Aquaculture 192 2001.

6784
www.elsevier.nlrlocateraqua-online

Zinc methionine and zinc sulfate as sources of

dietary zinc for juvenile abalone, Haliotis discus
hannai Ino
Beiping Tan, Kangsen Mai )
Aquaculture Research Laboratory, College of Fisheries, Ocean Uniersity of Qingdao, Qingdao 266003,
Peoples Republic of China
Received 31 January 2000; received in revised form 22 May 2000; accepted 22 May 2000

Abstract
A feeding experiment was conducted to determine the minimum dietary zinc requirement of
juvenile abalone, Haliotis discus hannai, with zinc methionine ZnMet. and zinc sulfate ZnSO4 P
7H 2 O. as the zinc sources and to compare the bioavailability of the two zinc sources using a
premium quality diet based on caseingelatin as the protein sources. Semipurified experimental
diets containing graded levels of dietary zinc 5.684.6 mg zincrkg. provided as either ZnMet or
ZnSO4 were fed to juvenile abalone in triplicate groups for 16 weeks. The results showed that the
average weight gain rate WGR, %., daily increment in shell length DISL, mmrday., soft-body
alkaline phosphatase activity SBAKP, Urg protein. and soft-body zinc concentration SB zinc,
mgrg. of the abalone were significantly ANOVA, P - 0.01. affected by dietary treatment, and
responded in broken-line models to increases in dietary zinc levels from the two zinc sources. The
requirements for dietary zinc using ZnMet and ZnSO4 as the supplemental zinc sources,
determined by broken-line regression analysis, on the basis of maximum WGR were 15.49 and
34.10 mgrkg, respectively, on maximum DISL were 15.16 and 33.99 mgrkg, respectively, on
maximum SBAKP were 15.54 and 31.91 mgrkg, and on maximum SB zinc deposition were
17.75 and 34.29 mgrkg, respectively. The shell zinc concentration, as well as iron concentration
in soft-body and shell of the abalone, however, was maintained relatively constant ANOVA,
P ) 0.05. regardless of dietary treatment. Based on these results, a minimum requirement for
dietary zinc was recommended to be 1618 mgrkg from ZnMet, and 35 mgrkg from ZnSO4 .
This experiment also showed that the bioavailability of dietary zinc with ZnMet was approxi-

Corresponding author. Tel.: q86-532-2032282; fax: q86-532-2032799.

E-mail address: kmai@mail.ouqd.edu.cn K. Mai..

0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 0 4 4 - 8 4 8 6 0 0 . 0 0 4 3 5 - X

68

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

mately three times as high as that of ZnSO4 to H. discus hannai Ino. q 2001 Elsevier Science
B.V. All rights reserved.
Keywords: Dietary zinc; Requirement; Bioavailability; Haliotis discus hannai; Feeding and nutrition
mollusks

1. Introduction
Zinc is known to be an essential micronutrient both in plants and animals. Zinc is
involved in various metabolic pathways. It serves as a specific cofactor of several
enzymes. In addition, zinc is an integral part of about 20 metalloenzymes, such as
alkaline phosphatase, alcohol dehydrogenase and carbonic anhydrase. Zinc is associated
with prostaglandin metabolism and also may have a structural role in nucleoproteins
reviewed by Watanabe et al., 1997.. Recent research on zincgene interactions has
assigned a basic role for this element in controlling growth Chesters, 1991.. Normal
zinc levels in freshwater Spry et al., 1988. and seawater Willis and Sunda, 1984. are
known to be insufficient to meet the requirement of growing aquatic species. Hence,
zinc is regarded as an essential nutrient in fish feeds e.g. Lall, 1989; NRC, 1993; Wei et
al., 1999. and shrimp feeds e.g. Li et al., 1995.. The zinc requirement of rainbow trout
was found to be between 15 and 30 mgrkg diet Ogino and Yang, 1978.. Gatlin et al.
1991. reported that dietary zinc requirement of the red drum was between 20 and 25
mgrkg diet. Whereas, the requirement of Atlantic salmon for zinc is G 67 mgrkg diet
Maage and Julshamn, 1993.. All these requirements were determined with zinc sulfate
ZnSO4 P 7H 2 O. as supplemental zinc source.
Amino acid-chelated zinc has been shown to have a higher absorption rate in the
animal intestine than inorganic forms of zinc, such as zinc sulfate, zinc carbonate, and
zinc oxide Ashmead, 1992.. Wedekind et al. 1992. reported that the bioavailability of
zinc from zinc methionine ZnMet. was greater than that of zinc sulfate ZnSO4 . and the
difference in bioavailability increased as complexity of the diet increased. The bioavailability of ZnMet relative to that of ZnSO4 was 117% in a crystalline amino acid purified
diet and 206% in a practical cornsoybean diet. Similar results have been reported by
Paripatananont and Lovell 1995. in channel catfish. They found that the relative
bioavailabilities of ZnMet, with ZnSO4 as the standard, were 352% for weight gain and
305% for bone zinc deposition in fish fed an egg-white diet, and 482% for weight gain
and 586% for bone zinc deposition in fish fed a soybean meal diet.
High dietary levels of zinc may, however, negatively affect the status of other
elements, such as iron Wekell et al., 1986.. Unnecessarily high additions of zinc and
other micronutrients also increase the price of feeds, as well as increase the input of
minerals to the aquatic environment Maage and Julshamn, 1993..
There is no information on the requirement of dietary zinc by any mollusks. Also, no
information is available on the bioavailabilities of organic and inorganic sources of
dietary zinc to these mollusk species. Declining abalone fisheries worldwide Clavier,
1992; Farlinger and Campbell, 1992; Guzman
del Proo,
1992; Johnson et al., 1992; Nie,
1992; Parker et al., 1992; Prince and Shepherd, 1992; Schiel, 1992; Tarr, 1992; Tegner

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

69

et al., 1992. have accelerated the development of abalone aquaculture. Haliotis discus
hannai is one of the most widely cultured and commercially important abalone species.
Therefore, the objectives of this study were to determine the dietary zinc requirement of
juvenile abalone, H. discus hannai Ino, with ZnMet and ZnSO4 as the zinc sources and
to compare the bioavailabilities of the two zinc sources using a premium quality diet
based on caseingelatin as the protein sources. This information will enable feed
formulators to determine which zinc sources and what amount of dietary zinc should be
used in the abalone diets.

