Aquaculture Nutrition
2008
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Nutrition Laboratory, Institute of Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen University, Guangzhou, China
This study was conducted to investigate the effect of die-
tary manganese (Mn) on growth, vertebrae and whole-body
Mn content of juvenile grouper, and to examine the effect
of dietary Mn on copper (Cu), iron (Fe), zinc (Zn), calcium
(Ca), phosphorus (P) and magnesium (Mg) content of
vertebrae and whole body. Seven casein-gelatin-based diets
were supplemented with 0, 5, 10, 15, 20, 50 and
1000 mg kg)1 of Mn from MnSO4ÆH2O. Grouper with an
initial weight of 12.9 ± 0.4 g were fed to satiation with one
of the seven diets for 8 weeks. Growth was not significantly
affected by dietary Mn supplements. Vertebrae Mn in-
creased from 31.7 to 118.1 mg kg)1 dry weight with dietary
Mn supplement increasing from 0 to 50 mg kg)1 (y =
)0.0002x3 + 0.0162x2 + 1.3903x + 26.27, R2 = 0.9561,
where y is the vertebrae Mn content and x is the dietary
Mn content). Whole-body Mn increased from 2.5 to
7.8 mg kg)1 wet weight with dietary Mn supplement
increasing from 0 to 50 mg kg)1 (y = 0.00001x3 )
0.00107x2 + 0.11054x + 2.24615, R2 = 0.9080, where y is
the whole-body Mn content and x is the dietary Mn con-
tent). Dietary Mn had no significant effect on vertebrae Fe,
Ca, P and Mg content, and whole-body Cu, Zn and Mg
content. However, vertebrae Zn and whole body Ca, P
were highest in fish fed diet supplemented with 15 mg kg)1
of Mn. Based on this, Mn supplement of 15 mg kg)1 might
be the optimum when the basal diet contained 4 mg kg)1
of Mn. Fish fed diet supplemented with 1000 mg kg)1 of
Mn did not show any gross abnormality or change in
feeding behaviour, but Mn contents of vertebrae and whole
body were as high as 695.1 mg kg)1 dry weight and
42.5 mg kg)1 wet weight, respectively. Also, whole body Fe
decreased significantly when Mn supplement was up to
1000 mg kg)1.
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 2008 Sun Yat-Sen University
doi: 10.1111/j.1365-2095.2008.00628.x
KEY WORDS: grouper, interactions, manganese, requirement,
toxicity
Received 5 March 2008, accepted 19 August 2008
Correspondence: Yong-Jian, Liu, Nutrition Laboratory, Institute of
Aquatic Economic Animals, School of Life Sciences, Sun Yat-sen Univer-
sity, Guangzhou 510275, China. E-mail: edls@mail.sysu.edu.cn
Manganese (Mn) is essential for normal growth, brain
function, reproduction and prevention of skeletal abnor-
malities in terrestrial animals and fish (Hurley & Keen 1987;
Lall 2002). The dietary Mn requirement in fish has been
reported to be 2.4 mg kg)1 diet for channel catfish (Gatlin &
Wilson 1984), 12–13 mg kg)1 diet for carp and rainbow trout
(Ogino & Yang 1980; Satoh et al. 1987), 15 mg kg)1 diet for
fingerling grass carp (Wang & Zhao 1994), 7.5–10.5 mg kg)1
diet for Atlantic salmon (Maage et al. 2000) and
13.77 mg kg)1 diet for gibel carp (Pan et al. 2008). Fish fed
low Mn diets may exhibit deficiency signs, such as reduced
growth, skeletal abnormalities (dwarfish), cataracts, low
copper-zinc superoxide dismutase (Cu, Zn-SOD) and man-
ganese-superoxide dismutase (Mn-SOD) activities, low tissue
Mn content and poor reproductive performance (Lall 2002).
To our knowledge, no previous studies have been reported
on dietary Mn requirement of grouper.
Grouper is considered an important candidate for inten-
sive aquaculture in the coastal areas of Southeast Asia, owing
to its excellent meat quality, high market value and efficient
feed conversion. Trash fish is still the main feed source for
grouper in China and other Asian countries. The commercial
feed for grouper is at the early stage of development and
there is a need for knowledge in the area of grouper nutrition.
The purpose of this study was to investigate the effect of
dietary Mn on growth, vertebrae and whole body Mn con-
tent of juvenile grouper, and to examine the effect of dietary
Mn on copper (Cu), iron (Fe), zinc (Zn), calcium (Ca),
phosphorus (P) and magnesium (Mg) content of vertebrae
and whole body.
The basal diet formulation and proximate analysis are given
in Table 1, and are similar to that of Ye et al. (2006), which
has been showed to be adequate for grouper. Casein
(Hulunbeier Sanyuan Milk Co., Ltd., Inner Mongolia,
