of 50% of red and

Table 1 Biochemical composition of experimental diets used to

feed juveniles of Strombus gigas
Biochemical composition (g kgÀ1)
Diets

Protein

Lipids

Crude fibre

Ash

NFE

1
2
3
4
5
6
7
8
9

193
190
190
277
278
327

365
373
381

26
66
79
35
55
69
45
56
82

04
04
04
08
08
14
09
13
10

71
61
66
87
90
97

111
111
109

706
679
661
593
569
493
470
447
418

NFE, nitrogen-free extract.

green algae and 50% of seagrass and sand) for 84 days.
The nine formulated diets were tested (Table 1), containing
levels of protein from 190 to 380 g kgÀ1 and levels of lipids
from 26 to 82 g kgÀ1. The nutritional status of queen
conchs was analysed using the following indices: siphonal
length, flesh weight and histological features of the digestive gland. Juveniles of S. gigas were measured and
weighed at the beginning of the experiment (T0) and at
days 21, 42, 63 and 84. At the beginning of the experiment,
juveniles from the wild, in the same size range as the
conchs from ORA, were dissected and prepared for the histological analysis of the digestive gland. Likewise, three
juvenile queen conch from the Florida hatchery were also
analysed before the start of the experiment, and another
three juveniles of each replicate receiving one of nine diets
(n = 9 conchs) were examined at the end of the experiment.

Histological examination involved cutting the visceral
mass of each individual into two sections: a distal part, containing only digestive gland and connective tissue, and a mid
part also containing stomach. Sampled sections were fixed in
alcoholic Bouin fluid and processed using standard histological techniques (Gabe 1968; Luna 1969). After dehydration
in ethanol series, and clearing with Clarene, the sections
were embedded in Paraplast wax. Sections of 5 lm thick
were stained with a trichrome stain following Gabe (1968)
which included Alcian blue (Hycel de Mexico, SA de Cv.
Zapopan, Jalisco, Mexico) at pH 2.5 to differentiate proteoglycans. Slides were also treated by the Periodic acid-Schiff
(PAS) reaction for glucide detection (Gabe 1968). The slides
were examined and pictures were taken with a Nikon DXm
1200F digital camera mounted on a Nikon microscope. All
the pictures were corrected for contrast and colour (Photoshop software, Adobe Photoshop CF 2. version 9.0, San
Jose, CA, USA). For every juvenile, two microscope slides
were prepared with five sections per slide (distal part). The

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd


incidence of blue granules (Gros et al. 2009) was obtained
by counting the total number observed in three fields of the
five sections under 409 magnification and calculating the
mean and standard deviation for each diet. A feed index was
established as the sum of blue granules counted in the large
cells of adenomers. These counts were transformed into
granule area (lm2), using the circle area formula for the
granules divided by the image area (lm2) observed on histological slides of digestive glands at 40 9 magnification
(which was always 37 368 lm2), and multiplied by 100. The

blue colour of the granules observed in the digestive cells
after staining with Alcian blue indicated that they were proteoglycan components. Such conspicuous secretory granules
are good markers that demonstrate the digestive cell secretion being delivered to the stomach. One microscope slide
was prepared with the mid part (containing stomach) as a
control of histochemical analysis. The stomach epithelium
that contains mucocytes stained blue. A non-parametric
Tukey test (Sokal & Rohlf 1995) was used to test for significant differences (P < 0.05) among diets in the feed index
and growth rates in siphonal length and whole body weight.

In wild juvenile conchs, the digestive gland has an array
of adenomers (Fig. 1a) similar to those described in

Figure 1 (a) Digestive gland from a
wild juvenile with primary ducts and
adenomers; (b) primary duct with two
types of epithelium; low and simple ( )
similar to the secondary ducts epithelium and plicate ciliated epithelium (Δ)
typical of primary ducts; (c) stomach
wall with mucocytes (◊) stained by
Alcian blue that constitutes a positive
control for various diets as mucocytes
are stained blue even if the digestive
gland granules are not; (d) adenomers
of a digestive gland section containing
with the two cell types. Large digestive
or secreting cells with large blue granules (star) and crypt cells with sporozoa-like microorganisms belonging to
the Apicomplexa group (arrow).

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

adults. All these secreting structures are connected to
small ducts, which join larger ducts attached to the stomach. The small secondary ducts are lined with a simple
epithelium composed of a single cell type. The larger primary ducts have two areas (Fig. 1b), one similar to the
small duct epithelium devoid of cilia and another composed of ciliated cells and mucocytes. The connective tissue surrounding the digestive gland possesses two
characteristic cell types, small round amoebocytes stained
red by PAS and blue granules in the large cells stained
blue by Alcian blue. Both may play a role in the transfer
of metabolites. The functional glandular structure comprises two cell types (Figs 1d & 2a): tall (80 lm) and narrow (<10 lm) digestive cells, which contained large
granules up to 10 lm in diameter stained by Alcian blue.
These cells alternate with vacuolated cells which are
always occupied by brown inclusions. These inclusions are
sporozoa-like microorganisms belonging to the Apicomplexa group. The observation of unstained sections demonstrates that the orange-brown colours of these
sporozoa-like microorganisms are their natural colours.
The mucous lining of the stomach as well as a number of
connective tissue cells are stained by Alcian blue (Fig. 1c)
giving a positive control to the histochemical reaction in
the digestive gland. The crystalline style is stained red by
the PAS reaction even after amylase digestion and stained
blue by Alcian blue, demonstrating that it contains glycoproteins as well as proteoglycans.

(a)

(c)

(b)

(d)

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2 Comparison of digestive glands of Strombus gigas juveniles among reared animals fed various formulated diets, including control
animals fed on a natural diet and wild juveniles. All the pictures are of digestive glands at the same magnification with obj 940. (a) Digestive gland of a wild juvenile showing large digestive or secreting cells with large blue granules (star) and crypt cells with sporozoa-like
microorganisms (arrow); (b) laboratory-reared individual from ORA showing smaller blue granules and a less homogenous structure than
the wild juvenile; (c) reared juvenile fed for 84 days on diet 8 showing that the digestive gland cells are completely destroyed although the
duct appears normal; (d) reared juvenile fed for 84 days on diet 2 showing intracellular unstained by Alcien blue, whereas the stomach
epithelium contains some mucocytes stained blue (black arrow); (e) reared juvenile fed for 84 days on diet 7 showing blue granules approximately half the size of those observed in the wild juveniles; (f) reared juvenile fed for 84 days on diet 7 and then on a natural food diet for
21 days showing blue granules typical of wild juveniles. Diet formulations are given in Table 1.

The status of the digestive gland from three individuals
immediately after being received from the hatchery was
assessed and considered as T0 status. These glands exhibited digestive cells with fewer and smaller blue granules
than in wild conchs (Fig. 2b). The blue granules were not
similar in structure to those of wild juveniles as the blue
colour was restricted to the centre of the granules. The sporozoa-like microorganisms were also different from those
described in wild juveniles, being a light creamy colour and
numerous.

Table 2 shows the initial and final siphonal length, weight
and daily growth of juvenile S. gigas fed on nine diets for
84 days, and values of a non-parametric Tukey test. The
highest growth rate of 0.23 mm dayÀ1 and weight gain
0.20 g dayÀ1 were observed for juveniles raised on diet 7.
The control group had an initial and final siphonal length
of 54.69 ± 7.3 and 75.98 ± 4.4 mm, respectively, (n = 60)
and a performance growth rate of 0.29 mm dayÀ1± 0.5.
The one-way ANOVA test showed significant differences
among diets in the mean growth per day in siphonal length

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd


Table 2 Growth parameters of the of juveniles of Strombus gigas fed for 84 days on one of nine formulated diets

Diets

Initial siphonal
length (mm)

End siphonal
length (mm)

Growth
rate dayÀ1
(mm)*

1
2
3
4
5
6
7
8
9

39.11
37.83
38.29
39.39
39.34
39.35
38.96
39.44
39.95

54.18
50.90
52.05
55.69
56.52
57.61
58.35
57.16
56.82

0.18
0.16
0.16
0.19
0.20
0.22
0.23
0.21
0.20

±
±
±
±
±
±
±
±
±

2.47
2.91
2.49
2.67
2.95
3.08
2.85
2.92
2.87

±
±
±
±
±
±
±
±
±

4.50
4.92
4.09
4.84
5.86
6.55
5.23
6.23
5.68

±
±
±
±
±
±
±
±
±

0.03
0.03
0.02
0.04
0.04
0.05
0.03
0.05
0.04

Tukey test
(P
0.05)
Initial
weight (g)

End
weight (g)

Growth
rate dayÀ1 (g)**

n

*

**

5.41
4.95

4.97
5.45
5.45
5.37
5.22
5.35
5.70

18.33
15.79
16.10
20.07
20.48
21.45
22.37
21.11
21.29

0.15
0.13
0.13
0.17
0.18
0.19
0.20
0.19
0.19

87
88

88
88
88
87
88
88
88

BC
A
AB
CD
CD
DE
E
DE
CD

AB
A
A
BC
BC
BC
C
BC
BC

±
±

±
±
±
±
±
±
±

1.05
1.03
0.84
1.07
1.18
1.09
1.09
1.01
1.19

±
±
±
±
±
±
±
±
±

3.61
4.11

3.24
4.67
5.88
6.23
5.73
5.73
6.03

Values are mean ± SD; letters indicate significant differences between dietary treatments (P

and flesh weight (P < 0.0001). Tukey test (P
0.05)
showed significant differences among diets 2, 3 and 7.
Survival was 100% for conchs fed with natural food and
diets 2, 3, 5, 6, 7, 8 and 9, and 97.9% for those fed with
diets 1 and 4. The digestive glands of juveniles reared with
various formulated diets have a different appearance. All
these experimental animals are characterized by the lack of
either blue granules or granules unstained by Alcian blue,
therefore, lacking their proteoglycan component. The PAS
reaction demonstrates that the granule glycoprotein in these
experimental conchs is similar to that of wild juvenile digestive granule content. For example, conchs fed diets 2 and 5
showed a normal structural appearance of the cells in the
digestive gland. However, the large granules of digestive
cells were not stained blue by the histochemical proteoglycan reaction (Fig. 2c). With diet 8, the digestive duct epithelium and the stomach contained a number of mucocytes
stained blue (Fig. 2d), and the digestive and crypt cells of
the digestive gland are completely destroyed. Here, the cell
membranes and intracellular granules have almost completely disappeared giving the cell a destroyed appearance
(Fig. 2d). Diet 7 appears to be the best of the 9 tested, as
the digestive gland structure still possesses some blue granules (Fig. 2e). However, these granules are much smaller

( 5 lm instead of 10 lm), less numerous and paler blue
than in the digestive gland of wild juveniles.
The highest occurrence of blue granules was registered in
conchs fed with natural food with a feed index of 30.0%.
Conchs fed with diet 7 showed a feed index of 14.5%,
conchs from ORA had a value of 10.0% on arrival and
those fed with diet 7 and then replaced with natural food
showed an increase in the mean value to 17.9%. The digestive gland was restored showing the digestive gland status
observed in the digestive gland of the queen conch control

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

±
±
±
±
±
±
±
±
±

0.04
0.04
0.03
0.05
0.06
0.06

0.06
0.06
0.06

0.05).

with digestive cells again containing numerous blue granules, although a little smaller (8 lm) than the wild ones
(Fig. 2f ). The lowest feed index was registered with diets 2,
6, 8 and 9 with no blue granules being found for any of
the diets (Fig 3). The one-way ANOVA test showed significant differences among diets in the median value of feed
index (P < 0.0001) and the Tukey test (P
0.05) showed
significant differences among feed index of diets 5, 7,
conchs fed with diet 7 plus natural food (Fig. 3). All the
individuals fed with formulated food have few sporozoalike microorganisms included in the vacuolated cells of the
digestive gland. These are generally light coloured without
evident sporulation.

