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.

KEY WORDSKEY 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

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� 2008 Sun Yat-Sen University

2008. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

doi: 10.1111/j.1365-2095.2008.00628.x

Aquaculture Nutrition

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

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-

gen, ammonia and pH were 28 ± 2 �C, 5.97 ± 0.03 mg L)1,

0.06 ± 0.02 mg L)1 and 8.04 ± 0.02, respectively. The Mn

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.

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

Choline chloride 5

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): LL-aspartic acid, 180; LL-threonine, 440;

LL-serine, 330; LL-glutamic acid, 530; LL-valine, 360; LL-methionine, 360;

LL-isoleucine, 290; LL-leucine, 550; LL-tyrosine, 220; LL-phenylalanine,

290; LL-lysine-HCl, 290; LL-histidine-HCl, 150; LL-proline, 14 560; LL-ala-

nine, 2730; LL-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).

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� 2008 Sun Yat-Sen University Aquaculture Nutrition

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,

Hoganas, 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 ANOVAANOVA (SPSS 12.0 for

Windows; SPSS Inc., Chicago, IL, USA). When the ANOVAANOVA

identified differences among groups, multiple comparisons

among means were made with Duncan�s 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.

Table 2 Mineral content of experimental diets1,2

Manganese

added

(Mn; mg kg)1)

Analysed mineral content

Calcium

(Ca; g kg)1)

Phosphorus

(P; g kg)1)

Mn

(mg kg)1)

Copper

(Cu; mg kg)1)

Iron

(Fe; mg kg)1)

Zinc

(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

ANOVAANOVA 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.

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� 2008 Sun Yat-Sen University Aquaculture Nutrition

Whole-body ash and mineral contents are shown in

Table 5. Whole-body ash, Mg, Cu and Zn were not signifi-

cantly affected by dietary Mn supplement. Whole body Ca

and P were highest in fish fed diet supplemented with

15 mg kg)1 of Mn. Whole body Fe was significantly lower

when dietary Mn supplement was 1000 mg kg)1.

Weight gain of grouper was not significantly affected by

dietary Mn supplement ranging from 0 to 1000 mg kg)1 in

this study, which indicates that Mn from the basal diet

(4 mg kg)1) was sufficient for the growth of grouper reared in

water containing 0.012 ± 0.001 mg L)1 of Mn, and the

extremely high dietary Mn had no detrimental effect on the

growth of grouper. It has also been reported that dietary Mn

supplement was not necessary for the growth of fish, and the

dietary Mn supplied by basal diet was: 1.3 mg kg)1 of Mn

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

from the casein diet (Maage et al. 2000; water

Mn = 2.9 lg L)1) or 4.8 mg kg)1 Mn from the fish meal

80.0

160.0

140.0

120.0

100.0

60.0

40.0

20.0

20

Ver

tebr

ae M

n co

nten

t (m

g kg

–1 d

ry w

eigh

t)

Dietary Mn content (mg kg–1 dry diet)

y = –0.0002x3 + 0.0162x2 + 1.3903x

40 60 8000.0

+ 26.27

R2 = 0.9561

Figure 1 Regression analysis of vertebrae manganese (Mn) content

of grouper fed diets containing various levels of Mn.

6.00

10.00

9.00

8.00

7.00

5.00

4.00

3.00

2.00

1.00

20

Who

le b

ody

Mn

cont

ent (

mg

kg–1

wet

wei

ght)

Dietary Mn content (mg kg–1 dry diet)

y = 0.00001x3 – 0.00107x2 + 0.11054x +

40 60 8000.00

2.24615

R2 = 0.9080

Figure 2 Regression analysis of whole body manganese (Mn) con-

tent of grouper fed diets containing various levels of Mn.

Table 3 Weight gain, feed efficiency,

survival, vertebrae and whole body

manganese (Mn) concentration of

grouper fed diets supplemented with

graded levels of Mn for 8 weeks

Mn added

(mg kg)1)

Weight

gain (%)2

Feed

efficiency3

Survival

(%)4

Vertebrae Mn

(mg kg)1 dry

weight)5

Whole body Mn

(mg kg)1 wet

weight)5

0 208 ± 11 1.04 ± 0.01abc 92 ± 3a 31.7 ± 0.6f 2.5 ± 0.2f

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

ANOVAANOVA 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.

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� 2008 Sun Yat-Sen University Aquaculture Nutrition

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

Table 4 Vertebrae mineral concentra-

tion (based on dry weight) of grouper

fed diets supplemented with graded lev-

els of manganese (Mn) for 8 weeks1

Mn added

(mg kg)1)

Calcium

(Ca; g kg)1)

Phosphorus

(P; g kg)1)

Magnesium

(Mg; mg kg)1)

Iron

(Fe; mg kg)1)

Zinc

(Zn; mg kg)1)

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

ANOVAANOVA 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

(mg kg)1)

Ash

(g kg)1)

Calcium

(Ca; g kg)1)

Phosphorus

(P; g kg)1)

Magnesium

(Mg; mg kg)1)

Copper

(Cu; mg kg)1)

Iron

(Fe; mg kg)1)

Zinc

(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

ANOVAANOVA 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).

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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).

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� 2008 Sun Yat-Sen University Aquaculture Nutrition