China) and gelatin (Rousselot Gelatin Co., Ltd., Guang-
dong, China) were used as the protein sources. Fish oil
(Gaolong Industrial Company Ltd., Fujian, China) and corn
oil (Defeng Starch Sugar Company, Guangdong, China)
were used as lipid sources, and corn starch (Langfang Starch
Factory, Hebei, China) was used as a carbohydrate source.
Seven experimental diets were formulated by supplementing
Table 1 Composition of the basal diet1
Ingredient g kg)1 diet
Casein 400
Gelatin 100
Fish oil 45
Corn oil 45
Corn starch 200
Attractant2 45
Mineral premix3 80
Vitamin premix4 20
Ascorbic phosphate ester 5 gen, ammonia and pH were 28 ± 2 C, 5.97 ± 0.03 mg L)1,
Choline chloride 5 0.06 ± 0.02 mg L)1 and 8.04 ± 0.02, respectively. The Mn
Cellulose 35
Sodium carboxymethyl cellulose 20
1
Proximate analysis of basal diet: moisture, 106 g kg)1; ash,
47.2 g kg)1; crude protein, 538 g kg)1; ether extract, 94.7 g kg)1.
2
Attractant (mg kg)1 diet): L-aspartic acid, 180; L-threonine, 440;
L-serine, 330; L-glutamic acid, 530; L-valine, 360; L-methionine, 360;
L-isoleucine, 290; L-leucine, 550; L-tyrosine, 220; L-phenylalanine,
290; L-lysine-HCl, 290; L-histidine-HCl, 150; L-proline, 14 560; L-ala-
nine, 2730; L-arginine, 2280; taurine, 3370; glycine, 8920; betain-
HCl, 9100; cellulose, 50 (modified from Mackie & Mitchell 1985).
3
Mineral premix (mg kg)1 diet): calcium lactate, 37 670; NaH2-
PO4Æ7H2O, 24 644; ferric citrate, 1476; CoCl2Æ6H2O, 42; KI, 6.8;
AlCl3Æ6H2O, 7.2; CuSO4Æ5H2O, 8.1; KCl, 4144; ZnSO4Æ7H2O, 140;
Na2SeO3, 0.66; cellulose, 11 870.
4
Vitamin mixture (mg g)1 mixture): thiamin hydrochloride, 2.5;
riboflavin, 10; calcium pantothenate, 25; nicotinic acid, 37.5; pyri-
doxine hydrochloride, 2.5; folic acid, 0.75; inositol, 100; menadione,
2; alpha-tocopheryl acetate, 20; retinol acetate, 1; cholecalciferol,
0.0025; biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted
with cellulose to 1 g (modified from Lin & Shiau 2003).
the basal diet with 0, 5, 10, 15, 20, 50 and 1000 mg kg)1 Mn
from MnSO4ÆH2O. The Mn concentrations of the experi-
mental diets were determined by inductively coupled plasma
atomic emission spectrophotometer after wet digestion and
found to be 4 ± 0, 10 ± 0, 16 ± 0, 23 ± 0, 29 ± 1,
72 ± 1 and 1349 ± 4 mg kg)1, respectively. Element anal-
ysis (Ca, P, Mn, Cu, Fe and Zn) of the experimental diets are
showed in Table 2.
All the dry ingredients were weighed and mixed for
15 min, and then fish oil and corn oil were added and mixed
for another 15 min. Deionized water was added and mixed
again for another 15 min. The wet mixture was transformed
into 2.5-mm pellets in an F-26 Pelleter (SCUT Factory,
Guangzhou, China). The resultant pellets were air-dried,
and stored at )20 C until used.
Juvenile grouper (Epinephelus coioides) were obtained from a
nursery in Dayawan, Huizhou, China, transported to the
laboratory and maintained in seven 300-L circular fibreglass
tanks for acclimatization. During the acclimatization period,
fish were fed the basal diet for 10 days until the fish accepted
purified diet totally.
At the beginning of the experiment, healthy fish with an
initial body weight of 12.9 ± 0.4 g (mean ± SE; n = 21)
were distributed randomly into 21 circular fibreglass tanks
(300 L, three tanks per diet, 20 fish per tank). Filtered sea-
water (salinity, 30 g L)1) was supplied to each tank at a flow
rate of 4 L min)1 in a flow through system. During the
experimental period, the water temperature, dissolved oxy-
content of the water was 0.012 ± 0.001 mg L)1. Natural
light cycle was maintained during the feeding trial. Fish were
fed to apparent satiation twice per day (09:00 and 16:00 h)
for 8 weeks.
At the beginning of the feeding trial, five fish were randomly
sampled for initial analysis of whole body mineral content.
Ten fish were killed and cooked in a microwave for 4 min.
Vertebrae were removed from the 10 fish for the initial
analysis of mineral content.
At the end of the 8-week experiment, 10 fish from each
tank were randomly collected, 2 for analysis of whole-body
ash and mineral content, and 8 were anaesthetized with
MS-222 and cooked in a microwave oven for 6 min.
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 2008 Sun Yat-Sen University Aquaculture Nutrition
 Table 2 Mineral content of experimental diets1,2