Most histological studies of the digestive gland structure of
molluscs deal with bivalves (Purchon 1977; Morse & Zardus 1997). Gastropod digestive glands have been described
by comparison with bivalve digestive glands (Fretter &
Graham 1962). However, feeding methods and nutritional
requirements are not similar in these two groups. Studied
bivalves are suspension feeders and ingest small unicellular
algae submitted to intracellular digestion in the digestive
gland cells (digestive cells), whereas the prosobranch gastropod S. gigas is a grazer, usually feeding on macro algae
when available and on biofilm by grazing on Thalassia
leaves or by ingesting sand. Stoner & Waite (1991) studied
natural feed of S. gigas in the Bahamas and observed feeding differences at two sites (one with high seagrass biomass
and one with bare sand). At the site with low seagrass biomass, these authors found that macroalgae made up

approximately 50% of the stomach content, with detritus
and small infauna making up the other 50%. Despite large


Figure 3 Box plots (n = 85–90 in each plot) showing feed index established of area of blue granules counted in the adenomers of a digestive
gland of juvenile queen conch divided by the image area (37 368 lm2) observed at 40 9 magnification, and multiplied by 100. In juvenile
queen conchs fed nine different diets (see Table 1) for 84 days, including control animals fed on a natural diet (a biofilm of 50% of red
and green algae and 50% of seagrass and sand), conchs fed diet 7 for 84 days and then returned to a natural food diet for 21 days and laboratory-reared. The box contains 50% of the data (90% of data when whiskers are included), while dots indicate extreme values. Data are
given as medians (horizontal line within each box) ± SD. Letters indicate significant differences between dietary treatments (Tukey test,
P
0.05).

amounts of seagrass detritus in the stomachs of conch from
all size classes, stable isotope ratios of carbon and nitrogen
indicated that the food of conchs cannot be based only
upon detritus at any of the habitats. Macroalgae, particularly Laurencia spp. and Batophora oerstedi, were the
principal sources of food identified by 13C, despite low
standing crops. Differences in stomach contents were
higher between sites than ontogenetic or seasonal variations. Serviere et al. (2009) studied natural feed in juveniles
and adults of S. gigas from San Pedro, Belize. These
authors observed different kinds of food in stomach contents from both categories of conchs. They reported a total
of 22 items in the stomach contents of juvenile and adult
conchs. The most diverse phylum was Rodophyta. The second largest groups present in the stomach contents were
Cyanophyta and Protozoa. Seagrass also constituted an
important component in the stomachs of adults. Aquaculture for queen conch has been established for several decades. However, there is a need to improve husbandry
techniques for the grow-out of juveniles with diets that
allow a growth rate equal to or higher than that of wild
juveniles, which typically show growth rates of 0.08–
0.16 mm dayÀ1 (De Jesu´s & Oliva 1997; Iversen & Jory
1997). Aldana Aranda et al. (1996) fed conchs (0.9 mm)

with Frippak No. 2 (480 g kgÀ1 of protein) and reported a
growth rate of 0.31 mm dayÀ1. Glazer et al. (1997) fed

conchs of 44–47 mm with Koi fish food and macroalgae
and obtained a growth rate of 0.34–0.35 mm dayÀ1, respectively. The highest growth rate obtained by Shawl et al.
(2008) when supplementing an artificial juvenile queen
conch diet with various macroalgae (Agardhiella added to
catfish chow) was 0.11 mm dayÀ1. Amber et al. (2011)
determined the growth of juvenile queen conchs fed on
artificial diets. The highest growth rates (0.10 and
0.11 mm dayÀ1) were found for conchs that were fed diets
containing a soy protein isolate protein substitution of
15% or less, showing the importance of the protein source
in the artificial diets of juvenile queen conchs (Table 3). In
this study, we obtained a growth of 0.16–0.23 mm dayÀ1
with formulated diets (190 g kgÀ1proteins/26 g kgÀ1 lipids
and 365 g kgÀ1 proteins/45 g kgÀ1 lipids, respectively).
Britz & Hecht (1997) studied a herbivorous Gastropod
(Haliotis midae) and concluded that diets containing the
highest level of dietary fat (100 g kgÀ1) produced significantly lower growth rates and efficiencies of protein deposition in comparison with abalone fed on diets containing
60 g kgÀ1 and 20 g kgÀ1 fat. However, these trends were
more marked among the small abalone size class. From the
analysis of data of Table 3, the growth of juvenile S. gigas
with natural food was between 0.08 and 0.23 mm dayÀ1
and growth with different diets used to fed juvenile queen
conch improved from 0.01 to 0.18 mm dayÀ1, which is

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

Table 3 Summary of available growth
rate estimates for Strombus gigas juveniles reared on various diets

Site

Food

Initial shell
length (mm)

Growth rate
(mm dayÀ1)

References

Puerto Rico.
Martinique
FWI
Laboratory

Macroalgae
–

25–75

0.10–0.40
0.23

Creswell (1984)
Rathier (1987)

Frippak No. 2

0.90

0.01

Laboratory
Bahamas
Punta Gavila´n,
Me´xico
Laboratory
Laboratory

Koi fish
Natural food
Natural food

44–47

0.01
0.08–0.16
0.14

Aldana Aranda
et al. (1996)
Glazer et al. (1997)
Iversen & Jory (1997)

De Jesu´s & Oliva (1997)

0.08
0.11

Shawl & Davis (2006)
Shawl et al. (2008)

0.10

Amber et al. (2011)

Laboratory

–
Agardhiella added
to catfish chow
Catfish chow (60%),
Agardhiella (22%),
soy protein (15%)
and fish oil (2%)

fairly similar to that obtained with natural food. However,
it has been shown that siphonal length is not a sufficient
measure to accurately determine the nutritional status of
molluscs (Lucas & Beninger 1985). This was also the case
in the present study, where data of growth rates demonstrate similar growth for conch fed with different diets. The
histological appearance of the digestive gland, particularly
the digestive cells, appears to be a more sensitive feed index
with which to evaluate the efficiency of diets for juvenile

queen conch. Histochemical analysis of the digestive gland
and the feed index proposed in this study will be a useful
tool to test micronutrients issued from algae to adapt the
formulated feed and feeding rate to the real needs of juveniles. The feed index proposed in this study, which provides
values ! 50% than those exhibited by wild juveniles, could
be considered adequate. The structure of the digestive
gland in wild adults of S. gigas appears more complex than
in wild juveniles. Crypt cells containing spherocristals
described by Gros et al. (2009) which have not been identified in the digestive gland of wild juveniles or coming from
ORA for this study. These cells may differentiate with age
and maturation of the nutritional or excretory function of
the digestive gland. However, an ultrastructural study of
the digestive gland will be necessary to ascertain the
absence of these crypt cells and spherocristals. Brown
inclusions observed in vacuolated cells in this study have
been described in the digestive gland of adult queen conchs
(Baqueiro Ca´rdenas et al. 2007; Gros et al. 2009; Volland
et al. 2010).
Feeding on formulated pellets is a very important modification of the feeding habits of queen conchs, although the
pellets are ingested and have been identified in the stomach.

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd

99
32.30

We have identified a number of synthetic foods stuck to
the crystalline style in histological sections. The food pellets

seem to be digested in the stomach and reduced to a fine
homogenous powder that is transported to the digestive
gland tubules where it may be absorbed. The presence in
the stomach of large quantities of secretion granules originating from the digestive cells demonstrates that these cells
have a secreting function different from the function
identified in suspension feeders which is only intracellular
digestion. Volland (2010) and Volland et al. (2010) demonstrated enzyme activity (arylsulphatase and acid phosphatase) associated with the apical region of digestive cells
which contained the blue granules in various Strombidae
fed with natural and artificial food.
For the experimental trial of nine formulated pellets, the
optimal nutritional status that resulted in the best digestive
gland structure as well as the best shell growth corresponded to diet 7 (45 g kgÀ1 of lipids and 365 g kgÀ1 proteins). However, the histological structure of the digestive
gland demonstrates that this diet, even though it appears
to be the best of the nine formulations tested, is not really
satisfactory and could be improved as demonstrated by the
improved appearance of the digestive gland of the individuals returned to a natural algal diet.

The authors wish to thank Xcaret aquarium facilities for
nursery work (G. Quintana, A. Cordova, R. Raigoza,
E. Briones, E. Rios, R. Torres), the Ichtyology laboratory
of CINVESTAV IPN Merida and Marine laboratory of
the University of French Antilles, for histology and digital


photomicroscope facilities, respectively, Adriana Zetina
and Teresa Cola´s for histology work, and Erick Baqueiro
and Gemma Franklin (a native English speaker) for English revision. Thanks to three anonymous reviewers for
their comments that enriched this work. Grant: SEPCONACYT 50094 Variacio´n espacio temporal del patro´n
reproductivo de, S. gigas en diferentes ha´bitats y su modelo
biofı´ sico de conectividad para el Caribe.

Aldana Aranda, D., Marı´ n, L. & Brito, N. (1996) Estudios preliminares sobre el crecimiento de postlarvas y juveniles del caracol rosa
Strombus gigas (Mollusca: Gastropoda), utilizando un alimento
microencapsulado. Proc. Gulf. Carib. Fish. Inst., 44, 470–482.
Aldana Aranda, D., Sa´nchez, M., Reynalga, P., Patin˜o, V. &
Baqueiro, E. (2005) Growth and reproductivity season of Queen
conch, Strombus gigas in Xel-Ha´ Park. Proc. Gulf. Carib. Fish.
Inst., 56, 741–754.
Amber, L.G., Acosta Salmo´n, H., Riche, M., Davis, M., Capo, T.
R., Haley, H. & Tracy, P. (2011) Growth and survival of juvenile queen conch Strombus gigas fed artificial diets containing
varying levels of digestible protein and energy. North Am. J.
Aquac., 73, 34–41.
Baqueiro Ca´rdenas, E., Frenkiel, L., Zetina Zarate, A. & Aldana
Aranda, D. (2007) Coccidian (Apicomplexa) parasite infecting
Strombus gigas Linne´, 1758 digestive gland. J. Shell. Res., 26, 1–3.
Bautista-Theurel, M. & Millamena, O.M. (1999) Diet development
and evaluation for juvenile abalone, Haliotis asinina: protein/
energy levels. Aquaculture., 178, 117–126.
Britz, P.J. & Hecht, T. (1997) Effect of dietary protein and energy
level on growth and body composition of South African abalone, Haliotis midae. Aquaculture, 156, 195–210.
Creswell, R.L. (1984) The influence of the experimental diets on
ingestion, assimilation, efficiency and growth of hatchery-reared
juvenile queen conch, Strombus gigas Linne´ fed experimental
diets. J. Shell. Res., 4, 23–30.
Davis, M. (2000) Queen conch (Strombus gigas) culture techniques
for research, stock enhancement and grow out markets. In:
Recent Advances in Marine Biotechnology, Vol 4 (Fingerman,
M. & Nagabhushanam, R. eds), pp. 127–159. Science Publishers
Inc., New Hampshire, USA.
De Jesu´s, A. & Oliva, J. (1997) Densidad, crecimiento y reclutamiento del caracol rosado Strombus gigas L. (Gasteropoda: Strombidae) en Quintana Roo, Me´xico. Rev. Biol. Trop., 45, 797–801.

FAO (2007) Regional workshop on the monitoring and management of queen conch, Strombus gigas. Kingston, Jamaica, 1–5
May 2006. FAO Fish. Rep. 832, Rome, FAO.2007. 174 pp.
Fretter, V. & Graham, A. (1962) British Prosobranch Mollusks
(Ray Society ed.). Bernard Quaritch Ltd., London.
Gabe, M. (1968) Techniques Histologiques. Masson, Paris.
Glazer, R.A., McCarthy, K.J., Anderson, L. & Kidney, J.A. (1997)
Recent advances in the culture of the queen conch in Florida.
Proc. Gulf. Carib. Fish. Inst., 49, 510–522.

Gros, O., Frenkiel, L. & Aldana Aranda, D. (2009) Structural
analysis of the digestive gland of the queen conch Strombus gigas
in relation with the presence of intracellular parasites. J. Moll.
Stud., 75, 59–68.
Iversen, E.S. & Jory, D.E. (1997) Mariculture and enhancement of
wild populations of queen conch (Strombus gigas) in the Western
Atlantic. Bull. Mar. Sci., 60, 929–941.
Lucas, A. & Beninger, P.G. (1985) The use of physiological condition indices in marine bivalve aquaculture. Aquaculture, 44, 187–
200.
Luna, G.L. (1969) Manual of Histologic Staining Methods of the
Armed Forces Institute of Pathology. McGraw-Hill, New York.
Morse, P. & Zardus, J.D. (1997) Bivalvia. In: Microscopic Anatomy of Invertebrates (Harrison, F.W. & Kohn, A.J. eds), Vol
6A: Mollusca II, pp. 7–118. Wiley-Liss, Inc., New York.
Nelson, L. & Morton, J.E. (1979) Cyclic activity and epithelium
renewal in the digestive gland tubules of the marine prosobranch
Maoricrypta monoxyla (Lesson). J. Moll. Stud., 45, 262–283.
Purchon, R.D. (1977) The Biology of the Mollusca. Pergamon
Press, New York.
Rathier, I. (1987) Etat d′avancement des recherches sur l′e´levage
du lambi Strombus gigas in Martinique. Proc. Gulf. Carib. Fish.
Inst., 38, 336–344.

Serviere, E., Villareal Mazariegos, A. & Aldana Aranda, D. (2009)
Natural feed of Strombus gigas. Proc. Gulf Carib. Fish. Inst., 61,
514–517.
Shawl, A. & Davis, M. (2006) Effects of dietary calcium and substrate on growth rate and survival of juvenile queen conch cultured for stock enhancement. Proc. Gulf Carib. Fish. Instit., 57,
955–962.
Shawl, A., Acosta Salmon, H., Davis, M., Cape, T. & Riche, M.A.
(2008) Effect of protein origin in artificial diets on growth and
survival of juvenile queen conch, Strombus gigas. Book of
Abstracts Aquaculture America, Florida, USA, 355 p.
Sokal, R.R. & Rohlf, F.J. (1995) Biometry: the principles and
practice of statistics. In: Biological Research, 3rd edn (Freeman,
W.H. ed.), pp. 887. W. H. Freeman and Co., San Francisco.
Stoner, A. & Waite, J. (1991) Trophic biology of Strombus gigas in
nursery habitats: diets and food sources in seagrass meadows.
J. Moll. Stud., 57, 451–460.
Theile, S. (2001) Queen Conch Fisheries and Their Management in
the Caribbean. TRAFFIC Europe. Technical Report to the
CITES Secretariat. TRAFFIC Europe, Brussels.
Volland, J.M. (2010) Interaction durable Strombidae Sporozoaires
et fonctionnement de l’organe hoˆte de la relation: La glande
digestive. PhD. Thesis, University des Antilles et de la Guyane,
Pointe a` Pitre, FWI, 237 pp.
Volland, J.M., Frenkiel, L., Aldana Aranda, D. & Gros, O. (2010)
Occurrence of Sporozoa-like microorganisms in the digestive
gland of various species of Strombidae. J. Moll. Stud., 76, 196–
198.
Wing, E.S. (2001) Native American use of animals in the Caribbean. In: Biography of the West Indies: Pattern and Perspectives, 2nd edn (Woods, C.E. & Sergile, F.E. eds), pp. 481–518.
CRC Press, NY.

