JOURNAL OF THE Vol. 28, No.

1
WORLD AQUACULTURE SOCIETY March, 1997

Comparative Net Absorption of Chelated and Inorganic

Trace Minerals in Channel Catfish Ictalurus punctatus Diets
TIPPAWAN PARIPATANANONT AND T. LOVELL’
RICHARD
Department of Fisheries and Allied Aquacultures, Auburn University, Alabama 36849 USA

Abstract
Coefficients of net absorption for copper, iron, manganese, selenium, and zinc were deter-
mined for chelated sources (copper proteinate, iron proteinate, manganese proteinate, sele-
nium proteinate, zinc proteinate) and inorganic sources (copper sulfate pentahydrate, fer-
rous sulfate heptahydrate, manganese sulfate monohydrate, sodium selenite, zinc sulfate hep-
tahydrate) of these elements with channel catfish Icfulurus puncfufus. Fish weighing approxi-
mately 60 g were placed into 40-L aquaria (12 fish/aquarium) a t a temperature of 28 f 2 C
and fed either an egg white-based, purified diet or a soybean meal-based, practical diet with
and without the test mineral sources for 6 wk then killed and feces collected from the hind-
gut. Treatments were arranged in a 2 X 2 factorial design. Absorption coefficients for the
elements in the basal and mineral supplemented diets were calculated by the indirect indica-
tor (chromic oxide) method and corrected for residual amounts of element in the basal diets.
Net absorption of the chelated minerals was significantly higher (f < 0.05) than net absorp-
tion of the inorganic minerals in both basal diets. Average percentage improvement in net
absorption of chelated minerals over inorganic minerals was 39.3% in the purified diets and
81.1% in the practical diets. These results may indicate that appreciably lower amounts of
chelated trace minerals than inorganic trace minerals can be used as supplements in catfish
feeds.

The nutritional value of dietary mineral
sources depends not only upon their con-
centration in the feedstuff but also upon the
amount of the element that is absorbed and
metabolized, or, the bioavailability of the el-
ement to the animal. Intrinsic (physiologi-
cal) and extrinsic (dietary) factors affect
bioavailability of trace elements to fish. In-
trinsic factors may include acidity of the di-
gestive tract in the fish species or nutritional
status of the fish. Extrinsic factors are those
affecting solubility of elements in the diges-
tive tract and include chemical forms of the
elements, competitive antagonism among
elements which may lead to formation of in-
soluble precipitates, adsorption of elements
on organic and inorganic surfaces such as
fiber or silica, and chelation effects which
may be positive or negative (O’Dell 1984;
Davis and Gatlin 1996).
Chelation has a marked influence on bio-
’ Corresponding author.
Q Copyright by the World Aquaculture Society 1997
availability of trace elements. A natural che-
lating agent in foods that decreases bio-
availability is inositol hexaphosphate, or
phytate, which forms insoluble complexes
with many cations, including zinc and iron
(Lo et al. 1981; Spinelli et al. 1983). There
are also naturally occurring chelating agents
that increase absorption of trace elements,
such as amino acids and peptides. Presently,
commercial trace mineral chelates are avail-
able for livestock in which the element is
sequested to an amino acid or a specific or
nonspecific peptide from a protein hydroly-
sate. This type of chelation increases ab-
sorption of the element by protecting it from
forming insoluble compounds or complexes
in the digestive tract or facilitating transfer
of the element across membranes (Ashmead
and Zunino 1992). Recently, Ashmead
(1992) proposed the metal chelate is ab-
sorbed from the gut intact, delivered to vari-
ous parts of the body, and degraded at the
site where the element is needed.
Livestock and poultry experiments have