Analysed mineral content

Manganese
added Calcium Phosphorus Mn Copper Iron Zinc
(Mn; mg kg)1) (Ca; g kg)1) (P; g kg)1) (mg kg)1) (Cu; mg kg)1) (Fe; mg kg)1) (Zn; mg kg)1)

0 6.8 ± 0.1 9.7 ± 0.1 4± 0 3.3 ± 0.1 478 ± 21 80 ± 4

5 7.4 ± 0.3 9.9 ± 0.0 10 ± 0 3.1 ± 0.1 501 ± 11 115 ± 24
10 7.2 ± 0.1 9.9 ± 0.1 16 ± 0 3.3 ± 0.0 502 ± 6 89 ± 2
15 7.3 ± 0.3 10.1 ± 0.1 23 ± 0 3.2 ± 0.0 513 ± 12 90 ± 0
20 8.4 ± 1.2 9.9 ± 0.3 29 ± 1 3.3 ± 0.0 510 ± 8 93 ± 1
50 10.3 ± 1.4 10.2 ± 0.1 72 ± 1 3.3 ± 0.1 542 ± 22 110 ± 7
1000 9.5 ± 0.1 10.3 ± 0.2 1349 ± 4 3.3 ± 0.0 532 ± 1 97 ± 1
ANOVA n.s. n.s. 0.000 n.s. n.s. n.s.
1
Mn was added as MnSO4ÆH2O.
2
No significant differences (P < 0.05) were observed in Ca, P, Mn, Cu, Fe and Zn contents of the diet.