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

Aquaculture Nutrition 18; 581–588 ª 2012 Blackwell Publishing Ltd


Aquaculture Nutrition
2012 18; 589–598

doi: 10.1111/j.1365-2095.2012.00937.x

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

1,2

1

1

1

1

1

1

1
1

School of Life Science, Sun Yat-Sen University, Guangzhou, China;
University, Guangzhou, China

A 63-day growth trial was undertaken to estimate the
effects of supplemented lysine and methionine with different dietary protein levels on growth performance and feed
utilization in Grass Carp (Ctenopharyngodon idella). Six
plant-based practical diets were prepared, and 32CP, 30CP
and 28CP diets were formulated to contain 320 g kgÀ1,
300 g kgÀ1 and 280 g kgÀ1 crude protein without lysine
and methionine supplementation. In the supplementary
group, lysine and methionine were added to formulate
32AA, 30AA and 28AA diets with 320 g kgÀ1, 300 g kgÀ1
and 280 g kgÀ1 dietary crude protein, respectively, according to the whole body amino acid composition of Grass
Carp. In the groups without lysine and methionine supplementation, weight gain (WG, %) and specific growth rate
(SGR, % dayÀ1) of the fish fed 32CP diet were significantly
higher than that of fish fed 30CP and 28CP diets, but no
significant differences were found between 30CP- and
28CP-diet treatments. WG and SGR of the fish fed 32AA
and 30AA diets were significantly higher than that of fish
fed 28AA diets, and the performance of grass carp was also
significantly improved when fed diets with lysine and
methionine supplementation (P < 0.05), and the interaction
between dietary protein level and amino acid supplementation was noted between WG and SGR (P < 0.05). Feed
intake (FI) was significantly increased with the increase in
dietary protein level and the supplementation of lysine and
methionine (P < 0.05), but feed conversion ratio (FCR)
showed a significant decreasing trend (P < 0.05). Two days
after total ammonia nitrogen (TAN) concentration test, the
values of TAN discharged by the fish 8 h after feeding

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

ª 2012 Blackwell Publishing Ltd

2

Animal Science College, South China Agricultural

were 207.1, 187.5, 170.6, 157.3, 141.3 and 128.9 mg kgÀ1
body weight for fish fed 32CP, 32AA, 30CP, 30AA, 28CP
and 28AA diets, respectively. TAN excretion by grass carp
was reduced in plant-based practical diets with the increase
in dietary protein level and the supplementation of lysine
and methionine (P < 0.05). The results indicated that
lysine and methionine supplementation to the plant protein
sources-based practical diets can improve growth performance and feed utilization of grass carp, and the dietary
crude protein can be reduced from 320 g kgÀ1 to
300 g kgÀ1 through balancing amino acids profile. The
positive effect was not observed at 280 g kgÀ1 crude
protein level.
KEY WORDS:

Ctenopharyngodon idella, lysine, methionine,
protein reduction, total ammonia nitrogen

Received 29 January 2011; accepted 13 November 2011
Correspondence: Yong-Jian Liu, School of Life Science, Sun Yat-Sen
University, Guangzhou 510275, China. E-mail:

Grass carp (Ctenopharyngodon idella) represent the second
largest aquaculture industry in the world inferior to silver
carp Hypophthalmichthys molitrix, constituting 14.7% of

the world aquaculture production, with an average annual
increase of 14% in China (FAO 1999). One of the major
reasons for the increase in production is the use of pelleted
feed, which enables the higher density or net–cage monoculture of this species to be achieved.
Protein is a major component in fish feeds, because it provides the essential and nonessential amino acids to synthesize


body protein and in part provides energy for maintenance.
Dabrowski (1977) found the protein requirement of grass
carp was between 410 g kgÀ1 and 430 g kgÀ1. Khan et al.
(2004) reported high WG in grass carp fed 300 g kgÀ1 and
350 g kgÀ1 crude protein diets. In China, grass carp are typically fed a commercial feed with 220–320 g kgÀ1 crude protein content. Protein, especially when derived from fishmeal
and soybean meal, is the most expensive nutrient in the preparation of diets for grass carp. The protein sources of grass
carp commercial diets almost are canola meal and cotton
meal. Lysine and methionine are the two most limiting
amino acids in the diets for grass carp (Wang et al. 2005;
Yang et al. 2010). It has been demonstrated that grass carp
can efficiently utilize crystalline amino acid (Yang et al.
2010). Supplementation of lysine-deficient diets with lysine
also improved WG in common carp (Viola et al. 1992a) and
channel catfish (Robinson et al. 1980; Robinson & Li 1994;
Zarate & Lovell 1997). Botaro et al. (2007) reported that
reducing 2.7% of dietary digestible crude protein (from
270 g kgÀ1 to 243 g kgÀ1) with crystalline amino acid had
no negative impact on growth performance of Nile tilapia.
Recently, Gaylord & Barrows (2009) also found dietary
crude protein content of plant-based diet for rainbow trout
can be reduced from 460 g kgÀ1 to 415 g kgÀ1 by supplementing lysine, methionine and threonine with no reduction
in growth. In addition, the imbalance of dietary amino acid
profile can lead to eutrophication of receiving water by nitrogen excretion (Cheng et al. 2003; Conceicao et al. 2003).

Therefore, the present studies were carried out to evaluate the effects of dietary protein reduction with lysine and
methionine supplementation on growth performance and
total ammonia nitrogen (TAN) excretion of grass carp
under laboratory conditions. These results could be useful
for the formulation of low-cost feed for grass carp.

Formulations of the experimental diets are shown in
Table 1. Three practical experimental diets (32CP, 30CP
and 28CP) that were formulated with 32% (DM), 30%
(DM) and 28% (DM) crude protein without supplementation of crystalline amino acid, lysine and methionine were
deficient for grass carp. In the other three experimental
diets (32AA, 30AA and 28AA), lysine and methionine were
added to the 32CP, 30CP and 28CP diets and were made
to equal the levels calculated to be present in the grass carp
whole body of 32% protein. All diets were made isoener-

getic. The amino acid compositions of six experimental
diets are shown in Table 2.
All dry ingredients were finely ground, weighed, mixed
manually for 5 min and then transferred to a Hobart mixer
(A-200T Mixer Bench Model unit, Resell Food Equipment
Ltd, Ottawa, ON, Canada) for another 15-min mixing.
Soya lecithin was added to a preweighed premix of soy oil
and mixed until homogenous. The oil mix was then added
to the Hobart mixer slowly while mixing was still continuing. All ingredients were mixed for another 10 min. Then,
distilled water (about 300 g kgÀ1 of diets) was added to the
mixture-form dough. The wet dough was placed in a dualscrew extruder (Institute of Chemical Engineering, South
China University of Technology, Guangzhou, China) and
extruded through an 1.25-mm die. The diets were dried
with forced air at 20 °C for 24 h, and the moisture was

reduced to about 100 g kgÀ1. The dry pellets were placed
in plastic bags and stored in a deep freezer at À20 °C until
used.

Grass carp (Ctenopharyngodon idella) juvenile from our facilities were used in this experiment, and their initial wet
weights were 2.10 ± 0.01 g. Before the experiment, the fish
were acclimated to the experimental conditions for 2 weeks
and fed a commercial diet containing 300 g kgÀ1 protein and
40 g kgÀ1 lipid to satiation. Twenty-five healthy fish were
randomly distributed to each of the 18 experimental fibreglass tanks (98 L 9 48 W 9 42 H cm, water volume of
200 L) connected to a recirculation system. Water exchange
in each tank was maintained at 10 L minÀ1. The water was
oxygenated, passed through artificial sponge (3 cm thickness), coral sand (25 cm thickness) and active-carbon filter
(25 cm thickness) to remove chlorine. During the trial period, the diurnal cycle was 12-h light/12-h dark. Water quality
parameters monitored weekly as follows: temperature,
29.1 ± 2.4 °C; dissolved oxygen, 7.4 ± 0.36 mg LÀ1; TAN,
0.089 ± 0.006 mgLÀ1; pH, 7.9 ± 0.09, respectively. Faeces
were collected daily during the last 2 weeks as described by
Wang et al. (2005). Faeces tankÀ1 was dried at 105 °C and
stored at À70 °C for determination of digestibility with
Y2O3 as indicator.
In the growth experiment, the fish were fed three times
per day and 7 days per week to apparent satiation for
9 weeks. After the growth experiment, ten healthy fish
(average wet weight 65.0 ± 0.25 g) were randomly distributed to each of the 12 experimental fibreglass tanks
(98 L 9 48 W 9 42 H cm, water volume of 200 L) and

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

Table 1 Formulation and approximate composition of practical diets for grass carp
Diet
Ingredients (g kgÀ1 diet)

32AA

30AA

28AA

32CP

30CP

28CP

Soybean meal1
Canola meal1
Cotton meal1
Rice bran meal1
Wheat flour1
Maize1
Mineral mix2
Vitamin mix3
Soy oil1
Choline chlorine (50%)1
Monocalcium phosphate1
51% L-Lysine SO45

84%MHA-Ca5
Phospholipid1
VC Ascorbic acid
Y2O36
Total

143.6
250.0
140.0
50.0
284.8
50.0
5.00
5.00
20.0
2.00
20.0
12.0
6.50
10.0
1.00
0.10
1000

86.6
250.0
140.0
50.0
284.8
104.3

5.00
5.00
20.0
2.00
20.0
14.5
6.70
10.0
1.00
0.10
1000

27.4
250.0
140.0
50.0
284.8
160.7
5.00
5.00
20.0
2.00
20.0
17.0
7.00
10.0
1.00
0.10
1000

143.6
250
140
50
284.8
68.5
5
5
20
2
20
0
0
10
1
0.1
1000

86.6
250. 0
140.0
50.0
284.8
125.5
5.00
5.00
20.0
2.00
20.0
0.00

0.00
10.0
1.00
0.10
1000

27.4
250.0
140.0
50.0
284.8
184.7
5.00
5.00
20.0
2.00
20.0
0.00
0.00
10.0
1.00
0.10
1000

(DM)
Moisture
Crude protein
Digestive Protein
Crude fat
Lysine

Methionine
Ash
Digestive energy kcal kgÀ1
Gross energy kcal kgÀ1

Approximate composition (g kgÀ1 dry matter)
105.7
97.6
101.1
333.5
311.2
285.5
264.0
258.9
231.4
44.8
44. 1
45. 4
19.906
19.558
19.502
10.250
10.121
10.210
83.8
81.3
80.1
3397
3334
3429

4792
4728
4751

113.3
319.7
261.7
45
13.509
4.802
83.1
3380
4841

106.5
293.9
249.2
45.0
12.404
4.588
78.7
3456
4839

108.1
273.5
234.1
44. 6
12.173
4.281

76.1
3436
4783

Digestible energy, gross energy, and digestive protein were measured (Wang et al. 2005).
Zhuhai Shihai Feed Corporation Ltd, Zhuhai, China.
2
Mineral mix(mg kgÀ1 of diet): MgSO4·7H2O,315; ZnSO4·7H2O,285; CaHPO4·2H2O,250; FeSO4·7H2O,200; MnSO4·H2O,25; CoSO4·7H2O,25;
CaIO3,25; CuSO4·5H2O,15; Na2SeO3,10. (Guangzhou Chengyi Aquatic Technology Ltd, Guangzhou, China).
3
Vitamin mix (mg kgÀ1 of diet): thiamin,3; riboflavin,8; vitmin A,1 500 IU; vitamin E,40; vitamin D3,2 000 IU; menadione,6; pyridoxine,4;
cyanocobalamin,2; biotin,2; calcium pantothenate,25; folic acid,2; niacin,12; inositol,50. (Guangzhou Chengyi Aquatic Technology Ltd,
Guangzhou, China).
4
L-Lysine SO4 contained L-Lysine ! 51% (CJ Co., Ltd., Liaocheng, China).
5
MHA-Ca contained DL-HMTBA (2-hydroxy-4-methylthio butanoic acid) ! 84% (Novus International Inc., Zhibo, China).
6
Y2O3 (Yttrium oxide), analytical pure (Weibo Chemical Ltd, Guangzhou, China).
1

fed with the diets from the growth experiment for 2 days.
After fish were fed at 0800 h with 2.5% of body weight, all
tanks were cleaned, the water supply to the tanks was shut
off, and oxygen was supplied to the tanks. At 0800 h and
1600 h, water samples were collected for TAN analyses for
2 days.

At the beginning of the feeding trial, 18 fish were randomly
sampled from the initial fish and killed for analyses of

whole body composition. At the end of the 63-day experiment, 12 fish from each tank were randomly collected for
proximate analysis, four for analysis of whole body compo-

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

sition and 8 were anaesthetized with tricaine methane sulphonate (MS222) (50 mg LÀ1) to obtain weights of
individual whole body, viscera, liver and mesenteric fat.
White muscle from both sides of the fillets without skin
and liver was dissected and frozen immediately in liquid
nitrogen and stored at À70 °C until used.
Diets and fish samples (including white muscle and liver)
were analysed in triplicate for proximate composition. Crude
protein, crude lipid, moisture, crude ash and gross energy
(GE) were determined following standard methods (AOAC
1984). Crude protein (N 9 6.25) was determined by the Kjeldahl method after acid digestion using an Auto Kjeldahl System (1030-Auto-analyzer, Tecator, Sweden). Crude lipid was
determined by the ether extraction method using a Soxtec


Diet
Amino acids

32AA

30AA

28AA

32CP

30CP

28CP

Essential amino acids
Lysine
Methionine
Phenylalanine
Histidine
Tryptophan
Arginine
Threonine
Isoleucine
Leucine
Valine

19.9
10.3
12.8
6.1
3.9
18.7
8.4
11.0
19.2
15.0

19.6
10.1

11.7
5.7
3.5
17.7
8.3
10.2
17.9
13.9

19.5
10.2
10.8
5.2
3.1
15.3
7.4
9.3
16.4
12.9

13.5
4.8
12.5
5.9
3.9
18.4
8.6
11.1
19.3
14.8

12.4
4.6
11.9
5.7
3.5
17.3
8.4
10.7
18.7
14.4

12.2
4.3
11.1
5.2
3.1
15.6
7.5
9.6
17.0
13.2

Non-essential amino acids
Serine
7.0
Proline
16.7
Cystine
1.8

Tyrosine
4.7
Aspartic acid
22.6
Glutamic acid
59.8
Glycine
13.2
Alanine
12.5

7.3
15.6
1.8
4.3
20.0
56.3
12.1
11.9

6.3
14.9
1.7
3.5
17.2
52.4
11.2
11.1

7.4

16.9
1.8
4.9
22.3
59.1
12.9
12.5

7.5
19.1
1.7
4.7
20.3
57.5
12.4
12.5

6.6
15.8
1.8
3.9
17.4
53.0
11.3
11.6

Table 2 Amino acid composition of
experimental diets for grass carp (g kgÀ1
dry diets)

Tryptophan is calculated from NRC.
As an analog of methionine, MHA-Ca cannot be detected by the amino acid analyser, so
methionine value was analysed the sum of MHA-Ca and Methionine.