2. Materials and methods

2.1. Feed formulation and manufacture
The basal diet formulation is given in Table 1. Casein and gelatin were used as
protein sources. Crude protein level of the experimental diets was 28%, which is
considered to be sufficient to maintain optimum growth of H. discus hannai Mai et al.,
1995b.. Soybean oil and menhaden fish oil 1:1. were used as the lipid sources. Dietary

Table 1
Composition of the basal diet dry weight basis.
Ingredient

Percent in diet

Casein Sigma, St. Louis, MO, USA.

Gelatin Sigma.
Dextrin Shanghai Chemical, Shanghai, China.
Sodium alginate Shanghai Chemical.
SOrMFO Food grade. a
Choline chloride Shanghai Chemical.
Carboxymethylcellulose Shanghai Chemical.
Zinc-free mineral mix b
Vitamin mix c

25.00
6.00
33.50
20.00
3.50
0.50
5.00
4.50
2.00

Proximate analysis (means of triplicate)

Crude protein %.
Crude lipid %.
Ash %.
Gross energy kJrg. d
Zinc mgrkg.

28.40
3.48
8.78
17.18
5.60

Soybean oil and menhaden fish oil 1:1. with 0.001% ethoxyquin.
Zn-free mineral mix, each 1000 g of diet contained: NaCl, 0.4 g; MgSO4P7H 2 O, 6.0 g; NaH 2 PO4P2H 2 O,
10.0 g; KH 2 PO4 , 20.0 g; CaH 2 PO4 . 2 PH 2 O, 8.0 g; Fe-citrate, 1.0 g; MnSO4PH 2 O, 64.8 mg; CuSO4P5H 2 O,
12.4 mg; CoCl 2 P6H 2 O, 0.4 mg; KIO 3 , 1.2 mg; Na 2 SeO 3 , 0.4 mg.
c
Vitamin mix, each 1000 g of diet contained: thiamin HCl, 120 mg; riboflavin, 100 mg; folic acid, 30 mg;
PABA, 400 mg; pyridoxine HCl, 40 mg; niacin, 800 mg; Ca pantothenate, 200 mg; inositol, 4000 mg; ascorbic
acid, 4000 mg; biotin, 12 mg; vitamin E, 450 mg; menadione, 80 mg; B12, 0.18 mg; retinol acetate, 100,000
IU; cholecalciferol, 2000 IU; ethoxyquin, 400 mg.
d
Estimated with an XYR-1 bomb calorimeter.
b

70

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

lipid level was 3.5%, which was sufficient to support optimum growth and provide
adequate essential fatty acids EFA. for abalone Mai et al., 1995a.. The compositions of
vitamin and mineral mixtures were modified from those used by Uki et al. 1985.. The
caseingelatin-based diet contained 5.60 " 0.79 mgrkg of intrinsic zinc. This semipurified formulation was employed because previous experiences in our laboratory indicated
limited acceptance of egg white-based purified diets by juvenile abalone. The basal diet
was supplemented with 0, 5, 10, 20, 30, 40, and 80 mg of zincrkg dry diet from either
zinc methionine ZnMet. Feed Additive, Ministry of Chemistry Industry of China,
Jinan. or zinc sulfate ZnSO4 P 7H 2 O. Sigma, St. Louis, MO, USA.. Final zinc
concentrations in the experimental diets n s 3. were found to be: 5.6 " 0.79, 11.2 "
0.87, 14.8 " 0.96, 26.1 " 1.22, 34.9 " 0.86, 43.3 " 1.13, and 83.9 " 1.48 mgrkg from
ZnMet, and 5.6 " 0.79, 10.7 " 0.69, 15.1 " 0.48, 25.2 " 0.76, 34.7 " 0.58, 45.3 " 1.06,
and 84.6 " 1.25 mgrkg from ZnSO4 as determined by ICP-AES Shearer, 1984..
Procedures for diet preparation were modified from those described by Mai et al.
1995a,b.. Casein, gelatin and some minerals were ground individually using a Pascal
Mill and then passed through a mesh with 125-mm pore size. Dry ingredients were
weighed on an electronic balance and thoroughly mixed. After adding water about
120%, vrw. to the mechanically mixed ingredients containing 20% sodium alginate, a
paste was made. The paste was shaped into 0.5-mm thick sheets, which were cut into
1-cm2 flakes. The flakes were dipped into an aqueous solution of CaCl 2 5%, wrv. for
1 min. By this treatment, sodium alginate was converted to an insoluble calcium alginate
gel, in which the nutrients were bound Uki and Watanabe, 1992.. The surplus solution
was drained, and then the flakes were sealed in a sample bag and stored at y208C until
use.
2.2. Animal rearing
Juvenile abalone, H. discus hannai used in this experiment was derived from a
spawning in June 1998, at Mashan Fisheries, Shandong, China. Before trial, shell length
was measured with calipers to the nearest 0.02 mm and the animals were weighed to the
nearest 0.01-g using an electronic balance.
A series of acrylic cages 1.5 = 1.0 = 0.5 m. were put into a rectangle cement tank
6 = 2 = 1.5 m.. Animals were kept in plastic baskets 20 = 20 = 20 cm.. Each rearing
unit basket. was stocked with 25 abalone juveniles mean weight 0.74 " 0.01 g; mean
shell length 16.41 " 0.04 mm.. There were 13 treatments, and each treatment was
conducted in three replicates. Thirty-nine baskets were assigned to the acrylic cages
using a completely randomized design. Seawater pumped from the coast adjacent to the
farm passed through sand filters filtered to 30 mm by primary sand filters, followed to
10 mm by secondary composite sand filters. into the tank continuously at a rate of
145 " 9 lrmin. During the experimental period, water temperature ranged from 18.5
22.08C, salinity 3034, pH 7.67.9. Dissolved oxygen was not less than 7.0 mgrl, and
there were negligible levels of free ammonia and nitrite. Zinc concentration in the water
flowing into the rearing system was 10.0 " 2.3 mgrl as determined by ICP-AES
n s 3..