Vertebrae were removed from the eight fish and pooled for
mineral analysis. Vertebrae were rinsed with deionized
water, dried and ground for mineral analysis.
Approximately 0.5 g of dried and finely ground samples
were digested with 20 mL of 65–68% nitric acid and 1 mL of
72% perchloric acid using Kjeldahl flasks. After digestion,
the samples were diluted to 25 mL and determined for Fe,
Cu, Mn and Zn contents by inductively coupled plasma
atomic emission spectrophotometer [ICP; model: IRIS
Advantage (HR); Thermo Jarrell Ash Corporation, Boston,
MA, USA]. Further dilutions were made to analyse Ca, P
and Mg contents. Detection limit was 0.001 mg L)1 for Fe,
Mn and Zn, and 0.002 mg L)1 for Cu. The standard solution
was provided by SPEX, CertiPrep, Inc., USA.
Moisture, crude protein and crude lipid of the experi-
mental diets were determined according to the AOAC (1984)
methods. Moisture was determined by drying in an oven at
105 C for 24 h; crude protein was analysed by the Kjeldahl
method after acid digestion (1030-Auto-analyzer; Tecator,
Höganäs, Sweden); crude fat was determined by the ether-
extraction method using a Soxtec System HT (Soxtec System
HT6; Tecator). Oven-dried feed were ashed at 550 C for
24 h in a muffle furnace.
Results were analysed by one-way ANOVA (SPSS 12.0 for
Windows; SPSS Inc., Chicago, IL, USA). When the ANOVA
identified differences among groups, multiple comparisons
among means were made with Duncans multiple-range test
at P < 0.05. Vertebrae and whole-body Mn contents to
graded levels of dietary Mn were plotted and tried with dif-
ferent models using curve estimation function of SPSS.
R-square of cubic model was highest and so cubic model
was chosen.
No gross abnormality or change in feeding behaviour were
observed in fish fed diet supplemented with 0 or
1000 mg kg)1 of Mn in this experiment. Weight gain, feed
efficiency and survival of juvenile grouper are shown in
Table 3. Weight gain was not significantly affected by
dietary Mn content. Feed efficiency was highest in fish fed
diet supplemented with 15 mg kg)1 Mn, and lowest in fish
fed diet supplemented with 1000 mg kg)1 of Mn. The
survival of the fish was not affected by the dietary Mn
supplement ranging from 0 to 50 mg kg)1, although
1000 mg kg)1 had a significantly negative effect on
survival.
Vertebrae and whole body Mn are presented in Table 3.
As dietary Mn supplement increased from 0 to 50 mg kg)1,
vertebrae Mn content increased from 31.7 to 118.1 mg kg)1
dry weight. The relationship between dietary and vertebrae
Mn content, determined by regression analysis, was y =
)0.0002x3 + 0.0162x2 + 1.3903x + 26.27 (R2 = 0.9561),
where y is the vertebrae Mn content and x is the dietary Mn
content (Fig. 1). Similarly, as dietary Mn supplement in-
creased from 0 to 50 mg kg)1, whole body Mn increased
from 2.5 to 7.8 mg kg)1 wet weight. The relationship be-
tween dietary Mn content and whole body Mn content was
y = 0.00001x3 – 0.00107x2 + 0.11054x + 2.24615, R2 =
0.9080, where y is the whole body Mn content and x is the
dietary Mn content (Fig. 2). When 1000 mg kg)1 of Mn was
incorporated in the diet, vertebrae and whole body Mn were
695.1 mg kg)1 dry weight and 42.5 mg kg)1 wet weight,
respectively.
Vertebrae mineral contents are shown in Table 4. Ca, P,
Mg and Fe of vertebrae were not significantly affected by
dietary Mn supplement. Vertebrae Zn was significantly
higher when dietary Mn supplement was 15 mg kg)1.

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 2008 Sun Yat-Sen University Aquaculture Nutrition

 Table 3 Weight gain, feed efficiency,
Vertebrae Mn Whole body Mn
survival, vertebrae and whole body
Mn added Weight Feed Survival (mg kg)1 dry (mg kg)1 wet
(mg kg)1) gain (%)2 efficiency3 (%)4 weight)5 weight)5
manganese (Mn) concentration of
grouper fed diets supplemented with
0 208 ± 11 1.04 ± 0.01abc 92 ± 3a 31.7 ± 0.6f 2.5 ± 0.2f graded levels of Mn for 8 weeks
5 223 ± 19 1.04 ± 0.03ab 92 ± 2a 41.9 ± 2.4ef 3.2 ± 0.1ef
10 206 ± 4 1.02 ± 0.02bc 80 ± 5ab 52.6 ± 1.9de 3.4 ± 0.1de
15 205 ± 23 1.10 ± 0.02a 85 ± 3ab 63.3 ± 3.0cd 4.3 ± 0.3cd
20 219 ± 1 1.06 ± 0.01ab 85 ± 5ab 75.4 ± 3.1c 4.9 ± 0.2c
50 237 ± 13 1.08 ± 0.02ab 87 ± 3a 118.1 ± 8.8b 7.8 ± 0.4b
1000 202 ± 16 0.97 ± 0.04c 73 ± 4b 695.1 ± 7.6a 42.5 ± 0.4a
ANOVA n.s. 0.023 0.042 0.000 0.000
1
Values are mean ± SE of three groups of fish (n = 3), with 20 fish per group for weight gain,
feed efficiency and survival, 8 fish per group for vertebrae Mn concentration and 2 fish per
group for whole body Mn concentration. Within a column, values not sharing a common
superscript are significantly different (P < 0.05).
2
Weight gain = 100 · (final body weight ) initial body weight)/(initial body weight). Initial
body weight of the fish was 12.9 ± 0.4 g (mean ± SE, n = 21).
3
Feed efficiency = wet weight gain (g)/dry feed intake (g).
4
Survival (%) = 100 · (final fish number)/(initial fish number).
5
Initial vertebrae Mn was 38.1 mg kg)1 dry weight and initial whole body Mn was 3.78 mg kg)1
wet weight.