System HT (Soxtec System HT6, Tecator, Sweden). Moisture
was determined by oven-drying at 105 °C for 24 h. Crude
ash was determined by incineration in a muffle furnace at
550 °C for 24 h. GE was determined using an adiabatic
bomb calorimeter. Amino acids were analysed following acid
hydrolysis using high-pressure liquid chromatography
(HPLC; Hewlett Packard 1090, Palo Alto, CA, USA). The
concentrations of dietary and faecal Y2O3 were determined
by inductively coupled plasma atomic emission spectrophotometer [ICP; model: IRIS Advantage (HR), Thermo Jarrel
Ash Corporation, Boston, MA, USA] after perchloric acid
digestion (Bolin et al. 1952).
Water samples were analysed for TAN concentration by
Nessler’s reagent colorimetric method (Zang 1991). Light
absorbance of water samples at 420 nm wavelength was
recorded on a UV-spectrophotometer (UV250), and TAN
concentration was determined using a standard curve.

All data are presented as means ± SEM The SPSS software
ver 13.0 for Windows of GLM procedure (SPSS Inc.,
Chicago, IL, USA) was used to conduct factorial ANOVA to
determine the effects of dietary protein content, crystal
amino acid supplementation and interaction of the two factors. When interaction between protein level and amino

acid supplementation was statistically significant for a particular response, differences among protein levels within
each diet type were determined using Tukey’s mean separation. Treatment effects and interactions were considered
significant at P < 0.05.

Fish readily accepted the experimental diets. At the end of
the growth trial, the survival were high (>94.67%), and there
were no significant differences in survival among fish fed all
the diets (Table 3). Weight gain, specific growth rate (SGR),
feed conversion ratio (FCR), FI, nitrogen retention (NR),
lipid retention (LR) for grass carp after 9-week feeding trial
are presented in Table 3. FI and NR were significantly
increased with the increase in dietary protein level and the
supplementation of lysine and methionine (P < 0.05), and
WG and SGR also showed the same trends, but there was
interaction found between dietary protein level and the
supplementation of lysine and methionine (P < 0.05). FCR
significantly decreased with the increase in dietary protein
level and the supplementation of lysine and methionine
(P < 0.05). LR was not significantly affected by the diet
treatments (P > 0.05).

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd


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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

2.10
13.7
96.0

554
2.98
1.31
1.75
28.0
184

IBW
FBW
Survival
WG%
SGR
FCR
FI
NR
LR

0.01
0.42C
2.31
22.0C
0.05C
0.03
0.02
0.91
8.48

2.09
10.6
97.3

405
2.56
1.39
1.75
27.2
179

±
±
±
±
±
±
±
±
±

30AA
0.00
1.01B
2.67
48.2B
0.15B
0.01
0.08
0.30
23.4

2.10
6.11

97.3
192
1.69
1.56
1.60
26.9
202

±
±
±
±
±
±
±
±
±

28AA
0.01
0.38A
2.67
18.6A
0.10A
0.02
0.06
0.29
13.2

2.10

9.25
94.7
340
2.34
1.40
1.66
26.8
211

32CP
±
±
±
±
±
±
±
±
±

0.01
1.13b
3.53
56.1b
0.20b
0.02
0.07
0.59
24.8

2.10
6.19
100
194
1.71
1.53
1.60
26.1
222

30CP
±
±
±
±
±
±
±
±
±

0.01
0.14ab
0.00
6.19ab
0.03ab
0.02
0.03
0.41
3.69

No Lys + met supplementation

2.09
5.05
98.67
142
1.40
1.58
1.44
23.9
222

28CP
±
±
±
±
±
±
±
±
±

0.01
0.16a
1.33
8.17a
0.05a
0.04

0.03
1.04
19. 6
<0.001
0.560
<0.001
<0.001
<0.001
0.028
0.030
0.801

Protein

Pr > F

<0.001
0.855
<0.001
<0.001
0.003
0.025
0.009
0.066

Lys + met

0.043
0.290
0.047

0.049
0.096
0.806
0.256
0.801

Protein*
(Lys + met)

AA
AA
AA
AA
AA

supply
supply
supply
supply
supply

> No AA supply
> No AA supply
< No AA supply
> No AA supply
> No AA supply

AA supply > No AA supply

Means ± SEM of three replicates. Probability associated with the F statistic for the factorial ANOVA. Within a row, capital letters indicated differences within diets with lys + met

supplementation and lowercase letters indicate differences within diets without Lys + met supplementation at P < 0.05 when interactions occurred for the full model.
IBW (g fishÀ1), initial body weight.
FBW (g fishÀ1), final body weight.
Survival (%) = 100 9 (final fish number)/(initial fish number).
Weight gain (WG, %) = 100 9 (final body weight-initial body weight)/initial body weight.
Specific growth rate (SGR, % dayÀ1) = 100 9 (ln final wt À ln initial wt)/63 days.
Feed conversion ratio (FCR) = Feed consumed/(FBW À IBW).
Feed intake (FI) = grams of dry feed consumed 9 100/100 g body mass/63 days.
Nitrogen retention (NR) = 100 9 retained nitrogen (g)/nitrogen fed (g).
Lipid retention (LR) = 100 9 retained lipid (g)/lipid fed (g).

±
±
±
±
±
±
±
±
±

32AA

Lys + met supplementation

Diet

Group

Table 3 Effect of with or without lysine and methionion supplementation in different protein level on growth and feed utilization of grass carp

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

±
±
±
±

1.90
1.71
2.83
0.02

±
±
±
±

10.3
1.01
10.9
0.21

±
±
±
±

0.45
0.11
0.40
0.06

11.3
2.11
4.54
2.13

0.43
0.10
0.27
0.07

10.1
1.79
3.58
2.09

±
±
±
±

518 ± 32.3
102 ± 2.51
310 ± 29.3

793 ± 3.62
169 ± 1.72
21.7 ± 1.81

732
121
117
21.9

30AA

553 ± 16.7
104 ± 2.72
214 ± 23.6

808 ± 2.14
170 ± 0.83
17.1 ± 2.00

741
123
106
21.9

32AA

Lys + met supplementation

±
±

±
±

7.20
0.92
0.13
0.64

12.9
2.37
6.02
2.30

±
±
±
±

0.52
0.17
0.42
0.08

478 ± 3.41
102 ± 3.62
311 ± 19.2

781 ± 6.13
172 ± 1.51
31.4 ± 5.42

728
123
122
23.2

28AA

±
±
±
±

5.70
3.93
11.9
0.33

11.4
2.42
4.83
2.20

±
±
±
±

0.55
0.18

0.39
0.07

524 ± 27.0
102 ± 2.51
294 ± 25.1

803 ± 21.1
157 ± 12.4
22.8 ± 6.81

729
122
124
23.0

32CP

±
±
±
±

5.80
2.14
0.53
0.64

13.1
2.61

5.83
2.24

±
±
±
±

0.40
0. 22
0.47
0.04

496 ± 16.6
106 ± 6.10
314 ± 23.2

787 ± 2.92
174 ± 2.41
28.6 ± 2.53

721
122
135
22.6

30CP

±
±

±
±

4.71
0.93
8.33
1.74

13.4
2.51
6.50
2.40

±
±
±
±

0.67
0.17
0.45
0.08

475 ± 24.2
99.2 ± 2.61
330 ± 31.6

779 ± 8.83
168 ± 2.61
38.6 ± 11.1

724
114
132
21.7

28CP

No Lys + met supplementation

0.000
0.109
0.000
0.007

0.023
0.568
0.019

0.159
0.379
0.073

0.214
0.161
0.408
0.948

Protein

Pr > F

0.005
0.002
0.003
0.053

0.537
0.879
0.442

0.895
0.412
0.200

0.290
0.098
0.068
0.863

Lys + met

0.403
0.237
0.535
0.986

0.544
0.585
0.157

0.854
0.295
0.990

0.287
0.112
0.902
0.241

Protein*
(Lys + met)

AA supply < No AA supply
AA supply < No AA supply
AA supply < No AA supply

Means ± SEM of three replicates. Probability associated with the F statistic for the factorial ANOVA. Within a row, capital letters indicated differences within diets with lys
+ met supplementation and lowercase letters indicate differences within diets without Lys + met supplementation at P < 0.05 when interactions occurred for the full model.
2
Means ± SEM of 24 replicates.
Viscerasomatic index (VSI) = 100 9 viscerasomatic weight (g)/body weight (g).
Hepatopancreasomatic index (HSI) = 100 9 liver weight (g)/body weight (g).
Intraperitoneal fat ratio (IPF) = 100 9 intraperitoneal fat weight (g)/body weight (g).
Condition factor (CF) = 100 9 body weight (g)/body length (cm)3.

1

Whole body
Moisture

Protein
Lipid
Ash
Muscle
Moisture
Protein
Lipid
Liver
Moisture
Protein
Lipid
Morphometry2
VSI
HSI
IPF
CF

Composition (g kgÀ1)1

Group

Table 4 Body composition and morphometry index of grass carp fed experimental diets for 63 days


The proximate compositions of the whole body, white muscle and liver of the grass carp are shown in Table 4. No
significant differences were found in the whole body moisture, protein and ash contents of fish among all the diet
treatments (P > 0.05). And the whole body lipid content
showed decreasing trends when fish was fed diets with
lysine and methionine supplementation (P = 0.068).
The highest muscle lipid content of fish was found in

28CP-diet treatment, while the lowest muscle lipid content
of fish was found in 32AA-diet treatment. 32CP, 30AA,
30CP and 28AA gave the intermediate results of muscle
lipid content. No significant differences were found in muscle moisture and protein contents of fish among all the diet
treatments (P > 0.05).
Liver moisture significantly increased with the increase in
the dietary protein level (P < 0.05), and liver lipid content
showed an opposite trend (P = 0.073). No significant differences were found in liver protein contents of fish among
all the diet treatments (P > 0.05).
Condition factor (CF), hepatopancreasomatic index
(HSI), intraperitoneal fat ratio (IPF) and viscerasomatic
index (VSI) of grass carp fed experimental diets are presented
in Table 4. VSI, IPF, CF and HSI decreased with increasing
dietary protein levels among diet treatments without amino
acids supplementation. VSI, IPF, CF and HSI showed a
decreasing trend among diet treatments with amino acids
supplementation.

The amount of TAN discharged into water among different
diet treatments are shown in Fig. 1. Fish fed 32CP diet discharged more TAN than that fed 30CP and 28CP diets,
indicating higher discharges of TAN at higher dietary
crude protein levels (P < 0.05). At the same dietary crude
protein level, fish fed lysine and methionine supplemented
diets discharged less TAN, demonstrating that lysine and
methionine supplementation reduced TAN discharge (P <
0.05), but the interaction was not found (P = 0.463).

Fish were fed to apparent satiation, and feed consumption
was affected by both the energy and protein content of the
diets. FI of grass carp was increased by dietary protein content. An increase in FI due to an increase in dietary protein

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

Ammonia nitrogen aŌer fed 8 h
(mg kg–1 body weight)

250
207.08
200

187.53
157.31

170.64

150

128.87

141.31

100
50
0

32AA

32CP

30AA
Diets

30CP

28AA

28CP

Figure 1 Total ammonia nitrogen discharged into water (mg kgÀ1
body weight, mean ± SD) after fed 8 h.

content, was also observed by Kim et al. (2001) for the haddock (Melanogrammus aeglefinus L), and by Luo et al.
(2004) for the grouper Epinephelus coioides. Compared with
most aquaculture fish species, grass carp has a low energy
requirement, grass carp preferentially adjusted intake to
protein before energy (Du et al. 2005). FI of grass carp was
also significantly increased with addition of lysine and
methionine. Amino acids deficiency causes loss of appetite,
resulting in low FI as shown in Chanos chanos (Borlongan
& Benitez 1990), Labeo rohita (Khan & Jafri 1993),
Oncorhynchus mykiss (Yamamoto et al. 2000) and Cirrhinus
mrigala (Ahmed & Khan 2004). Yamamoto et al. (2000)
indicated that Oncorhynchus mykiss showed a preference
for the balanced amino acid diet over the imbalanced amino
acid, and Oncorhynchus mykiss can also discriminate
deficiency of lysine in diets and show a rapid reduction in the
consumption of the amino acid imbalanced diet (Yamamoto
et al. 2001).