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

71

Prior to initiation of the experiment, the abalone underwent a 2-week conditioning

period during which they readily adjusted to a zinc-depleted caseingelatin-based diet
Table 1. and standardized environmental conditions. The feeding trial was run for 16
weeks. Abalone were hand-fed with the test diets at a rate equaling 510% of wet body
weightrday, once daily at 17:00. Every morning, uneaten feed and feces were cleaned
to maintain water quality.
2.3. Sample collection and analyses
At the termination of the experiment, animals were not fed for 3 days, then all
abalone were removed from the cages, weighed, measured and counted. Then, 15
abalone from each replicate were frozen y708C. for subsequent analyses. Growth was
expressed as weight gain rate WGR, %. and daily increment in shell length DISL,
mmrday.. The calculation formulae are as follows:
WGR % . s Wt y Wi . rWi = 100
DISL s SL t y SL i . rt = 1000
where Wt , Wi are final and initial mean weight g., respectively; SL t , SL i are final and
initial mean shell length mm., respectively; t is the feeding trial period day..
Proximate analyses of soft-body samples to determine protein, lipid, ash and moisture
levels were conducted using standard procedures AOAC, 1984..
Thawed soft bodies were weighed, and homogenized in cold 48C. 0.01 M Tris buffer
pH 7.5. at a ratio of 1:20 wrv.. The crude extract was obtained by centrifuging the
homogenate at 13,500 = g for 20 min at 48C, and filtering the supernatant. Soft body
alkaline phosphatase SBAKP. activity in the crude extract was determined spectrophotometrically using a r-nitrophenyl-phosphate substrate assay Tietz, 1986.. Protein was
estimated by a modification of the Lowry procedure Hartee, 1972. with bovine serum
albumin as the calibration standard. SBAKP activity was expressed as specific activity
Urg protein., where one unit U. is equal to the amount of enzyme necessary to
produce 1 mmol of nitrophenol from r-nitrophenyl-phosphate.rmin at 378C. Elemental
analyses of the shells and soft bodies of the abalone were modified from the method
described by Shearer 1984.. The shell samples were digested in a mixture of equal parts
of hydrochloric acid 37%, ACS reagent. and nitrite acid 70%, ACS reagent. at a ratio
of 1:20 wrv.. The soft body samples were digested in perchloric acid HClO4 , 70%,
ACS reagent. at a ratio of 1:20 wrv.. Then, the digests were appropriately diluted with
Milli-Q water within the analytical capabilities of the ICP atomic emission spectrophotometer JY 70plus, Jobin Yvon.. Elemental concentrations of the samples are
expressed on a wet-weight basis as recommended by Shearer 1984..
2.4. Leaching
A leaching test for zinc was carried out according to the method used by Coote et al.
1996.. Pre-weighed diet was placed onto 100-mm mesh screens and allowed it to settle
to the bottom of experimental cages without abalone. Temperature and flow rate were

72

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

adjusted to match those of the experiment, the values being 20 " 0.88C, and about
0.5-lrmin per cage, respectively. At the end of allotted time 0, 6, and 12 h,
respectively., the remaining diet was removed from the cages and dried overnight at
608C in an oven. Dried diet was submitted for analysis of total zinc with an ICP-atomic
emission spectrophotometer.
2.5. Statistical analysis
Data from each treatment were subject to one-way ANOVA. When overall differences were significant at less than 5% level, Tukeys test was used to compare the mean
values between individual treatments. Statistical analysis was performed using the
STATISTICAe package. Dietary zinc requirements of juvenile abalone were estimated
by broken-line regression analysis Robbins et al., 1979; Robbins, 1986.. The linear
segments of the regression lines below the breakpoints were used to compare the
bioavailability of dietary zinc from ZnMet with that from ZnSO4 by deriving the ratio of
the slopes of the lines Forbes and Parker, 1977; Paripatananont and Lovell, 1995..

3. Results
3.1. Leaching
The results of the leaching test with experimental diets is illustrated in Fig. 1. The
zinc content of the diets supplemented with the two zinc sources all declined during the
test period. After 6 h in seawater, the remaining zinc content of the diets ranged from
4.78 to 52.45 mgrkg for ZnMet diets Fig. 1A., and from 4.78 to 50.08 mgrkg for
ZnSO4 diets Fig. 1B.. After 12 h of immersion in seawater, the dietary zinc content
ranged from 3.96 to 29.35 mgrkg for ZnMet diets Fig. 1A., and from 3.96 to 31.05 for
ZnSO4 diets Fig. 1B.. There was a similar leaching rate between the two zinc sources
P ) 0.1, as determined by analysis of co-variance.. After 6 h of immersion in seawater,
the leached zinc accounted for approximately 40% of the total zinc in the diets for the
two sources of zinc, and this value increased to approximately 60% after 12 h of
immersion in seawater.
3.2. Surial and growth
There were no significant differences in survival of abalone fed the dietary treatments
ANOVA, P ) 0.05., which ranged from 94.7% to 100.0% for both zinc sources
Tables 2 and 3.. The mean weight gain rate WGR, %. and daily increment in shell
length DISL, mmrday. of the animals, however, were significantly affected by the
various levels of dietary zinc from the two zinc sources Tables 2 and 3.. WGR ranged
from 53% to 143% for ZnMet Table 2., and from 53% to 136% for ZnSO4 Table 3..
DISL ranged from 62 to 89 mmrday for ZnMet Table 2., and from 62 to 87 mmrday
for ZnSO4 Table 3.. Both WGR and DISL responded in broken-line models to increases
in dietary zinc levels from both sources of zinc Figs. 2 and 3.. The breakpoint in the

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

73

Fig. 1. Changes of zinc content in the diets containing various levels of supplemental zinc to the basal diet
from either ZnMet A. or ZnSO4 B. with increasing immersion time 0, 6 and 12 h, respectively. in seawater.
Error bars are the SD, values significantly different ANOVA, Tukeys test. from the controls 0 h. are
indicated with asterisks ) for P - 0.05 and ) ) for P - 0.01..

regression line, which was considered to be the minimum dietary level for optimum
response, was 15.5 mg zincrkg diet for WGR, and that was 15.2 mg zincrkg diet for
DISL when using ZnMet as the zinc source Fig. 2.. However, the breakpoint values for
WGR and DISL were much higher when using ZnSO4 as the zinc source compared to
ZnMet, and reached 34.1 and 34.0 mg zincrkg diet, respectively Fig. 3.. On the basis

74

Supplemental zinc
mgrkg.