Whole body Mn content (mg kg–1 wet weight)

160.0 10.00
y = 0.00001x3 – 0.00107x2 + 0.11054x +
y = –0.0002x3 + 0.0162x2 + 1.3903x 9.00 2.24615
Vertebrae Mn content (mg kg–1 dry weight)

140.0 + 26.27
8.00 R2 = 0.9080
R2 = 0.9561
120.0 7.00
6.00
100.0
5.00
80.0 4.00
3.00
60.0
2.00
40.0 1.00
0.00
20.0 0 20 40 60 80
Dietary Mn content (mg kg–1 dry diet)
0.0
0 20 40 60 80
Figure 2 Regression analysis of whole body manganese (Mn) con-
Dietary Mn content (mg kg–1 dry diet) tent of grouper fed diets containing various levels of Mn.

Figure 1 Regression analysis of vertebrae manganese (Mn) content

of grouper fed diets containing various levels of Mn. this study, which indicates that Mn from the basal diet
(4 mg kg)1) was sufficient for the growth of grouper reared in
Whole-body ash and mineral contents are shown in water containing 0.012 ± 0.001 mg L)1 of Mn, and the
Table 5. Whole-body ash, Mg, Cu and Zn were not signifi- extremely high dietary Mn had no detrimental effect on the
cantly affected by dietary Mn supplement. Whole body Ca growth of grouper. It has also been reported that dietary Mn
and P were highest in fish fed diet supplemented with supplement was not necessary for the growth of fish, and the
15 mg kg)1 of Mn. Whole body Fe was significantly lower dietary Mn supplied by basal diet was: 1.3 mg kg)1 of Mn
when dietary Mn supplement was 1000 mg kg)1. from casein diet (Knox et al. 1981) or 4.4 mg kg)1 from fish
meal diet (Yamamoto et al. 1983) for rainbow trout,
2.4 mg kg)1 from casein diet for channel catfish (Gatlin &
Wilson 1984; water Mn = 2 lg L)1) and 1.1 mg kg)1 Mn
Weight gain of grouper was not significantly affected by from the casein diet (Maage et al. 2000; water
dietary Mn supplement ranging from 0 to 1000 mg kg)1 in Mn = 2.9 lg L)1) or 4.8 mg kg)1 Mn from the fish meal

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 2008 Sun Yat-Sen University Aquaculture Nutrition

 Table 4 Vertebrae mineral concentra-
Mn added Calcium Phosphorus Magnesium Iron Zinc
tion (based on dry weight) of grouper
(mg kg)1) (Ca; g kg)1) (P; g kg)1) (Mg; mg kg)1) (Fe; mg kg)1) (Zn; mg kg)1)
fed diets supplemented with graded lev-
els of manganese (Mn) for 8 weeks1 0 188 ± 7 109 ± 4 4400 ± 321 18 ± 3 97 ± 2b
5 196 ± 4 112 ± 2 4333 ± 145 11 ± 2 94 ± 1b
10 187 ± 8 109 ± 3 4233 ± 167 13 ± 1 96 ± 3b
15 191 ± 10 110 ± 5 4500 ± 153 12 ± 1 139 ± 10a
20 189 ± 9 109 ± 3 4300 ± 115 17 ± 4 112 ± 9b
50 181 ± 5 107 ± 2 4333 ± 88 17 ± 2 110 ± 4b
1000 181 ± 11 107 ± 5 4267 ± 133 22 ± 8 100 ± 3b
ANOVA n.s. n.s. n.s. n.s. 0.001
1
Values are mean ± SE of three groups of fish (n = 3), with eight fish per group. Within a
column, values not sharing a common superscript are significantly different (P < 0.05). Mineral
contents of initial vertebrae were: Fe (16.8 mg kg)1), Zn (114 mg kg)1), Ca (179 g kg)1),
P (107 g kg)1) and Mg (4275 mg kg)1).