Dabrowski (1977) found there were a linear relationship
between dietary protein level and body protein and growth
and up to optimum at dietary protein levels of 410 g kgÀ1
and 430 g kgÀ1, respectively. Our results also indicated that
low-protein diets induced adverse effects on growth performance of grass carp, there were significant differences in
growth performance of fish fed 32CP, 30CP and 28CP
diets, which indicated that grass carp need to be fed diet
with higher protein content.
In the present experiment, the growth performance of fish
fed 32AA and 30AA diets were significantly higher than
that of fish fed 32CP and 30CP diets. Results of the present
investigation demonstrate significant improvement of
growth and feed utilization of grass carp can be achieved by
L-Lysine sulphate and MHA-Ca supplementation. Some


researchers also have demonstrated that Indian Major Carp
(Mukhopadhayay & Ray 1999; Sardar et al. 2009), Nile
tilapia (Furuya et al. 2004), Rainbow trout (Cheng et al.
2003), Red sea bream (Pagrus major) (Takagi et al. 2002)
and Pacu (Abimorad et al. 2009) fed diets supplemented
with lysine and methionine had better growth performance.
Addition of multiple amino acids to reduce dietary protein
content have been widely studied and used in the production animal industry. The dietary digestible crude protein of
nile tilapia can be reduced from 270 g kgÀ1 to 243 g kgÀ1
(Botaro et al. 2007). Gaylord & Barrows (2009) found that
plant-based dietary protein content for rainbow trout could
be reduced from 460 g kgÀ1 to 415 g kgÀ1 by supplementing lysine, methionine and threonine without growth reduction. The dietary protein content for common carp also
could be reduced from 300 g kgÀ1 to 250 g kgÀ1 by supplementing lysine without growth reduction (Viola et al.
1992a). In the present experiment, fish fed 30AA diet had a

higher growth than fish fed 32CP diets, 32CP diet was deficient of lysine and methionine for grass carp, and 30AA
diet was balanced by providing lysine and methionine. So
we can slightly reduce the dietary crude protein in the practical diets through balancing amino acids profile. But the
growth performance of grass carp fed 32AA diet was significantly higher than that of fish fed 30AA diet, which indicated that dietary protein could not be reduced by
supplementing lysine and methionine if amino acids meet or
exceed the requirement. The present results indicated that
no significant differences were found in growth performance
of grass carp fed 28CP and 28AA diets. Research on the
Blue catfish Ictalurus furcatus also demonstrated that the
growth performance of catfish fed low-protein diets was not
improved by supplementing lysine and methionine (Webster
et al. 2000). But essential amino acid supplementation to
fish meal-based diets with low protein to energy ratios can
improve the protein utilization in rainbow trout (Yamamoto et al. 2005), because the relative proportion of the ingredients containing protein, that is, fish meal, soybean meal and
wheat flour, were the same between diets having different
protein to energy ratios, and therefore, the essential amino
acid balance of the test diets excluding the supplemental
amino acids is the same. In our study, 28CP diet fed to the
grass carp had lower soybean meal content and higher
maize meal content than in the high-protein diets, which
resulted in inferior essential amino acid balances. The effect
of supplemental limited amino acids to the lower-protein
diets for grass carp as well as for pig noted above was considered to be the result from the improvement in dietary
amino acid balance, not the same effect as found in the

low-protein diets with the same essential amino acid balance
as the high-protein diet (Kerr & Easter 1995).
Dietary protein levels also affected the morphological
measurements of grass carp (Table 4), VSI, IPF and HSI
were inversely related to dietary protein levels. This relationship has also been reported in several other studies

(Brown et al. 1992; Yang et al. 2002; Gaylord & Barrows
2009). In our study, VSI, IPF and HSI showed a significant decreasing trend with lysine and methionine supplementation. Gaylord & Barrows (2009) also found HSI and
IPF of rainbow trout (Oncorhynchus mykiss) were significantly reduced with multiple amino acid supplementation
in plant-based feeds. Brown et al. (1992) suggested that
IPF and HSI reflect the proportional accumulation of
energy in both the abdominal cavity and the liver, and it
has been widely acknowledged that feeding diets deficient
in amino acid results in excess energy deposition as fat in
the liver, fillet or abdominal cavity. The results indicated
that muscle, liver and whole body lipid contents of fish fed
32CP diet were slightly lower than that of fish fed 30CP
and 28CP diets, and muscle, liver and whole body lipid
contents showed a reducing trend with increasing lysine
and methionine supplementation. Several studies have
demonstrated that supplementation of lysine-and methionine-deficient diets with lysine and methionine reduced carcass lipid content of Indian Major Carp (Labeo rohita H.)
and rainbow trout (Cheng et al. 2003; Sardar et al. 2009).
But there was no differences in the body composition of
channel catfish fed diet with supplemental lysine and
methionine (Li & Robinson 1998). The reduction in carcass
lipid content of fish may be related to enhanced protein
synthesis as a result of lysine and methionine supplementation. The NR was increased with lysine and methionine
supplementation.
Total ammonia nitrogen was directly related to dietary
nitrogen and protein intake in teleosts (Rychly 1980; Beamish & Thomas 1984; Engin & Carter 2001; Yang et al.
2002). Our results indicated TAN excretion of grass carp
increased with increasing dietary protein levels regardless
of amino acid supplementation. In the present experiment,
the diets were isoenergenic, the diets with less dietary protein levels had higher dietary carbohydrate levels (i.e.
maize; Table 1), and conversely, the diets with higher dietary protein levels had lower dietary carbohydrate levels.
Some researchers found that increasing the dietary level

of non-protein digestible energy could increase NR by
decreasing nitrogen losses (Kaushik & Oliva Teles 1985;
Me´dale et al. 1995). In the present study, high crude
protein diets resulted in higher excretion of ammonia,

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd


which is in agreement with other reports (Savitz et al.
1977; Chakraborty et al. 1992; Cheng et al. 2003). TAN
excretion of grass carp was also reduced with lysine and
methionine supplementation in every dietary CP levels.
Viola & Lahav (1991) reported that feeding common carp
a diet containing a 250 g kgÀ1 CP supplemented with
5 g kgÀ1 lysine could reduce the amount of nitrogen excretion per unit WG by 20%. Viola et al.(1992b) further
reported that common carp could reduce nitrogen excretion
by about 20% by reducing dietary CP from 300 g kgÀ1 to
250 g kgÀ1 along with supplementing lysine (0.5%) and
methionine (0.3%). Cheng et al. (2003) also found that
TAN excretion by rainbow trout was reduced with lysine
supplementation. In this study, protein retention of grass
carp was slightly increased with lysine and methionine supplementation, and ammonia is the major end product of
protein catabolism (Elliott 1976), so TAN excretion of
grass carp was reduced with lysine and methionine supplementation.
In conclusion, results of the present investigation indicated
the growth performance of grass carp can be improved with
supplementation of lysine and methionine in practical diets,
and we can reduce the dietary crude protein from 320 g kgÀ1

to 300 g kgÀ1 in the practical diets through balancing amino
acids profile. TAN excretion of grass carp was also reduced
with lysine and methionine supplementation.

Abimorad, E.G., Favero, G.C., Castellani, D., Garcia, F. & Carneiro, D.J. (2009) Dietary supplementation of lysine and/or
methionine on performance, nitrogen retention and excretion in
pacu Piaractus mesopotamicus reared in cages. Aquaculture, 295,
266–270.
Ahmed, I. & Khan, M.A. (2004) Dietary lysine requirement of
fingerling Indian major carp, Cirrhinus mrigala (Hamilton).
Aquaculture, 235, 499–511.
AOAC (1984) Official Methods of Analysis, pp. 1141, Association
of Official Analytical Chemists, Arlington, VA.
Beamish, F. & Thomas, E. (1984) Effects of dietary protein and
lipid on nitrogen losses in rainbow trout, Salmo gairdneri. Aquaculture, 41, 359–371.
Bolin, D.W., King, R.P. & Klosterman, E.W. (1952) A simplified
method for the determination of chromic oxide when used as an
index substance. Science, 116, 634–635.
Borlongan, I. & Benitez, L. (1990) Quantitative lysine requirement
of milkfish (Chanos chanos) juveniles. Aquaculture, 87, 341–347.
Botaro, D., Furuya, W., Silva, L., Santos, L., Silva, T. & Santos,
V. (2007) Dietary protein reduction based on ideal protein concept for Nile Tilapia (Oreochromis niloticus) cultured in net pens.
Rev. Bras. Zootec., 36, 517–525.
Brown, M.L., Nematipour, G.R. & Gatlin, D.M. (1992) Dietary
Protein Requirement of Juvenile Sunshine Bass at Different
Salinities. Prog. Fish-Cult., 54, 148–156.

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd

Chakraborty, S., Ross, L. & Ross, B. (1992) The effect of dietary
protein level and ration level on excretion of ammonia in common carp, Cyprinus carpio. Comp. Biochem. Physiol. A Physiol.,
103, 801–808.
Cheng, Z.J., Hardy, R.W. & Usry, J.L. (2003) Plant protein ingredients with lysine supplementation reduce dietary protein level in
rainbow trout (Oncorhynchus mykiss) diets, and reduce ammonia
nitrogen and soluble phosphorus excretion. Aquaculture, 218,
553–565.
Conceicao, L.E.C., Grasdalen, H. & Ronnestad, I. (2003) Amino
acid requirements of fish larvae and post-larvae: new tools and
recent findings. Aquaculture, 227, 221–232.
Dabrowski, K. (1977) Protein requirements of grass carp fry (Ctenopharyngodon idella Val.). Aquaculture, 12, 63–73.
Du, Z.-Y., Liu, Y.-J., Tian, L.-X., Wang, J.-T., Wang, Y. & Liang,
G.-Y. (2005) Effect of dietary lipid level on growth, feed utilization and body composition by juvenile grass carp (Ctenopharyngodon idella). Aquacult. Nutr., 11, 139–146.
Elliott, J.M. (1976) The energetics of feeding, metabolism and
growth of brown trout, Salmo trutta, in relation to body weight,
water temperature and ration size. J. Anim. Ecol., 45, 923–948.
Engin, K. & Carter, C. (2001) Ammonia and urea excretion rates of
juvenile Australian short-finned eel (Anguilla australis australis) as
influenced by dietary protein level. Aquaculture, 194, 123–136.
FAO (1999) The FAO Yearbook of Fishery Statistics: Aquaculture
Production. FAO, Rome.
Furuya, W.M., Pezzato, L.E., Barros, M.M., Pezzato, A.C.,
Furuya, V.R.B. & Miranda, E.C. (2004) Use of ideal protein
concept for precision formulation of amino acid levels in fishmeal-free diets for juvenile Nile tilapia (Oreochromis niloticus
L.). Aquacult. Res., 35, 1110–1116.
Gaylord, T.G. & Barrows, F.T. (2009) Multiple amino acid supplementations to reduce dietary protein in plant-based rainbow
trout, Oncorhynchus mykiss, feeds. Aquaculture, 287, 180–184.
Kaushik, S. & Oliva Teles, A. (1985) Effect of digestible energy on
nitrogen and energy balance in rainbow trout. Aquaculture, 50,

89–101.
Kerr, B. & Easter, R. (1995) Effect of feeding reduced protein,
amino acid-supplemented diets on nitrogen and energy balance
in grower pigs. J. Anim. Sci., 73, 3000–3008.
Khan, M.A. & Jafri, A. (1993) Quantitative dietary requirement
for some indispensable amino acids in the Indian major carp,
Labeo rohita (Hamilton) fingerling. J. Aquac. Tropics, 8, 67–80.
Khan, M., Jafri, A. & Chadha, N. (2004) Growth, reproductive
performance, muscle and egg composition in grass carp, Ctenopharyngodon idella (Valenciennes), fed hydrilla or formulated
diets with varying protein levels. Aquacult. Res., 35, 1277–1285.
Kim, J.D., Lall, S. & Milley, J. (2001) Dietary protein requirements of juvenile haddock (Melanogrammus aeglefinus L.). Aquacult. Res., 32, 1–7.
Li, M. & Robinson, E. (1998) Effects of supplemental lysine and
methionine in low protein diets on weight gain and body composition of young channel catfish Ictalurus punctatus. Aquaculture,
163, 297–307.
Luo, Z., Liu, Y., Mai, K., Tian, L., Liu, D. & Tan, X. (2004)
Optimal dietary protein requirement of grouper Epinephelus coioides juveniles fed isoenergetic diets in floating net cages. Aquacult. Nutr., 10, 247–252.
Me´dale, F., Brauge, C., Valle´e, F. & Kaushik, S. (1995) Effects of
dietary protein/energy ratio, ration size, dietary energy source
and water temperature on nitrogen excretion in rainbow trout.
Water Sci. Technol., 31, 185–194.