Dietary zinc
mgrkg.

Initial shell
length mm.

Initial weight
g.

Final shell
length mm.

0
5
10
20
30
40
80

5.6
11.2
14.8
26.1
34.9
43.1
83.9

16.41
16.39
16.44
16.48
16.41
16.37
16.39

0.74
0.73
0.75
0.75
0.73
0.73
0.74

23.27 a
24.71b
26.00 c
26.15 c
26.06 c
26.21c
25.91c

0.1
0.36
0.89

0.03
0.62
0.71

0.2
75.05
0.00

ANOVA
Pooled SEM
F value
P value

Final weight
g.
1.13 a
1.49 b
1.76 c
1.79 c
1.76 c
1.78 c
1.74 c

0.05
110.27
0.00

WGR
%.

DISL
mmrday.

Survival
%.

53.2 a
102.6 b
137.0 c
138.8 c
140.3 c
142.8 c
137.0 c

62.3 a
76.6 b
86.9 c
87.9 c
87.7 c
89.4 c
86.5 c

94.7
98.7
100.0
100.0
100.0
98.7
100.0

8.3
89.01
0.00

5.2
70.51
0.00

Means in the same column sharing a common superscript letter were not significantly different P ) 0.05. as determined by Tukeys test.

4.5
1.67
0.20

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

Table 2
Weight gain rate WGR., daily increment in shell length DISL. and survival of abalone fed graded levels of dietary zinc from zinc methionine ZnMet. for 16 weeks

Supplemental zinc
mgrkg.

Dietary zinc
mgrkg.

Initial shell
length mm.

Initial weight
g.

Final shell
length mm.

0
5
10
20
30
40
80

5.6
10.7
15.1
25.2
34.7
45.3
84.6

16.41
16.36
16.47
16.57
16.33
16.46
16.41

0.74
0.72
0.75
0.76
0.73
0.75
0.74

23.27 a
23.67 ab
24.48 bc
25.19 cd
25.87 d
25.83 d
25.80 d

0.1
0.80
0.59

0.03
1.49
0.25

0.2
32.43
0.00

ANOVA
Pooled SEM
F value
P value

Final weight
g.
1.13 a
1.24 a
1.43 b
1.60 c
1.72 cd
1.74 cd
1.75d

0.03
77.27
0.00

WGR
%.

DISL
mmrday.

Survival
%.

53.2 a
71.8 ab
90.7 b
112.2 c
136.2 d
132.4 d
135.8 d

62.3 a
66.5ab
72.8 b
78.4 c
86.6 d
85.2 d
86.0 d

94.7
98.7
100.0
98.7
100.0
98.7
100.0

9.4
64.40
0.00

4.8
35.26
0.00

4.9
1.26
0.34

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

Table 3
Weight gain rate WGR., daily increment in shell length DISL. and survival of abalone fed graded levels of dietary zinc from zinc sulfate ZnSO4P7H 2 O. for 16
weeks

Means in the same column sharing a common superscript letter were not significantly different P ) 0.05. as determined by Tukeys test.

75

76

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

Fig. 2. Regression of weight gain rate WGR, %., daily increment in shell length DISL, mmrday., soft-body
alkaline phosphatase activity SBAKP, Urg protein. and soft-body zinc concentration mgrkg, wet-weight
basis. on dietary zinc levels and breakpoints bkpt. in the lines for juvenile abalone fed diets containing graded
levels of zinc methionine ZnMet. for 16 weeks. The term ACriteria valuesB represents the values of selected
criteria, including WGR, DISL, SBAKP and SB zinc.

of WGR or DISL, the bioavailability of dietary zinc mainly from ZnMet to abalone was
significantly higher P - 0.01, as determined by analysis of co-variance. than that from
ZnSO4 . The ratios of the slopes of the broken-line equations were 3.28 9.1039r2.774.
for WGR and 3.27 2.6776r0.8189. for DISL with ZnMet as zinc source compared to
ZnSO4 Figs. 2 and 3.. Thus, the bioavailability of dietary zinc mainly from ZnMet
seemed to be about three times as high as that mainly from ZnSO4 .
3.3. Carcass composition and SBAKP actiity
There were no significant differences P ) 0.05. in carcass composition of the
abalone fed various levels of dietary zinc from the two zinc sources Table 4.. Soft body
alkaline phosphatase SBAKP. activity ranged from 30.0 to 85.4 Urg protein for
abalone fed diets containing graded levels of supplemental zinc from ZnMet, and from
30.0 to 86.2 Urg protein for abalone fed diets containing graded levels of supplemental
zinc from ZnSO4 Table 4.. Enzyme activity responded in broken-line models to

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

77

Fig. 3. Regression of weight gain rate WGR, %., daily increment in shell length DISL, mmrday., soft-body
alkaline phosphatase activity SBAKP, Urg protein. and soft-body zinc concentration mgrkg, wet-weight
basis. on dietary zinc levels and breakpoints bkpt. in the lines for juvenile abalone fed diets containing graded
levels of zinc sulfate ZnSO4P7H 2 O. for 16 weeks. The term ACriteria valuesB represents the values of
selected criteria, including WGR, DISL, SBAKP and SB zinc.

increases in dietary zinc levels with both sources of zinc. The breakpoints in the
regression lines were 15.5-mg zincrkg diet for ZnMet Fig. 2., and 31.9-mg zincrkg
diet for ZnSO4 Fig. 3.. On the basis of SBAKP activity, the bioavailability of dietary
zinc mainly from ZnMet to abalone was significantly higher P - 0.01, as determined
by analysis of co-variance. than that from ZnSO4 .The bioavailability of dietary zinc
mainly from ZnMet in relation to that mainly from ZnSO4 was 278% 100 =
5.6278r2.0234..
3.4. Elemental concentrations
The levels of soft body ash, zinc, and iron are shown in Table 5. After 16 weeks of
feeding trial, no significant differences P ) 0.05. were observed in the levels of soft
body ash and iron among dietary treatments Table 5.. The zinc content, however, was
significantly affected P - 0.01. by the various levels of dietary zinc from the two zinc
sources. The zinc content ranged from 16.6 to 54.6 mgrg for ZnSO4 , and from 16.6 to
54.3 mgrg for ZnMet. Zinc content also responded in a broken-line model to increases
in dietary zinc levels with both sources of zinc Figs. 2 and 3.. The breakpoints in the

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

78

Table 4
Carcass composition and alkaline phosphatase SBAKP. activity in abalone fed graded levels of supplemental
zinc from ZnSO4 or ZnMet for 16 weeks )
Supplemental
zinc
mgrkg. ) )

Moisture %.