Table 5 Whole-body ash and mineral concentration (based on wet weight) of grouper fed diets supplemented with graded levels of manganese
(Mn) for 8 weeks1

Mn added Ash Calcium Phosphorus Magnesium Copper Iron Zinc

(mg kg)1) (g kg)1) (Ca; g kg)1) (P; g kg)1) (Mg; mg kg)1) (Cu; mg kg)1) (Fe; mg kg)1) (Zn; mg kg)1)

0 46 ± 1 13.6 ± 0.5ab 9.7 ± 0.3ab 508 ± 10 0.63 ± 0.05 10.5 ± 0.9a 17 ± 0

5 46 ± 2 13.1 ± 0.2bc 9.4 ± 0.1abc 498 ± 9 0.61 ± 0.04 11.5 ± 0.2a 17 ± 0
10 46 ± 1 13.1 ± 0.1bc 9.3 ± 0.0bc 490 ± 6 0.65 ± 0.09 10.8 ± 0.7a 22 ± 4
15 48 ± 1 14.3 ± 0.6a 10.1 ± 0.3a 518 ± 9 0.67 ± 0.05 11.1 ± 0.3a 23 ± 5
20 45 ± 1 13.0 ± 0.1bc 9.4 ± 0.1abc 489 ± 4 0.67 ± 0.02 10.3 ± 0.4a 20 ± 2
50 46 ± 0 12.9 ± 0.4bc 9.2 ± 0.3bc 490 ± 15 0.64 ± 0.04 9.8 ± 0.2ab 18 ± 1
1000 45 ± 1 12.3 ± 0.3c 8.9 ± 0.2c 476 ± 15 0.66 ± 0.07 8.6 ± 0.5b 22 ± 4
ANOVA n.s. 0.033 0.045 n.s. n.s. 0.023 n.s.
1
Values are mean ± SE of three groups of fish (n = 3), with two fish per group. Within a column, values not sharing a common superscript
are significantly different (P < 0.05). Mineral contents of initial whole body were: Cu (0.45 mg kg)1), Fe (8.10 mg kg)1), Zn (24 mg kg)1), Ca
(15.6 g kg)1), P (10.2 g kg)1) and Mg (537 mg kg)1).

diet (Lorentzen et al. 1996) for Atlantic salmon. However,
some studies demonstrated depressed growth in fish receiving
Mn-deficient diets, such as rainbow trout (Ogino & Yang
1980), carp (Ogino & Yang 1980; Satoh et al. 1983, 1987),
fingerling grass carp (Wang & Zhao 1994) and gibel carp
(Pan et al. 2008). Mn is often considered to be among the
least toxic of trace elements for domestic animals (McDowell
2003). Maximum Mn dietary tolerable levels (NRC 2005) for
common livestock species are for sheep and cattle
(1000 mg kg)1), poultry (2000 mg kg)1), and for swine,
horses and rabbits (400 mg kg)1). Our data shows that
grouper grew normally when dietary Mn supplement was as
high as 1000 mg kg)1.
Bone and whole body Mn responded readily to dietary Mn
in rainbow trout (Ogino & Yang 1980; Knox et al. 1981),
carp (Ogino & Yang 1980; Satoh et al. 1983, 1987), channel
catfish (Gatlin & Wilson 1984), grass carp (Wang & Zhao
1994), Atlantic salmon (Lorentzen et al. 1996; Lorentzen &
Maage 1999; Maage et al. 2000) and gibel carp (Pan et al.
2008), and they are widely used as Mn status indicators in
fish. In the present experiment, both vertebrae and whole
body Mn concentration increased with increasing dietary Mn
supplement from 0 to 50 mg kg)1 (Figs 1 & 2). It has also
been reported that bone Mn concentration increased linearly
with increasing dietary Mn from 2.4 to 62.4 mg kg)1 in
channel catfish (Gatlin & Wilson 1984). However, in grass
carp (Wang & Zhao 1994), Atlantic salmon (Lorentzen et al.
1996; Maage et al. 2000) and gibel carp (Pan et al. 2008),
vertebrae and whole body Mn increased by dietary Mn
increments and reached a plateau when the requirement was
met. The differences between the studies may be because of
dietary Mn supplement level, duration of the experiment, fish
species or fish size. Different fish species may have different
abilities in regulating Mn uptake and excretion. The authors
used a broken-line regression model to estimate the Mn
requirement in those studies (Lorentzen et al. 1996; Maage
et al. 2000; Pan et al. 2008). In this study, the broken-line
regression model was not considered suitable to estimate the
Mn requirement because the Mn concentration continued to
increase and it did not reach a plateau. Using the equation