Mukhopadhayay, N.R. & Ray, A.K. (1999) Improvement of quality of sal (Shorea robusta) seed meal protein with supplemental
amino acids in feed for rohu, Labeo rohita (Hamilton), fingerlings. Acta Ichthyol. Fiscal., 29, 25–39.
Robinson, E.H. & Li, M.H. (1994) Use of plant proteins in catfish
feeds: replacement of soybean meal with cottonseed meal and
replacement of fish meal with soybean meal and cottonseed
meal. J. World Aquac. Soc., 25, 271–276.
Robinson, E.H., Wilson, R.P. & Poe, W.E. (1980) Re-evaluation
of the lysine requirement and lysine utilization by fingerling

channel catfish. J. Nutr., 110, 2313–2316.
Rychly, J. (1980) Nitrogen balance in trout: II. Nitrogen excretion
and retention after feeding diets with varying protein and carbohydrate levels. Aquaculture, 20, 343–350.
Sardar, P., Abid, M., Randhawa, H.S. & Prabhakar, S.K. (2009)
Effect of dietary lysine and methionine supplementation on
growth, nutrient utilization, carcass compositions and haematobiochemical status in Indian Major Carp, Rohu (Labeo rohita
H.) fed soy protein-based diet. Aquacult. Nutr., 15, 339–346.
Savitz, J., Albanese, E., Evinger, M. & Kolasinski, P. (1977) Effect
of ration level on nitrogen excretion, nitrogen retention and efficiency of nitrogen utilization for growth in largemouth bass
(Micropterus salmoides). J. Fish Biol., 11, 185–192.
Takagi, S., Shimeno, S., Hosokawa, H. & Ukawa, M. (2002) Effect of
lysine and methionine supplementation to a soy protein concentrate diet for red sea bream Pagrus major. Fish. Sci., 67, 1088–1096.
Viola, S. & Lahav, E. (1991) Effects of lysine supplementation in
practical carp feeds on total protein sparing and reduction of
pollution. Isr. J. Aquacult./Bamidgeh, 43, 112–118.
Viola, S., Angeoni, H. & Lahav, E. (1992a) Limits of protein sparing by lysine supplementation of pondfish feeds. Israeli Journal
of Aquaculture/Bamidgeh, 44, 125–126.
Viola, S., Lahav, E. & Angconi, H. (1992b) Reduction of feed protein
levels and of nitrogenous N-excretions by lysine supplementation
in intensive carp culture. Aquat. Living Resour., 5, 277–285.
Wang, S., Liu, Y.H., Tian, L.X., Xie, M.Q., Yang, H.J., Wang, Y.
& Liang, G.Y. (2005) Quantitative dietary lysine requirement of

juvenile grass carp Ctenopharyngodon idella. Aquaculture, 249,
419–429.
Webster, C., Tiu, L., Morgan, A. & Gannam, A. (2000) Differences in Growth in Blue Catfish Ictalurus furcatus and Channel
Catfish Z. punctatus Fed Low-Protein Diets With and Without
Supplemental Methionine and/or Lysine. J. World Aquac. Soc.,
31, 195–205.
Yamamoto, T., Shima, T., Furuita, H., Shiraishi, M., Sa´nchezVa´zquez, F. & Tabata, M. (2000) Self-selection of diets with different amino acid profiles by rainbow trout (Oncorhynchus mykiss). Aquaculture, 187, 375–386.

Yamamoto, T., Shima, T., Furuita, H., Suzuki, N., Sanchez-Vazquez, F.J. & Tabata, M. (2001) Self-selection and feed consumption of diets with a complete amino acid composition and a
composition deficient in either methionine or lysine by rainbow
trout, Oncorhynchus mykiss (Walbaum). Aquacult. Res., 32, 83–
91.
Yamamoto, T., Sugita, T. & Furuita, H. (2005) Essential amino
acid supplementation to fish meal-based diets with low protein
to energy ratios improves the protein utilization in juvenile
rainbow trout Oncorhynchus mykiss. Aquaculture, 246, 379–
391.
Yang, S., Liou, C. & Liu, F. (2002) Effects of dietary protein level
on growth performance, carcass composition and ammonia
excretion in juvenile silver perch (Bidyanus bidyanus). Aquaculture, 213, 363–372.
Yang, H., Liu, Y., Tian, L., Liang, G. & Lin, H. (2010) Effects of
Supplemental Lysine and Methionine on Growth Performance
and Body Composition for Grass Carp (Ctenopharyngodon idella). Am. J. Agric. Biol. Sci., 5, 222–227.
Zang, W.-l. (1991) Water quality analyses for fish culture [M].
Agriculture Press, Beijing.
Zarate, D.D. & Lovell, R.T. (1997) Bioavailability of free vs. protein-bound lysine in practical diets for young channel catfish
(Ictalurus punctatus). Aquaculture, 159, 87–100.

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

Aquaculture Nutrition 18; 589–598 ª 2012 Blackwell Publishing Ltd


Aquaculture Nutrition
2012 18; 599–609

doi: 10.1111/j.1365-2095.2012.00944.x

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

1
1

2

2

National Institute of Nutrition and Seafood Research (NIFES), Bergen, Norway;

Atlantic salmon fed diets devoid of fishmeal but added
0.5 g kg−1 fish protein concentrate (FPC) showed reduced
growth and lipid deposition without affecting protein accretion as compared to fish fed a fishmeal-based control diet.
The aim of the current study was to assess whether higher
inclusion of FPC improved the growth and lipid deposition
of Atlantic salmon (initial body weight 380 g) fed high
plant protein diets. Quadruplicate groups of fish were fed
diets containing 200 g kg−1 fishmeal of which was replaced
with FPC (150, 112, 75, 38 and 0 g kg−1) for a period of
79 days. The rest of the diet protein was a mixture of plant
proteins. The lipid source used was fish oil. A fishmealbased diet was included as a positive control for growth
performance. None of the test diets differed from the positive control-fed fish in voluntary feed intake, growth performance or nutrient accretion. Thus, the test diets were
found appropriate to assess the effect of FPC inclusion.
Replacement of fishmeal with increasing concentration of
FPC did not affect voluntary feed intake (P = 0.56), but
growth performance decreased (P = 0.02) resulting in an
increased feed conversion ratio (P = 0.003). Viscerosomatic
index decreased as diet FPC inclusion increased (P = 0.012)
without affecting the dress out weight (P = 0.08). Thus, the

apparently improved growth in fish fed the diets with the low
FPC inclusion was because of a higher visceral mass. Possible
reasons for the reduced visceral mass following addition of
FPC to high plant protein diets are discussed.
KEY WORDS:

Atlantic salmon, fish protein concentrate, growth,
plant proteins, taurine, visceral mass

Received 15 June 2011; accepted 13 November 2011
Correspondence: Marit Espe, National Institute of Nutrition and Seafood
Research (NIFES), PO Box 2029, Nordnes N-5817, Norway. E-mail:


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

ª 2012 Blackwell Publishing Ltd

2

EWOS Innovation AS, Dirdal, Norway

Any increase in farmed fish production requires the use of
alternative protein sources as the wild fish catch will not
increase to any extent (Tacon 1995; Tacon et al. 2006). Generally, fish fed diets with high inclusions of plant protein
ingredients shows poorer performance than fish fed marine
ingredients (Gomes et al. 1995; Kaushik et al. 1995, 2004;
Mambrini et al. 1999; de Francesco et al. 2004). When the
total or a major proportion of diets are replaced by plant
proteins and the diets are added hydrolysed fish protein concentrate (FPC), voluntary feed intake and growth improve

(Fournier et al. 2004; Espe et al. 2006, 2007). Low inclusion
of FPC in fishmeal-based diets has been reported to improve
the voluntary feed intakes and growth, while higher inclusion levels decreased the growth (Espe et al. 1999; Refstie
et al. 2004; Hevrøy et al. 2005). When Atlantic salmon were
offered diets without any fishmeal, but added 50 g kg−1
FPC, the growth was reduced because of a reduced lipid
accretion, while protein accretion was unaffected (Espe
et al. 2006). The improved voluntary feed intakes following
the addition of FPC to plant-based diets might be due to
that FPC also supplies non-amino acid nitrogen compounds
having an attractive smell and supplies taurine that is not a
constituent of plant ingredients (Espe & Lied 1999; Liaset &
Espe 2008). Atlantic salmon synthesize taurine provided
that they are fed adequate methionine (Espe et al. 2008,
2010, 2011). Recently, Espe et al. (2012) reported that supplementation with 1 g kg−1 taurine to a high plant protein
diet reduced the body lipid without affecting the body protein in juvenile Atlantic salmon and increased both liver and
muscle pools of free amino acids. In addition, liver concentration of polyamines increased in fish fed the taurine supplemented diet as compared to fish fed diets not
supplemented with taurine. Atlantic salmon fed methioninelimiting diets had reduced liver taurine and stored more triacylglycerol (TAG) in liver as compared to fish fed diets


being adequate in methionine (Espe et al. 2010). On the
other hand, Dias et al. (2005) reported lower activity of the
lipogenic enzymes and decreased TAG accumulation in the
marine teleost European seabass (Dicentrarchus labrax) fed
soy protein containing low methionine as compared to a
fishmeal-fed control. Gaylord et al. (2007) reported that dietary methionine but not taurine affected lipid metabolism
and viscera mass in juvenile rainbow trout (Oncorhynchus
mykiss). Rats fed proteins containing low taurine and glycine contained more visceral fat as compared to rats fed protein sources with higher concentrations of these amino acids
(Liaset et al. 2009). As our previous experiments in which
Atlantic salmon were fed high plant protein diets only tested

an inclusion of 50 g FPC kgÀ1 diet (Espe et al. 2006, 2007),
and growth improved by higher FPC inclusion in fishmealbased diets (Espe et al. 1999), the current study aimed to
test whether FPC replacement of the 200 g kg−1 dietary
fishmeal present affected voluntary feed intake and growth.
As a positive control for voluntary feed intake and growth,
a diet containing 350 g fishmeal kg−1 diet without any FPC
was used.

Five 6-mm extruded diets were prepared in which the 200 g
fishmeal present kg−1 diet was replaced with graded levels
of FPC such that the final diets contained 0, 37.5, 75,
112.5, or 150 g FPC kgÀ1 diet. The FPC used was made
from blue whiting (Micromesisticus poutasou). FPC mirrors
fishmeal protein and amino acid composition, while its solubility may differ dependent on storage time and temperature (Espe & Lied 1999). Therefore, the protein solubility
was analysed in the diets, and the higher the inclusion of
FPC the higher is the solubility of dietary proteins. The
lowest fishmeal inclusion was set to 5% as Atlantic salmon
fed diets without any fishmeal grew less than the fishmealfed control (Espe et al. 2006), but when 50 g kgÀ1 diet fishmeal was included to such diets, growth did not differ from
the fishmeal-based control diet (Espe et al. 2007). The
remaining diet protein was a blend of plant protein ingredients. As a control for growth performance and nutrient
accretion, a diet containing 350 g fishmeal kgÀ1 diet without any FPC was included in the trial. The lipid source
used in all diets was fish oil. All test diets contained the
same energy content as well as an amino acid profile to fulfil the requirement of Atlantic salmon. The diet composition and amino acid profiles are listed in Tables 1 & 2.

Table 1 Composition of diets (g kgÀ1)
Fishmeal (g kgÀ1)
FPC (g kgÀ1)

350
Control

50
150

87.5
112.5

125
75

162.5
37.5

200
0

Ingredients
Fishmeal
FPC
Wheat gluten
Plant protein
Micronutrients
Fish oil

354
0
40
323
16
267

50
150
120
381
25
274

87.5
112.5
120
382
25
273

125
75
120
382
25
273

162.5
37.5
120
382
25
273

200

0
120
382
25
273

Composition
Dry matter
Protein
Lipid
Ash
Gross energy
DE (MJkgÀ1)
DP/DE (gMJÀ1)

948
440
287
85
24.0
20.2
18.9

936
423
285
77
23.7
19.8
18.8

942
418
290
78
23.9
20.0
18.6

940
416
276
79
23.7
19.2
18.6

947
441
276
80
24.1
20.0
19.6

946
444
272
78
24.1

19.9
19.9

Micronutrient mixture is proprietary of EWOS Innovation but
contains vitamins and minerals added to fulfil the requirement of
Atlantic salmon (NRC 1993) in addition to the inert marker, 0.1%
yttrium oxide. Plant protein was soy protein concentrate and pea
protein concentrate blended 1 : 1.14 by weight.
FPC, fish protein concentrate.

Fifty Atlantic salmon with a mean body weight (BW) of
380 g were used in each of 24 tanks (size of the tanks of quadrangular fibreglass tanks with a water volume of 13 m3).
Each tank was supplied with running seawater (salinity
30 g LÀ1 and ambient temperature, ranged from 11.4 to
7.2 °C from November 2006 to February 2007) at a flow rate
of 1.5 L kgÀ1 biomass minÀ1. A 24-h light regime was used
to maximize growth. Each experimental diet was randomly
allocated among the tanks, and each diet was fed to quadruplicate tanks of fish. The fish were fed three times daily to
apparent satiation using automatic belt feeders (Hølland
Teknologi, Sandnes, Norway). All tanks were equipped with
feed collectors (Excess Fish feed collector; Hølland Teknologi) to measure the actual feed intake. Uneaten feed was collected daily. At the start and end of the experiment, fish were
individually weighed. A pooled sample of 10 fish was collected at the start of the experiment to be analysed for chemical composition. At the end of the growth experiment, six
fish from each tank were sampled 5 h postprandial and killed
with a sharp blow to the head. Individual BW to the nearest
g and fork length to the nearest 0.5 cm were recorded in each
of the sampled fish and the condition factor (CF = BW in
grams/length in cm3) calculated. Blood was drawn from the
caudal vein into heparinized syringes and centrifuged at
1800 g for 10 min before plasma was collected. Viscera and
liver were collected and weighed for calculation of indexes

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd


Table 2 Amino acid composition (g per 16 g N) of the experimental diets
Diet amino acids
Fishmeal
(g kgÀ1)
FPC (g kgÀ1)
IAAs
Met
Lys
Thr
Leu
Ile
Val
Phe
His
Arg
Trp
DAAs
Glu
Asp
Ser
Gly
Pro
Ala
Tyr

OH-Pro
ΣAA-N
IAA/DAA
Tau
Solubility
NAA-N

350
Control

50
150

87.5
112.5

125
75

162.5
37.5

200
0

2.7
7.5
3.5
7.1
4.2

4.6
4.4
1.9
6.4
0.98

2.5
6.4
3.5
6.8
4.1
4.4
4.4
1.8
6.4
0.95

2.6
6.3
3.4
6.9
4.1
4.5
4.5
1.8
6.4
0.98

2.7
6.8

3.6
7.3
4.2
4.6
4.7
1.9
6.6
1.03

2.7
6.6
3.4
7.1
4.0
4.4
4.6
1.9
6.3
1.04

2.6
6.4
3.4
7.4
3.5
4.3
4.6
1.9
6.1
1.08

16.7
9.0
4.5
5.1
5.0
5.0
3.5
0.5
92.9
0.87
0.3
24.5
7.1

19.0
7.9
4.5
4.5
6.3
4.3
3.4
0.4
92.0
0.82
0.4
45.8
8.0

19.5

8.3
4.8
4.7
6.6
4.4
3.4
0.5
94.0
0.79
0.3
40.0
6.0

20.2
8.8
4.8
4.8
6.7
4.7
3.6
0.5
97.8
0.80
0.3
30.8
2.2

19.9
8.7
4.7

4.7
6.3
4.5
3.4
0.5
95.0
0.79
0.3
25.7
5.0

19.5
8.5
4.6
4.5
6.4
4.4
3.5
0.5
93.4
0.79
0.2
15.8
6.6

Tau is taurine, and NAA-N (non-amino acid N) is nitrogen not
accounted for by the amino acid analyses. Tau is not included in
the IAA : DAA ratios. Solubility was analysed as the % free of total
alpha amino acid N in diets analysed after reaction with TNBS.
FPC, fish protein concentrate.

relative to BW. Liver and white epaxial trunk muscle was
dissected and flash frozen in liquid nitrogen. Remaining fish
in the tanks were then fed their respective diets for a period
of 1 week after which faeces was collected by stripping.
Thereafter, feed was withheld for 2 days until the rest of the
fish were individually weighed and the length recorded. Eviscerated fish were also weighed and used to calculate dress
out weight (10 fish per tank). Before handling, fish were
sedated with AQUI-S (4 ppm) and fully anaesthetized with
MS222 (50 mg LÀ1). The experimental protocol was
approved by the Norwegian Board of Experiments with Living Animal.