Protein %.

ZnSO4

ZnMet

ZnSO4

ZnMet

ZnSO4

ZnMet

0
5
10
20
30
40
80

77.52
77.14
77.19
76.76
77.29
77.36
77.13

77.52
76.67
77.25
77.20
76.81
77.15
76.88

53.41
53.56
53.44
52.86
53.48
53.29
53.87

53.41
53.57
53.52
53.74
53.52
53.96
53.92

7.17
7.36
7.28
7.45
7.48
7.29
7.39

1.0
0.94
0.52

0.9
1.65
0.26

0.4
0.70
0.66

0.3
0.45
0.82

0.2
0.61
0.72

ANOVA
Pooled SEM
F value
P value

Lipid %.

SBAKP Urg protein.

ZnSO4

ZnMet

7.17
7.21
7.30
7.29
7.24
7.33
7.39

30.0 a
38.5a
60.3 b
80.4 c
85.5 c
86.2 c
85.5 c

30.0 a
51.8 b
83.3 c
85.1c
83.2 c
84.4 c
85.4 c

0.1
0.83
0.58

4.2
238.77
0.00

4.0
123.74
0.00

Means in the same column sharing a common superscript letter were not significantly different P ) 0.05. as
determined by Tukeys test.
)
Values are means of three groups of abalone, with eight abalonergroup ns 3. for determining moisture,
protein and lipid, and three abalonergroup ns 3. for measuring SBAKP.
))
The basal diet contained 5.60 mg of zincrkg diet, and the measured total dietary zinc levels are the
same as those in Tables 2 and 3.

Table 5
Ash and selected elemental concentrations in the soft bodies of abalone fed various levels of supplemental zinc
from ZnSO4 or ZnMet for 16 weeks )
Supplemental zinc
mgrkg. ) )
0
5
10
20
30
40
80
ANOVA
Pooled SEM
F value
P value

Ash %.a

Zinc mgrg.aa

ZnSO4

ZnMet

11.50
11.49
11.52
11.48
11.49
11.59
11.48

11.50
11.54
11.51
11.53
11.46
11.56
11.59

0.9
0.22
0.96

1.0
0.48
0.80

ZnSO4
16.56 a
24.15 b
33.31c
41.25d
52.28 e
52.81e
54.56 e

1.3
127.16
0.00

Iron mgrg.aa

ZnMet
16.56 a
28.04 b
46.56 c
53.18 c
53.12 c
52.40 c
54.27 c

1.1
115.33
0.00

ZnSO4

ZnMet

486
515
516
521
504
509
522

486
499
520
506
512
504
524

21.7
0.72
0.65

18.5
0.83
0.58

Means in the same column sharing a common superscript letter were not significantly different P ) 0.05. as
determined by Tukeys test.
)
Values are means of three groups of abalone,with the soft bodies of four abalonergroup. ns 3..
))
The basal diet contained 5.60 mg of zincrkg diet, and the measured total dietary zinc levels are the
same as those in Tables 2 and 3.
a
Dry-weight basis.
aa
Wet-weight basis.

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

79

Table 6
Ash and selected elemental concentrations in the shells of abalone fed various levels of supplemental zinc
from ZnSO4 or ZnMet for 16 weeks )
Supplemental zinc
mgrkg. ) )

Ash %.a
ZnSO4

0
5
10
20
30
40
80

74.54
74.51
74.64
75.58
74.49
74.68
74.54

0.1
0.34
0.89

ANOVA
Pooled SEM
F value
P value

Zinc mgrg.aa

Iron mgrg.aa

ZnMet

ZnSO4

ZnMet

ZnSO4

ZnMet

74.54
74.47
74.58
74.52
74.49
74.55
74.52

28.20
29.56
28.64
29.63
30.24
29.11
28.44

28.20
28.33
29.72
30.51
30.08
30.97
29.23

117
126
123
121
118
125
130

117
121
120
126
122
127
123

0.2
0.21
0.96

1.0
0.67
0.68

1.2
1.72
0.25

8.6
0.44
0.83

9.1
0.31
0.91

Values are means of three groups of abalone, with the shells of 4 abalonergroup ns 3..
The basal diet contained 5.60 mg zincrkg diet, and the measured total dietary zinc levels are the same
as those in Tables 2 and 3.
a
Dry-weight basis.
aa
Wet-weight basis.
))

regression lines were 17.8-mg zincrkg diet for ZnMet Fig. 2., and 34.3-mg zincrkg
diet for ZnSO4 Fig. 3.. On the basis of SB zinc deposition, the bioavailability of dietary
zinc mainly from ZnMet to abalone was significantly higher P - 0.01, as determined
by analysis of co-variance. than that from ZnSO4 . The bioavailability of zinc from

Table 7
The dietary zinc requirements on the basis of weight gain rate WGR, %., daily increment in shell length
DISL, mmrday., soft body alkaline phosphatase SBAKP, Urg protein. and soft body zinc concentration SB
zinc, mgrg. determined with ZnMet and ZnSO4 as the zinc sources and the relative bioavailability %. of
dietary zinc with ZnMet as compared with ZnSO4 in juvenile abalone
Criteria

Zinc
sources

Broken-line equation

Breakpoints
mgrkg. a, )

Requirements )
mgrkg.