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 2008 Sun Yat-Sen University Aquaculture Nutrition

y = 0.00001x3 ) 0.00107x2 + 0.11054x + 2.24615 (R2 =
0.9080, y is the whole body Mn content and x is the dietary
Mn content), we can easily estimate whole body Mn content
of grouper according to Mn content of the fish feed. Verte-
brae play an important role in Mn accumulation of fish. It
has been reported that dietary Mn had significant effects on
Mn concentration in vertebrae and whole fish, but not in
liver (Lorentzen et al. 1996). Gatlin & Wilson (1984) also
reported that Mn supplement had a significant effect on Mn
concentration in bone, but not in liver. The highest Mn
concentration is found in bone (Lall 2002).
Mineral interactions are important in trace mineral
metabolism (review by Hilton 1989). In this study, vertebrae
Ca, P, Mg and Fe contents were not affected by dietary Mn
supplement ranging from 0 to 1000 mg kg)1, but vertebrae
Zn was highest in fish fed diet supplemented with 15 mg kg)1
of Mn. It has been reported that dietary Mn supplement in a
white fish meal diet had no significant effect on vertebrae Ca,
P or Mg concentrations, but vertebrae Zn concentration in-
creased with increasing dietary Mn and reached a plateau at
dietary Mn levels above 10 lg g)1 diet (Satoh et al. 1991).
Zinc can influence bone mineralization either directly, as
divalent cation acting on nucleation and mineral accumu-
lation, or indirectly, as a cofactor of enzymes or other
metalloenzymes involved in the process (Gomez et al. 1999).
It is possible that the decreased bone Zn may partly explain
skeletal deformities of fish fed Mn-deficient diets. Although
vertebrae Zn responded to dietary Mn, whole body Zn did
not show significant differences among treatments. Maage
et al. (2000) reported that dietary Mn had no significant
effect on whole body Zn. In this study, whole body Ca and P
were also higher in fish fed diet supplemented with
15 mg kg)1 Mn, and decreased when dietary Mn supplement
was above 15 mg kg)1. A negative correlation between whole
body P and dietary Mn levels was observed by Maage et al.
(2000). In this study, the interaction between dietary Mn, Ca
and P was observed in whole body but not in vertebrae. The
toxicity of excessive Mn appears to cause antagonism in
mineral metabolism particularly with Fe in some animals
(McDowell 2003). Grouper fed 1000 mg kg)1 of Mn had
significantly lower whole body Fe. No previous studies have
been reported about dietary Mn toxicity on fish.
In summary, dietary Mn did not significantly affect growth
of grouper. Vertebrae and whole body Mn increased readily
with dietary Mn supplement increasing from 0 to
50 mg kg)1. Based on vertebrae Zn and whole body Ca and
P content, Mn supplement of 15 mg kg)1 might be optimum
when the basal diet contained 4 mg kg)1 of Mn. Grouper fed
diet supplemented with 1000 mg kg)1 of Mn did not show
any gross abnormality or change in feeding behaviour, except
that Mn content of vertebrae and whole body were very high
and whole body Fe decreased significantly.
The authors thank the staff of the Guangdong Evergreen
Group for providing the experimental base and for their
logistic support during this study. They would also like to
thank S.X. Deng, Y.G. Qiao for their assistance with sam-
pling and Q.Y. Cui for skilled technical assistance with the
mineral analyses. This work was funded by Key Technologies
R&D Program during the 10th Five-Year Plan, China (grant
no. 2001DA505D/06).
AOAC (Association of Official Analytical Chemists) (1984) Official
Methods of Analysis, 14th edn., 1141 pp. AOAC, Arlington, VA.
Gatlin, D.M. III & Wilson, R.P. (1984) Studies on the manganese
requirement of fingerling channel catfish. Aquaculture, 41, 85–92.
Gomez, S., Rizzo, R., Pozzi-Mucelli, M., Bonucci, E. & Vittur, F.
(1999) Zinc mapping in bone tissues by histochemistry and syn-
chrotron radiation-induced X-ray emission: correlation with the
distribution of alkaline phosphatase. Bone, 25, 33–38.
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