All chemical analyses were carried out in duplicate. Nitrogen was determined after total combustion using a Nitrogen

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

Analyser (Perkin Elmer, 2410 Ser. II, Norwalk, CT, USA).
The total lipid in feed and faeces was determined gravimetrically as the sum of free and bound lipid. Free or loosely
bound lipid was extracted with petroleum ether and dried at
103 ± 1 °C. The samples were thereafter hydrolysed with
HCl in a Tecator Soxtec Hydrolysing unit to release the
bound lipid, which was extracted with petroleum ether and
dried at 103 ± 1 °C. Dry weight and ash content were determined gravimetrically after freeze-drying the samples and
dried to constant weight in an oven at 550 °C, respectively.
Gross energy was analysed by Parr Bomb Calorimetry
(Moline, IL, USA). Muscle lipid content was analysed gravimetrically after extraction with ethyl acetate. Yttrium was
determined in both the feed and faeces by the use of ICPMS as described (Espe et al. 2006). Dietary and faecal
amino acid concentration was determined after acidic

hydrolysis in 6 N HCl at 110 °C for 22 h and prederivatization with phenylisothiocyanate (PITC®) and analysed
according to the study by Cohen & Strydom (1989). Dietary
tryptophan was determined after basic hydrolyses in Ba
(OH)2 for 20 h at 110 °C using HPLC (Supelcosil LC-18
column) as described (Liaset et al. 2003). Amino acid composition in de-proteinized plasma, liver and muscle was
determined on the Biochrom 20 plus Amino Acid Analyzer
(Amersham Pharmacia Biotech, Uppsala, Sweden) equipped
with a lithium column using postcolumn derivatization with
ninhydrin as described (Espe et al. 2006). The degree of
hydrolysis in the feeds was determined as the free alpha
amino groups relative to the total after being hydrolysed in
6 N HCl at 110 °C for 22 h as described (Espe et al. 1999).
Lipid classes in liver were determined as described by Bell
et al. (1993) with modification as described (Rathore et al.
2010). S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) were extracted from liver in 0.4 mol LÀ1
HClO4, separated on a reverse phase C18 column and
detected at 254 nm as described (Wang et al. 2001). The
polyamines putrescine, spermine and spermidine were
extracted in 0.4 mol LÀ1 HClO4 precolumn derivatized with
dansyl chloride and separated on a C18 reverse phase column and detected at 254 nm as described (Liaset & Espe
2008). The quantification of SAM, SAH and the polyamines
was performed by standards from Sigma (Sigma Aldrich,
Munich, Germany).

Feed conversion ratio (FCR) was calculated from the
amount of diet fed (kg dry matter) and the total biomass
(kg) gained:


FCR ¼ ðkg diet fedÞ Â ðkg final biomass

À kg initial biomass þ kg dead fishÞÀ1 :
Specific growth rate (SGR) was calculated as % daily
growth increase
SGR ¼ ðln BW2 À ln BW1 Â days of experimentÀ1 Þ Â 100
where BW1 and BW2 represent the initial and final biomass
in grams, respectively.
Protein efficiency ratio (PER) was calculated as weight
gain (g) for each gram protein fed/consumed
PER ¼ ðBW2 À BW1 Â protein fedÀ1 Þ Â 100:
Protein productive value (PPV) and energy productive
value (EPV) were calculated as retained nutrient (g) of fed
nutrient (g)
PPV ¼ ðfinal protein content À initial protein contentÞ
 protein fedÀ1 :
EPV ¼ ðfinal energy content À initial energy contentÞ
 energy fedÀ1 :

ANOVA was used to evaluate any diet-induced changes in
intakes and performance between fish fed the test diets and
the fishmeal-fed control. Homogeneity in variation was
tested using Levenes test. To determine the effect of diet
(i.e. FPC inclusion) on performance and tissues amino acid
concentrations, regression analyses were applied according

to the statistical design of the experiment. Any differences
between the liver lipids, SAM, SAH and polyamines in fish
fed the control feed and those fed the test diet added 0 or
150 g FPC kgÀ1 diets were evaluated by ANOVA followed by
Tukey’s test. All tests were performed using the statistical
program STATISTICA version 9.0 (Statsoft Inc, Tulsa, OK,

USA), and P < 0.05 was accepted significantly different.

To determine whether FPC improved voluntary feed intake
and growth, Atlantic salmon were fed high plant protein
diets where up to 75% of the dietary fishmeal present was
replaced with FPC. Growth increase relative to initial BW
was 187% in the fishmeal-fed control, while the corresponding values in test diets were 159–183% (Table 3). No
significant differences existed between fish fed the fishmeal
control diet and any of the test diets in either mean voluntary feed intake or growth performance. Neither did the
test diets differ from the control-fed fish in protein accretion (PER: range 2.46–2.76, PPV: range 0.49–0.52), in
energy accretion (EPV: range 0.52–0.56), in relative liver
size (hepatosomatic index (HSI): range 1.02–1.10) or in viscerosomatic index (VSI: range 9.72–10.57). All test diets
were digested equally well as was the reference fishmeal
diet (ADC-N: range 86–89, ADC-energy: range 81–84,
Table 4). Thus, the test diets supported the growth and
nutrient accretion equally well as did the fishmeal-based
control diet containing from two to seven times more fishmeal. Based on these results, the test diets were found
acceptable to be used to evaluate the effect of increasing
the FPC inclusion in the Atlantic salmon using the regression design.

Table 3 Start and end BW (g), growth increase (DG,% of start wt), mean feed intake (MFI, g per fish per day), feed conversion ratio
(FCR), hepatosomatic index (HSI), visceasomatic index (VSI), condition factor (CF), protein efficiency ratio (PER) and protein productive
value (PPV) in Atlantic salmon fed the respective diets for a period of 79 days
Fishmeal (g kgÀ1)
FPC (g kgÀ1)

350
Control

Start wt

End wt
DG
MFI
FCR
HSI
VSI
CF
PER
PPV

371
1065
187
7.2
0.83
1.02
9.94
1.31
2.76
0.52

±
±
±
±
±
±
±
±
±

±

50
150
5
47
13
0.3
0.05
0.02
0.12
0.02
0.16
0.01

379
981
159
7.1
0.93
1.05
9.72
1.35
2.55
0.50

87.5
112.5
±
±

±
±
±
±
±
±
±
±

3
25
6
0.3
0.01
0.03
0.24
0.03
0.02
0.02

382
991
159
7.1
0.92
1.06
10.03
1.37
2.59
0.51

125
75
±
±
±
±
±
±
±
±
±
±

3
32
9
0.2
0.02
0.01
0.28
0.01
0.05
0.01

380
1001
164
7.0
0.89

1.08
9.92
1.36
2.71
0.51

162.5
37.5
±
±
±
±
±
±
±
±
±
±

3
34
10
0.3
0.02
0.03
0.11
0.03
0.05
0.01

387
1027
165
7.1
0.87
1.04
10.01
1.36
2.59
0.51

200
0
±
±
±
±
±
±
±
±
±
±

3
23
6
0.1
0.01
0.02

0.16
0.02
0.04
0.01

380
1077
183
7.3
0.87
1.10
10.57
1.39
2.46
0.49

±
±
±
±
±
±
±
±
±
±

2
37
9

0.3
0.02
0.02
0.12
0.01
0.12
0.02

Values are tank means ± SE, n = 4.
BW, body weight; FPC, fish protein concentrate.

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd


Table 4 Apparent digestibility (%) of crude compounds and amino acids in Atlantic salmon fed the test diets. The control diet is listed for
comparison
Fishmeal (g kgÀ1)
FPC (g kgÀ1)

350
Control

50
150

87.5
112.5

125
75

162.5
37.5

200
0

Nitrogen
Lipids
Energy
IAA’s

88 ± 0.3
95 ± 0.2
84.2 ± 0.4

88 ± 1
95 ± 0.3
83.6 ± 0.6

89 ± 0.2
96 ± 0.2
83.7 ± 0.3

86 ± 1
94 ± 1
81.01.2

89 ± 1
95 ± 0.5
83.1 ± 1.3

89 ± 1
95 ± 0.4
82.4 ± 1.4

Met
Lys
Arg
Leu
Val
Ile
His
Thr
Phe
Trp
DAA’s

90.4
94.1
93.2
92.9
91.3
92.0
88.3
90.3
91.7
na

±
±
±
±
±
±
±
±
±

0.1
0.1
1.2
0.1
0.1
0.3
0.5
0.4
0.1

90.8
93.0
94.4
92.6
90.6
91.9
81.7
89.3
92.5

na

±
±
±
±
±
±
±
±
±

0.5
0.3
0.3
0.3
0.3
0.3
2.3
0.5
0.2

90.8
92.4
94.2
92.6
90.7
91.9
85.0
88.6

92.4
na

±
±
±
±
±
±
±
±
±

0.4
0.1
0.1
0.2
0.3
0.3
1.6
0.1
0.2

88.2
92.2
93.0
91.0
88.5
89.9
81.3

87.1
90.6
na

±
±
±
±
±
±
±
±
±

0.9
0.4
0.8
0.9
0.8
0.8
2.9
1.2
0.8

90.3
92.9
94.1
92.3
89.9
91.2

85.1
88.6
91.7
na

±
±
±
±
±
±
±
±
±

0.7
0.8
1.0
1.1
1.3
1.2
3.6
1.6
1.0

88.7
93.0
94.3
92.5
89.8

90.4
82.7
88.5
90.5
na

±
±
±
±
±
±
±
±
±

1.1
0.8
0.8
1.0
1.2
1.3
2.9
1.4
1.5

94.2
88.5
65.6
84.3

86.6
91.3
91.0
91.1
na

±
±
±
±
±
±
±
±

0.2
0.9
2.9
0.4
0.2
0.1
0.4
0.1

94.3
88.8
65.3
85.6
87.5
91.2

91.5
91.0
na

±
±
±
±
±
±
±
±

0.4
1.0
2.2
0.7
0.6
0.3
0.3
0.2

94.4
89.5
67.1
85.8
87.6
91.0
91.9
90.7

na

±
±
±
±
±
±
±
±

0.1
0.5
0.8
0.3
0.2
0.2
0.3
0.5

93.3
88.4
64.2
83.9
84.9
89.1
89.8
88.9
na

±
±
±
±
±
±
±
±

0.7
0.9
1.5
1.1
1.1
0.9
0.8
0.8

94.8
90.4
66.1
85.9
86.5
90.2
91.4
90.0
na

±
±

±
±
±
±
±
±

1.0
0.7
3.1
1.9
1.5
1.2
1.1
1.5

94.6
89.3
62.4
85.5
85.5
90.1
91.4
90.0
na

±
±
±
±

±
±
±
±

0.8
0.9
2.0
1.2
1.4
1.2
1.0
1.1

Crude components

Glu
Asp
OH-pro
Ser
Gly
Ala
Pro
Tyr
Cys

Values are tank means ± SE.
na, not analysed; FPC, fish protein concentrate.

Table 5 Effect of dietary FPC inclusion (x, %) on growth performance

and accretion (y)

Performance
y

y = ax + b

P-values

r

Start BW (g)
End BW (g)
VFI (g kgÀ1 ABW per day)
FCR
PPV
PER
Dress out wt (g kgÀ1 ABW)
Daily protein gain (g kgÀ1 ABW)
Daily lipid gain (g kgÀ1 ABW)
HSI
VSI
CF
Body lipid/protein ratio

y
y
y
y
y

y
y
y
y
y
y
y
y

0.48
0.02
0.56
0.003
0.99
0.50
0.08
0.27
0.18
0.25
0.012
0.23
0.64

À0.17
À0.51
0.14
0.64
0.005
0.16
À0.41

À0.26
À0.31
À0.27
À0.56
À0.28
À0.11

=
=
=
=
=
=
=
=
=
=
=
=
=

383.0 À 0.2x
1061.5 À 6.1x
7.9 + 0.01x
0.86 + 0.004x
0.51 + 0.0002x
2.6 + 0.04x
844.8 À 5.2x
1.8 À 0.01x
1.6 À 0.01x

1.08 À 0.02x
10.4 À 0.05x
1.38 À 0.002x
0.81 À 0.01x

ABW is arbitrary body weight (initial BW+end BW/2). VFI is the mean voluntary feed intake.
Dress out weight is the BW-viscera weight. The other abbreviations are given in Material and
methods, and bold letters indicate statistical difference (P < 0.05).