Relative
bioavailability
%. b

WGR

ZnMet
ZnSO4
ZnMet
ZnSO4
ZnMet
ZnSO4
ZnMet
ZnSO4

Y s142.76y9.103915.49y X .
Y s136.19y2.77434.10y X .
Y s87.67y2.677615.16y X .
Y s86.02y0.818933.99y X .
Y s83.22y5.628715.54y X .
Y s86.15y2.023431.91y X .
Y s 53.18y3.158517.75y X .
Y s 52.28y1.186734.29y X .

15.49"0.3
34.10"0.7
15.16"0.2
33.99"0.7
15.54"0.2
31.91"0.6
17.75"0.3
34.29"0.7

15.5"0.3
34.1"0.7
15.2"0.2
34.0"0.7
15.5"0.2
31.9"0.6
17.8"0.3
34.3"0.7

328

DISL
SBAKP
SB zinc

327
278
266

Values are means"SEM ns 3. as determined by broken-line regression analysis Robbins et al., 1979..
Breakpoint in the regression line.
b
The ratio of the slope of ZnMet broken-line equation to the slope of ZnSO4 broken-line equation=100.
a

80

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

ZnMet in relation to that from ZnSO4 was 266% 100 = 3.1585r1.1867.. Both the ash
content and the selected elemental concentrations in the shells were relatively constant
P ) 0.05. regardless of dietary treatment Table 6..
The dietary zinc requirements for different criteria determined with ZnMet and
ZnSO4 and the relative bioavailabilities of ZnMet compared to ZnSO4 in juvenile
abalone are summarized in Table 7.

4. Discussion
The diets used in this experiment supported satisfactory abalone growth. After 16
weeks of feeding, all groups which obtained sufficient dietary zinc from the two zinc
sources grew well both in mean WGR and in mean DISL, in comparison to those
reported by other authors e.g. Uki et al., 1985; Uki and Watanabe, 1992; Mai et al.,
1995a,b; Mai, 1998..
To our knowledge, there is only one published paper pertaining to the dietary mineral
nutrition of abalone H. laeagata. Coote et al., 1996.. This is probably in part due to
the problems associated with incredible leaching of minerals added to soft moist diets. In
the present experiment, the special feed manufacturing technology was adopted so as to
improve the water stability of the feed. The leaching of supplemental zinc from the basal
diet, however, was still high Fig. 1.. The leaching trials indicated that the remaining
dietary zinc from the two zinc sources was approximately 60% of the total dietary zinc
after 6 h of immersion in seawater, and this value decreased to 3040% after 12 h of
immersion. We observed the fact that the digestive tracts of most abalone were full of
food within 2 h of feeding with the premium quality diets Mai et al., 1998.. This,
together with the fact that a series of criteria responded in broken-line models to
increases in dietary zinc levels with the two zinc sources makes the requirement of
dietary zinc of juvenile abalone recommended in the present study acceptable. If the
water stability of dietary zinc can be further improved, however, the requirement of
dietary zinc for this species may be further reduced to a certain extent.
In the present study, the WGR and DISL were the two responsive parameters to
dietary zinc levels from both ZnMet and ZnSO4 , and responded in broken-line models to
increases in dietary zinc levels. Significantly depressed growth was noticed after 16
weeks of feeding abalone low-zinc diets. Impaired growth also has been observed in
fishes fed low-zinc diets Ogino and Yang, 1978, 1979; Gatlin and Wilson, 1983; Gatlin
et al., 1991; Paripatananont and Lovell, 1995; Wei et al., 1999.. The present result
indicates that supplementation of zinc to the basal diet is necessary to obtain normal
growth of abalone, H. discus hannai.
The activity of the zinc-containing enzyme, alkaline phosphatase, in soft-body was
strongly influenced by dietary zinc concentration from the two zinc sources. This
implies that the abalone did experience zinc deficiency, which would lead to depressed
enzyme activity. Thus, the alkaline phosphatase activity was a useful criterion in
estimating dietary zinc requirements and evaluating bioavailability of zinc for H. discus
hannai.

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

81

Many studies examining the dietary elemental requirements of aquatic species have
shown that depressed whole-body or tissue levels of essential elements could result from
insufficient dietary intake Lovell, 1978; Ogino and Yang, 1978, 1979; Gatlin et al.,
1982; Wilson et al., 1982; Paripatananont and Lovell, 1995.. Baker 1986. also indicated
that studies on the mineral requirements of animals should include measurement of body
stores of the test element. Mineral analyses at the end of the feeding trial indicated that
soft-body zinc concentrations of the abalone increased linearly until dietary zinc reached
17.8 mgrkg for ZnMet Fig. 2., and 34.3 mgrkg for ZnSO4 Fig. 3.. The reduced zinc
reserves were becoming depleted and deficiency signs would most likely become
apparent if those diets were fed for an extended period of time. Therefore, the soft-body
zinc concentration was also a responsive criterion for estimating the dietary zinc
requirement of abalone. However, similar responses were not observed in shell zinc
concentrations of the abalone. This suggests that shell zinc deposition of the abalone was
not a useful criterion for determining the zinc requirement of the abalone, especially
when the experimental duration was not long enough. Shellfish have special formation
mechanisms for biomineralization of their hard tissues. Sakai 1980. found that the
accumulation of organic acids in the rearing water could lead to severe shell erosion in
young abalone, and then cause the shell to split along the respiratory apertures. Chen
1989. reported that there was a marked depression in calcium and zinc concentration in
split H. diersicolor supertexta. These results, along with the data obtained in the
present study, imply that the rearing water quality, such as pH, perhaps plays a more
significant role than the dietary mineral concentration in shell mineralization and shell
mineral deposition of abalone.
Many studies with fish have shown elevated iron levels in zinc deficiency Ogino and
Yang, 1978; Wekell et al., 1986; Spry et al., 1988.. The zinc deficiency sign such as
depressed growth occurred in this experiment, but no elevated iron levels were observed
either in the shells or in the soft bodies from the abalone fed the low-zinc diets with
ZnMet or ZnSO4 . The interaction among minerals such as zinciron should be further
studied in mollusks.
The minimum level of dietary zinc for juvenile abalone varied with zinc sources and
criteria used Table 7.. Data of growth, alkaline phosphatase and soft-body zinc
concentration showed that about 1618 mgrkg of dietary zinc from ZnMet could
maintain optimum responses. However, when using ZnSO4 as the supplemental zinc
source, the minimum level of dietary zinc increased to 3235 mgrkg. We therefore,
estimate that the dietary zinc requirement of juvenile abalone is 1618 mgrkg when
using ZnMet as zinc. This estimated requirement is higher than that for channel catfish
6.58 mg zincrkg; Paripatananont and Lovell, 1995.. Using ZnSO4 as the supplemental
zinc sources, the dietary zinc requirement of the abalone was found to be 3235 mgrkg.
This value is higher than those reported for rainbow trout 1530 mgrkg; Ogino and
Yang, 1978., carp 1530 mgrkg; Ogino and Yang, 1979., channel catfish 20 mgrkg;
NRC, 1993., tilapia 20 mgrkg; McClain and Gatlin, 1988. and red drum 2025
mgrkg; Gatlin et al., 1991..
Data from the present study indicate that the juvenile abalone required approximately
three times as much zinc in the inorganic form as in organic form. The relative
bioavailability values of ZnMet to ZnSO4 for weight gain rate, 328%, daily increment in