62
 ABSORPTION OF TRACE MINERALS BY CATFISH 63

shown an improvement in growth and dis- TABLE1 . Composifion of the basal diets.
ease resistance when chelated minerals Egg white Soybean
were compared with inorganic sources diet meal diet
(Sanford 1976; Brethour 1984; Wedekind et Ingredients" (g/kg)
al. 1992). Paripatananont and Lovell (1995)
showed that chelated zinc reduced the di- Spray-dried egg white 400.0
Dextrin 370.0
etary zinc requirement of channel catfish Cellufil 107.5
three to five times when compared to inor- Biotin 0.0005
ganic zinc. No reports in the literature com- Corn oil 30.0 30.0
pare net absorption of chelated trace ele- Cod liver oil 30.0 30.0
ments with inorganic sources of the ele- Cu, Fe, Mn, Se, Zn-free
minerd mixb 40.0 40.0
ments in fish. If net retention of chelated el- Vitamin mix' 14.0 14.0
ements is significantly higher than that of Ascorbic acid 3.5 3.5
traditional inorganic sources, less of the el- polyphosphate
ement in chelated form can be used in fish Chromic oxide 5.0 5.0
feeds, which could improve economics and Soybean meal 550.0
Cottonseed meal 145.0
reduce the amount of trace elements dis- Corn 110.0
charged from aquaculture systems. The ob- Wheat 47.5
jective of this research was to compare net Carboxymethylcellulose 25.0
absorption of copper (Cu), iron (Fe), man-
Residual trace minerals
ganese (Mn), selenium (Se), and zinc (Zn)
(mgikg)
from chelated and inorganic sources in pu-
rified (without phytate) and practical type Copper 0.001 6.76
Iron 10.27 91.25
(with phytate) diets for channel catfish. Manganese 2.04 29.72
Se1enium 0.83 1 0.322
Materials and Methods Zinc 7.42 40.21
a Source of ingredients: spray-dried egg white, ICN
Experimental Design and Diets
Nutritional Biochemicals, Irvine, California, USA;
Absorption coefficients for Cu, Fe, Mn, other purified ingredients, United States Biochemical
Se, and Zn from chelated and inorganic Corporation, Cleveland, Ohio, USA, ascorbic acid
sources were determined in two basal diets, polyphosphate (15% ascorbic acid), Hoffman La
Roche (Basel, Switzerland).
an egg white-based diet and a soybean bContains (as g/kg of premix): CaCO,, 300.0;
meal-based diet (Table 1). The egg white MgSO4 . 7H20, 132.0; KzHP04, 240.0; NaHzPO, .
diet was formulated from purified ingredi- H,O, 231.0; NaCI, 45.0; AICI, . 6H,O, 0.15; KI, 0.15;
ents to contain 34% crude protein and 3.1 CoCI, . 6H20, 1.0; cellulose, 50.35.
kcal of digestible energy (DE)/g based on 'Provides the following diluted in cellulose (mg/kg
of diet): thiamin, 10; choline, 500; niacin, 1 5 0
tabular values of the diet ingredients pro- riboflavin, 2 0 pyridoxine, 20; calcium pantothenate,
vided by the National Research Council 200; vitamin B,,, 0.06; retinyl acetate (500,000 IU/g),
(NRC 1993). The soybean meal diet was 12; alpha-tocopherol, 50; cholecalciferol ( 1 ,000,000
formulated to contain 32% crude protein ICU/g), 1; menadione Na-bisulfite, 80; inositol, 400;
and 2.9 kcal of DE/g, based on tabular val- biotin. 1.
ues for the diet ingredients (NRC 1993).
The diets were prepared as 3-mm diameter, cept Cu, Fe, Mn, Se, and Zn were added to
semi-moist (approximately 30% moisture) both basal diets to meet the channel cat-
pellets as described by El Naggar and fish's dietary requirements (NRC 1993).
Lovell (1991). Additional biotin was in- The organic sources of the tested minerals
cluded in the egg white diet to compensate were copper proteinate, iron proteinate,
for biotin binding effect of avidin (Scarpa manganese proteinate, selenium proteinate,
and Gatlin 1992).All inorganic minerals ex- and zinc proteinate (Chelated Minerals Cor-
64 PARIPATANANONT AND LOVELL