The dietary amino acid profiles were similar in all of the
test diets where the fishmeal present was replaced with
increasing amount of FPC (Table 2). However, the

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

increased FPC inclusion increased dietary taurine and
decreased tryptophan as these amino acids were higher
and lower in the FPC as compared to the fish meal,


respectively. The higher the FPC inclusion, the higher the
nitrogen solubility becomes (Table 2). Apparent amino
acids digestibility of all diets were high (Table 4), and
replacement of the dietary fishmeal with FPC had no effect
on digestibility. Replacement of the fishmeal with FPC had
no significant effect on voluntary feed intake (P = 0.56),
but reduced the end BW (P = 0.02). As a consequence,
these fish had a higher feed conversion (FCR, P = 0.003,

Table 5). Replacement of fishmeal with increasing amounts
of FPC had no significant effect on protein utilization
(PER and PPV, Table 5). The body lipid-to-protein ratio
was not affected by treatment (P = 0.64) neither was the
lipid nor protein gain. Increasing the dietary FPC inclusion
significantly reduced VSI (P = 0.012), while HSI and CF
did not significantly decreased (Table 5). The dress out

weight was not significantly affected (P = 0.08) by the dietary FPC inclusion (Table 5).
Plasma, liver and muscle pools of free amino acids generally decreased when dietary fishmeal was replaced with
FPC (Table 6). However, the decrease in amino acid concentration was only significant for methionine and glutamine in plasma and muscle, histidine in plasma and liver,
phenylalanine in plasma, and glycine and proline in muscle,
while tyrosine was significantly reduced in plasma, liver
and muscle (Table 6). Free tryptophan was only found in
plasma, while the tryptophan concentration in both liver
and muscle was below the detection level. In plasma, tryptophan decreased as dietary FPC increased. The only free
amino acids that increased when dietary FPC inclusion
increased were arginine, serine and alanine (Table 6). These

Table 6 Effects of dietary FPC inclusion (x, % inclusion) on postprandial amino acids and nitrogen metabolites in plasma, liver and white
muscle (y, lmol per 100 g or lmol dLÀ1)
Plasma (lmol dLÀ1)

Liver (lmol per 100 g)

White trunk muscle (lmol per 100 g)

y

y = ax + b

P

r

y = ax + b

P

r

y = ax + b

P

r

Met
Cys
Lys
Arg
Leu
Val
Ile
His
Trp
Thr
Phe
Glu
Gln

Asp
Ser
Gly
Ala
Pro
Tyr
Metabolites
Taurine
P-ethanolamine
Cystathionine
Urea
Ammonia
Ornithine
Citrulline
Carnosine
Anserine
1 methyl histidine
b-Alanine
Arg/Lys ratio
Tau/Met ratio

y=
nd
y=
y=
y=
y=
y=
y=
y=

y=
y=
y=
y=
y=
y=
y=
y=
y=
y=

0.050
–
0.54
0.18
0.19
0.18
0.13
0.02
0.006
0.05
0.009
0.13
0.005
0.25
0.21
0.23
0.003
0.52
0.003

À0.46
–
À0.14
0.32
À0.30
À0.31
À0.35
À0.51
À0.60
À0.48
À0.57
À0.36
À0.62
À0.28
0.30
À0.29
0.64
À0.16
À0.62

y=
y=
y=
y=
y=
y=
y=
y=
nd

y=
y=
y=
y=
y=
y=
y=
y=
y=
y=

45.9 À 0.2x
3.3 + 0.2x
132.1 À 1.4x
44.7 + 0.3x
134.3 À 1.3x
100 À 0.6x
51.4 À 0.3x
93.4 À 1.1x

0.53
0.17
0.07
0.49
0.02
0.17
0.14
0.004
–
0.20

0.22
1.0
0.7
0.88
0.37
0.07
0.40
0.25
0.007

À0.15
0.35
À0.42
0.16
À0.52
À0.33
À0.36
À0.62
–
À0.31
À0.30
0.001
À0.11
À0.04
0.21
À0.42
0.21
0.28
À0.62

y=
nd
y=
y=
y=
y=
y=
y=
nd
y=
y=
y=
y=
y=
y=
y=
y=
y=
y=

90.9 À 1.1
14.5 À 0.2x
72.6 À 0.04x
120.5 À 2.6x
6.1 À 0.1x
38.3 + 2.5x
428.3 À 7.7
207.0 + 1.9x
122.4 À 2.2x
31.4 À 0.3x

0.049
–
0.07
0.005
0.33
0.19
0.17
0.10
–
0.051
0.08
0.93
0.017
0.06
0.0001
0.0001
0.048
0.010
0.009

À0.47
0.41
0.62
À0.24
À0.31
À0.32
À0.39
–
À0.45

À0.40
À0.02
À0.56
À0.42
0.78
À0.77
0.46
À0.57
À0.58

0.54
0.93
0.00002
0.003
0.004
0.35
0.03
–
–
0.03
0.10
0.01
0.10

À0.15
À0.02
À0.80
À0.65
À0.62
À0.22

À0.50
–
–
0.50
0.38
0.58
0.40

y=
y=
y=
y=
y=
y=
nd
nd
nd
nd
y=
y=
y=

2674 À 5x
19.0 À 0.2x
46.2 À 1.5x
1087 À 32x
382.5 À 3.6x
36.5 À 0.1x

0.18

0.15
0.002
0.02
0.15
0.62
–
–
–
–
0.98
0.012
0.69

À0.31
À0.34
À0.66
À0.54
À0.33
À0.12
–
–
–
–
À0.05
0.55
0.10

83.1 + 0.2x
5.06 À 0.04x
9.1 À 0.2x

112.5 À 3.7x
360.3 À 0.6x
3.86 + 0.05x
7.42 + 0.02x
24.0 À 0.89x
1093.8 À 4.9x
5.79 À 0.06x
166.5 À 1.2x
0.15 + 0.02x
4.73 + 0.08x

0.56
0.18
0.0005
0.0001
0.10
0.28
0.90
0.0004
0.004
0.36
0.45
0.18
0.14

0.14
À0.32
À0.70
À0.79
À0.38

0.25
0.03
À0.73
À0.61
À0.22
À0.18
0.32
0.38

y=
y=
y=
y=
y=
y=
y=
nd
nd
y=
y=
y=
y=

30.7 À 0.4x
36.1 À 0.1x
17.7 À 0.1x
73.5 À 0.6x
78.4 À 0.6x
39.2 À 0.4x
8.1 À 0.1x

2.29 À 0.05x
40.4 À 0.5x
26.7 À 0.4x
37.8 À 0.3x
56.8 À 0.9x
6.5 À 0.05x
18.7 + 0.2x
50.7 À 0.2x
46.5 + 0.8x
26.5 À 0.2x
24.2 À 0.4x
131.6 À 0.3x
6.3 À 0.0003x
1.9 À 0.06x
115.5 À 2.1x
67.1 À 1.2x
2.0 À 0.01x
4.6 À 0.09x

3.7 + 0.2x
9.5 + 0.3x
0.48 + 0.07x
4.28 + 0.09x

10.3 À 0.9x
39.6 À 0.3x
765 À 0.03x
1149 À 15x
160 À 0.08x
199 + 1x

283 À 2x
452 + 1x
106 + 2x
56.8 À 0.6x

61.0 À 0.01x
0.34 + 0.01x
58.98 + 0.17x

y
y
y
y
y
y
y
y
y
y
y
y
y

=
=
=
=
=
=
=

=
=
=
=
=
=

18.2 À 0.3x
55.5 + 1.0x
8.1 + 0.3x
34.9 À 0.1x
30.3 À 0.2x
12.3 À 0.09x
7.9 À 0.2x

Bold letters indicate statistical differences.
FPC, fish protein concentrate.

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd


Table 7 Neutral lipid classes, taurine-to-methionine ratio, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), SAM-to-SAH
ratio, arginine-to-lysine ratio, the polyamines putrescine, spermine and spermidine and the spermidine-to-spermine ratio as occurring 5 h
postprandial in liver of fish fed the control diet and the test diets containing 0 and 150 g FPC kgÀ1
Diets

0 g FPC kgÀ1 diet

150 g FPC kgÀ1 diet

Control

P-value

TAG (mg gÀ1 liver, wet wt)
DAG (mg gÀ1 liver, wet wt)
NEFA (mg gÀ1 liver, wet wt)
Cholesterol (mg gÀ1 liver, wet wt)
Taurine/methionine
SAM (nmol gÀ1 wet wt)
SAH (nmol gÀ1 wet wt)
SAM/SAH
Arginine/lysine
Putrescine (lmol gÀ1 wet wt)
Spermine (lmol gÀ1 wet wt)
Spermidine (lmol gÀ1 wet wt)
Spermidine/spermine

24.9
1.03
1.15
2.93
59.4
56.8
55.4
1.04
0.33
1.09

0.39
0.58
1.47

27.4
0.84
1.18
2.88
62.9
67.2
37.1
1.83
0.41
0.95
0.46
0.57
1.25

19.9
0.89
1.17
3.07
64.0
57.2
49.2
1.16
0.33
1.12
0.39
0.58

1.47

0.18
0.25
0.98
0.06
0.87
0.35
0.30
0.001
0.07
0.23
0.055
0.95
0.02

±
±
±
±
±
±
±
±
±
±
±
±
±

2.4
0.08
0.12
0.05
1.8
4.2
4.8
0.11B
0.03
0.05
0.02
0.04
0.04A

±
±
±
±
±
±
±
±
±
±
±
±
±

4.2
0.08

0.10
0.05
6.7
6.7
4.0
0.14A
0.02
0.09
0.02
0.02
0.05B

±
±
±
±
±
±
±
±
±
±
±
±
±

1.3
0.06
0.12
0.06

7.2
4.9
1.6
0.07B
0.02
0.06
0.01
0.02
0.05A

SAH was not homogeneous in variance, and Kruskall–Wallis test was used. Values are mean ± SE, n = 4.
DAG, diacylglycerol; FPC, fish protein concentrate; NEFA, non-esterified fatty acids; SAH, S-adenosylhomocysteine; TAG, triacylglycerol.
Row means followed by different letters are significantly different (p < 0.05).

changes in amino acid profiles occurred even though the
dietary concentration of these amino acids were similar
(Table 2) and the mean voluntary feed intake was unaffected by treatment (Table 5). The nitrogen-containing
metabolites generally declined as dietary FPC increased
(Table 6). However, the postprandial taurine concentration
in plasma, liver or white trunk muscle was not affected by
the FPC inclusion (Table 6) even though FPC inclusion in
the diets increased the diet taurine (Table 2). Cystathionine
and urea were significantly reduced in plasma, liver and
white trunk muscle, while citrulline only was significantly
decreased in the plasma (Table 6). The imidazole dipeptides, anserine (b-alanyl-1-methylhistidine) and carnosine
(b-alanylhistidine) were significantly reduced in white trunk
muscle rendering a significant increase in plasma 1-methyl
histidine (Table 6).
The altered amino acid profiles thus indicate that the
inclusion of FPC in the diets caused changes in the metabolism. Therefore, the control-fed fish and the fish fed the

diets containing 150 or 0 g FPC kgÀ1 diet were analysed
further to determine any differences in lipid classes, polyamines or the methylation status (i.e. SAM, SAH, SAM/
SAH ratio) in the liver. The dietary FPC inclusion did not
affect the liver TAG, diacylglycerol (DAG) or non-esterified fatty acids (Table 7). Neither did these values differ
from the control-fed fish. Total cholesterol concentration
was lower in liver of fish fed the diet containing 150 g
FPC kgÀ1 diet but did not show a statistical difference
(Table 7, P = 0.06). SAM and SAH in liver were not significantly different in fish fed either the fishmeal control

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

Aquaculture Nutrition 18; 599–609 ª 2012 Blackwell Publishing Ltd

diet or the test diets containing 150 or 0 g FPC kgÀ1 diet.
However, the higher mean SAM and lower SAH in fish fed
the diet containing 150 g FPC kgÀ1 diet resulted in a significantly higher (P = 0.001) ratio of SAM to SAH in liver
of fish fed this diet as compared to the control-fed fish and
those fed the diet without any FPC (i.e. the diet containing
200 g fishmeal kgÀ1 diet, Table 7). Fish fed the diets with
higher FPC inclusion showed an increased ratio of arginine
to lysine in plasma and liver (Table 6), and as arginine
availability might affect the polyamines, these were analysed in the liver. Fish fed the diet containing 150 g
FPC kgÀ1 diet tended to have a higher concentration of
spermine (P = 0.055), but did not differ significantly in
putrescine and spermidine concentrations (Table 7). However, the higher mean spermine resulted in a significantly
lower ratio (P = 0.02) of spermidine to spermine in the
liver of fish fed the diet containing 150 g FPC kgÀ1 diet as
compared to fish fed both the control diet and the diet
without FPC (Table 7).

Fish fed higher levels of FPC apparently spent more energy
on growth and metabolism compared with fish fed the diets
with lower FPC inclusion as the fish consumed equal
amounts of feed, but grew less. Increasing the dietary FPC
increased the solubility of the dietary proteins. Previously
we reported that lower inclusion of FPC improved growth
performance, while higher inclusion of FPC reduced
growth in Atlantic salmon fed fishmeal-based diets (Espe