82

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

shell length, 327%, alkaline phosphatase activity, 278%, and soft-body zinc deposition,
266%, accurately described the differences in the two sources of zinc. These results
agree with those reported by Paripatananont and Lovell 1995.. They indicated that the
relative bioavailabilities of ZnMet, with ZnSO4 as the standard, were 352% for weight
gain and 305% for bone zinc deposition with egg-white diets.
The nutritional value of dietary mineral sources depends not only upon their content
in the feedstuff but also upon the bioavailability of the element to the animal Paripatananont and Lovell, 1997.. Studies with mammals have shown that chelation of
minerals to amino acids may increase their absorption rate in the intestine Ashmead,
1992.. Ashmead 1992. indicated that the higher bioavailability of amino acid-bound
trace elements to animals is because chelation protects the element from forming
insoluble complexes in the digestive tract and facilitates zinc transport across the
intestinal mucosa. He also suggested that the chelate could remain intact until it reaches
the site in the body where the element is needed. The results obtained in this study
clearly indicated that the bioavailability of ZnMet is much higher than that of ZnSO4 to
juvenile abalone, H. discus hannai. Thus, in the formulation of diets for the abalone, the
dietary allowance of zinc could be reduced by using the chelated-zinc instead of the
inorganic form. This may lower the cost of feed and reduce the water pollution.
Acknowledgements
The authors are grateful for financial support by grant No. 39670572 from the
National Natural Science Foundation of China NNSFC.. We also thank Mr. Fulong Liu
of Shandong Commodity Inspection Bureau for assistance in elemental analyses.
References
Ashmead, H.D., 1992. The Roles of Amino Acid Chelates in Animal Nutrition. Noyes Publications, NJ, 479
pp.
Association of the Official Analytical Chemists, 1984. Method of Analysis. AOAC, Washington, DC, 1141 pp.
Baker, D.H., 1986. Problems and pitfalls in animal experiments designed to establish dietary requirements for
essential nutrients. J. Nutr. 116, 22392249.
Chen, H.C., 1989. Farming the small abalone, Haliotis diersicolor supertexta, in Taiwan. In: Hahn, K.O.
Ed.., Handbook of Culture of Abalone and Other Marine Gastropods. CRC Press, Boca Raton, FL, pp.
265283.
Chesters, J.K., 1991. Trace elementgene interactions with particular reference to zinc. Proc. Nutr. Soc. 50,
123129.
Clavier, J., 1992. The ormer Haliotis tuberculata. fishery of France and the Channel Islands. In: Shepherd,
S.A., Tegner, M.J., Guzman
del Proo,
S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 454460.
Coote, T.A., Hone, P.W., Kenyon, R., Maguire, G.B., 1996. The effect of different combinations of dietary
calcium and phosphorus on the growth of juvenile Haliotis laeigata. Aquaculture 145, 267279.
Farlinger, S., Campbell, A., 1992. Fisheries management and biology of northern abalone, Haliotis
kamtschatkana, in the northerneast Pacific. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,
S.A.
Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 395405.
Forbes, R.M., Parker, H.M., 1977. Biological availability of zinc in and as influenced by whole fat soy flour in
rat diets. Nutr. Rep. Int. 15, 681689.

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

83

Gatlin, D.M. III, Wilson, R.P., 1983. Zinc supplementation of practical channel catfish diets. Aquaculture 52,
191198.
Gatlin, D.M. III, OConnell, J.P., Scarpa, J., 1991. Dietary zinc requirement of the red drum, Sciaenops
ocellatus. Aquaculture 92, 259265.
Gatlin, D.M. III, Robinson, E.H., Poe, E.E., Wilson, R.P., 1982. Magnesium requirement of channel catfish. J.
Nutr. 112, 11971202.
Guzman
del Proo,
S.A., 1992. A review of the biology of abalone and its fishery in Mexico. In: Shepherd,
S.A., Tegner, M.J., Guzman
del Proo,
S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 341360.
Hartee, E.F., 1972. Determination of protein: a modification of the Lowry method that gives a linear
photometric response. Anal. Biochem. 48, 422427.
Johnson, D.W., Al-Harassy, A., Al-Harthy, M., 1992. The sultanate of oman abalone fishery. In: Shepherd,
S.A., Tegner, M.J., Guzman
del Proo,
S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 448453.
Lall, S.P., 1989. The minerals. In: Halver, J.E. Ed.., Fish Nutrition. Academic Press, San Diego, CA, pp.
219257.
Li, A., Li, J., Lei, Q., Wang, F., Chuang, J., 1995. Application of different forms of trace minerals in feed for
fish and prawn. In: Li, A., Shiau, S. Eds.., The Proceeding of the Second Symposium of Worlds Chinese
Scientists on Nutrition and Feeding of Finfish and Shellfish. Ocean University of Qingdao Press, Qingdao,
China, pp. 3346, in Chinese with English abstract..
Lovell, R.T., 1978. Dietary phosphorus requirements of channel catfish Ictaluras punctatus .. Trans. Am.
Fish. Soc. 107, 617621.
Maage, A., Julshamn, K., 1993. Assessment of zinc status in juvenile Atlantic salmon Salmo salar . by
measurement of whole body and tissue levels of zinc. Aquaculture 117, 179191.
Mai, K., 1998. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and H.
discus hannai Ino: VII. Effects of dietary vitamin C on survival, growth and tissue concentration of
ascorbic acid. Aquaculture 161, 383392.
Mai, K., He, G., Xu, W., 1998. Studies on postprandial changes of digestive status and free amino acids in the
viscera of Haliotis discus hannai Ino. J. Shellfish Res. 17, 717722.
Mai, K., Mercer, J.P., Donlon, J., 1995a. Comparative studies on the nutrition of two species of abalone,
Haliotis tuberculata L. and H. discus hannai Ino: III. Response of abalone to various levels of dietary
lipids. Aquaculture 134, 6580.
Mai, K., Mercer, J.P., Donlon, J., 1995b. Comparative studies on the nutrition of two species of abalone,
Haliotis tuberculata L. and H. discus hannai Ino: IV. Optimum dietary protein level for growth.
Aquaculture 136, 165180.
McClain, W.R. III, 1988. Dietary zinc requirement of Oreochromis aureus and effects of dietary calcium and
phytate on zinc bioavailability. J. World Aquacult. Soc. 19, 103108.
Nie, Z., 1992. A review of abalone culture in China. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,
S.A.
Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 592602.
NRC National Research Council., 1993. Nutrient Requirements of Fish. National Academy Press, Washington, DC, 114 pp.
Ogino, C., Yang, G., 1979. Requirement of carp for dietary zinc. Bull. Jpn. Soc. Sci. Fish. 45, 967969.
Ogino, C., Yang, G.Y., 1978. Requirement of rainbow trout for dietary zinc. Bull. Jpn. Soc. Sci. Fish. 44,
10151018.
Paripatananont, T., Lovell, R.T., 1995. Chelated zinc reduces the dietary zinc requirement of channel catfish,
Ictalurus punctatus. Aquaculture 133, 7382.
Paripatananont, T., Lovell, R.T., 1997. Comparative net absorption of chelated and inorganic trace minerals in
channel catfish Ictalurus punctatus diets. J. World Aquacult. Soc. 28, 6267.
Parker, D.O., Haaker, P.L., Togstadt, H.A., 1992. Case histories for three species of California abalone:
Haliotis corrugata, H. fulgens and H. cracherodii. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,

S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 384394.
Prince, J.D., Shepherd, S.A., 1992. Australian abalone fisheries and their management. In: Shepherd, S.A.,
Tegner, M.J., Guzman
del Proo,
S.A. Eds.., Abalone of the World. Blackwell Scientific Publications,
Cambridge, MA, pp. 407426.

84

B. Tan, K. Mai r Aquaculture 192 (2001) 6784

Robbins, K.R., 1986. A method, SAS program, and examples for fitting the broken line to growth data. Univ.
Tenn. Res. Rep. 8690. Univ. Tenn Agric. Exp. Sta., Knoxville, TN.
Robbins, K.R., Norton, H.W., Baker, D.H., 1979. Estimation of nutrient requirements from growth data. J.
Nutr. 109, 17101714.
Sakai, H., 1980. A method to prevent erosion in the shells of young abalone. Aquaculture 28, 102106.
Schiel, D.R., 1992. The paua abalone. fishery of New Zealand. In: Shepherd, S.A., Tegner, M.J., Guzman
del
Proo,
S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 427437.
Shearer, K.D., 1984. Changes in elemental compositions of rainbow trout of associated with growth and
reproduction. Can. J. Fish. Aquat. Sci. 41, 15921600.
Spry, D.J., Hodson, P.V., Wood, C.M., 1988. Relative contributions of dietary and waterborne zinc in the
rainbow trout Salmo Gairdneri .. Can. J. Fish. Aquat. Sci. 45, 3241.
Tarr, R.J.Q., 1992. The abalone fishery of South Africa. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,

S.A. Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 438447.
Tegner, M.J., DeMartini, J.D., Karpov, K.A., 1992. The California red abalone fisheries: a case study in
complexity. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,
S.A. Eds.., Abalone of the World.
Blackwell Scientific Publications, Cambridge, MA, pp. 370383.
Tietz, N.W., 1986. Textbook of Clinical Chemistry. W.B. Saunders, Philadephia, PA, 1919 pp.
Uki, N., Watanabe, T., 1992. Review of the nutritional requirements of abalone Haliotis spp.. and
development of more efficient artificial diets. In: Shepherd, S.A., Tegner, M.J., Guzman
del Proo,
S.A.
Eds.., Abalone of the World. Blackwell Scientific Publications, Cambridge, MA, pp. 504517.
Uki, N., Kemuyama, A., Watanabe, T., 1985. Development of semipurified test diets for abalone. Bull. Jpn.
Soc. Sci. Fish. 51, 18251833, in Japanese, with English abstract..
Watanabe, T., Kiron, V., Satoh, S., 1997. Trace minerals in fish nutrition. Aquaculture 151, 185207.
Wedekind, K.J., Hortin, A.E., Baker, D.H., 1992. Methodology for assessing zinc bioavailability: efficiency
estimates for zincmethionine, zinc sulfate, and zinc oxide. J. Anim. Sci. 70, 178187.
Wei, W., Li, A., Li, D., 1999. Effect of dietary supplemented zinc on the growth and some biochemical
parameters of juvenile flounder Paralichthys oliaceus .. J. Ocean Uni. Qingdao 18, 6066, in Chinese
with English abstract..
Wekell, J.C., Shearer, K.D., Gauglitz, E.J. III, 1986. Zinc supplementation of trout diets: tissue indicators of
body zinc status. Prog. Fish-Cult. 48, 205212.
Willis, J.N., Sunda, W.G., 1984. Relative contributions of food and water in the accumulation of zinc by two
species of marine fish. Mar. Biol. 80, 273279.
Wilson, R.P., Robinson, E.H., Gatlin, D.M. III, Poe, W.E., 1982. Dietary phosphorus requirement of channel
catfish. J. Nutr. 112, 11971202.