poration, Salt Lake City, Utah, USA). The the guidelines of the Animal Care and Use
inorganic sources were copper sulfate pen- Committee of Auburn University. At the end
tahydrate (CuSO, . 5H20), ferrous sulfate of the feeding period, fish were killed ap-
eptahydrate (FeSO, . 7H@), manganese proximately 8 h after last feeding by expos-
sulfate monohydrate (MnSO, . H20), so- ing them to an overdose (500 mg/L) of an-
dium selenite (Na2Se03), and zinc sulfate esthetic (tricane methane sulfonate, MS-
heptahydrate (ZnSO, . 7H20) (Fisher Sci- 222) and feces were immediately collected
entific, Norcross, Georgia, USA). The con- from the hindgut posterior to the intestinal
centrations of the elements added to each sphincter as described by Smith and Love11
basal diet from organic or inorganic sources (1973) and kept frozen until analyzed. Fe-
were 10, 60, 5 , 0.5, and 40 m a g of diet ces were dried to constant weight and
for Cu, Fe, Mn, Se, and Zn, respectively. pooled from 12 fish from each aquarium.
Chromic oxide (0.5%) was added to each Copper, iron, manganese, zinc, selenium,
diet as an indicator for net absorption mea- and chromic oxide contents of feces were
surement. Diets were stored at -18 C and analyzed by the same procedures used for
thawed in a refrigerator for 24 h before diets.
feeding. The basal and experimental diets Net absorption coefficients for each min-
were analyzed for Cu, Fe, Mn, and Zn eral in the basal and test (supplemented) di-
with an inductively-coupled argon plasma ets were calculated by the formula:
(ICAP) spectrometer (Model 9000, Jarrell-
Ash Division, Fisher Scientific Company,
Waltham, Massachusetts, USA). Se was
analyzed by a fluorometric method (Asso- where NA = percentage net absorption of
ciation of Official Analytical Chemists mineral in the diet. Net absorption of the
1990). Chromic oxide was analyzed by the supplemented mineral was corrected for re-
procedure described by Furukawa and Tsu- sidual amounts of the element in the basal
kahara (1966). diet. Corrected net absorption, CNA, was
calculated as follows:
Digestibility Trials
Channel catfish weighing approximately CNA = 100
60 g from the Alabama Agricultural Experi-
ment Station were brought into the fish nu-
trition laboratory at the Auburn University Three corrected net absorption coeffi-
Fisheries Research Unit, placed into a cients were determined for each element in
trough and fed an egg white basal diet each diet. The data were analyzed by 2 X
supplemented with all minerals (inorganic 2 (2 basal diets, 2 mineral sources) facto-
sources) for 7 d to acclimate the fish to the rial arrangement of treatments using the
laboratory environment. The fish were sub- SAS ANOVA procedure (SAS 1987) to de-
sequently stocked randomly into 40-L termine if there were differences between
aquaria (12 fisWaquarium) which were sup- diets or mineral sources, or inter-
plied with a continuous flow of sand- action. Specific mean comparisons were
filtered water from a 10-ha reservoir. Water made using Fisher's least significant differ-
flow rate through each aquarium was 1 ence test (Sokal and Rolf 1981). Differences
L/min, temperature was maintained at 28 2 were considered significant at P < 0.05.
2 C and a 12-h lightll2-h dark photoperiod
was maintained. Three aquaria of fish were
randomly assigned to each of the basal and Results and Discussion
experimental diets. The fish were fed twice Table 2 presents average corrected net ab-
daily to satiation for 6 wk. Handling proce- sorption coefficients for each mineral from
dures and facilities were in compliance with chelated and inorganic sources in the two
 ABSORPTION OF TRACE MINERALS BY CATFISH 65

TABLE2. Means ( 2 SD) of net absorption of supplemented minerals from organic and inorganic sources in
egg white-based and soybean meal-based diets for channel carfish.'

Net absorption (%)

Basal diet Mineral source Copper Iron Manganese Selenium Zinc
Egg white Chelated 89.9" 87.8" 93.1" 90.8" 91.3"
Inorganic 64.1 65.3b 66.6b 62.gb 66.4b
Soybean meal Chelated 89.3" 83.6" 87.1" 88.9" 90.4'
Inorganic 39.7' 5 1.3' 40.3' 69.7b 5 1.9'
Pooled SEM 2.1 I 3.67 3.89 3.74 2.57

ANOVA
Source of Variation: Probability' - - - - - - - - --- - _ _ - - -
Diet o.Oo0 1 0.0018 0.000I 0.3452 0.0033
Mineral source o.Oo0 I o.Oo01 o.Oo01 o.Oo01 o.Oo01
Diet*Mineral o.Oo01 0.0404 0.0036 0.1142 0.0065
' Means in columns with different letters are significantly different (P < 0.05). Means of pooled samples of
feces from 12 fish in each of three aquaria.
* Probability of treatment differences as determined by ANOVA.
types of diets. Both the type of diet and the Average percentage improvement of all
source of mineral had a significant effect on minerals except Se was 94.6% in the
net absorption of all minerals except Se. Net soybean meal diet as compared to 38% in
absorption of all minerals was significantly the egg white diet.
higher for chelated minerals than for inor- Results of this study agree with those
ganic minerals in both the purified and prac- from studies with dogs (Lowe et al. 1994),
tical diets. Net absorption of chelated min- lambs (Spears 1989), chickens (Suso and
erals was not different between the purified Edwards 1968) and rats (Ashmead and
and practical diets, but net absorption of in- Zunino 1992) which have shown that net
organic minerals, with the exception of Se, absorption of chelated trace elements is
was significantly higher in the purified diet higher than that of inorganic forms. Hardy
than in the practical diet. Net absorption of and Shearer (1992) found in rainbow trout
Se in neither chelated nor inorganic forms Oncorhynchus mykiss that feeding a zinc-
was significantly different between the pu- amino acid chelate resulted in greater zinc
rified and practical diets. There was a sig- deposition in body tissue than zinc sulfate
nificant interaction between diet and min- in a low calcium-phosphorus diet but not in
eral source for all minerals except Se. high calcium-phosphorus diets. Bell and
Chelation improved net absorption of all Cowey (1989) found in purified diets of At-
minerals except Se more in the practical lantic salmon Safmo safar that net absorp-
diet than in the purified diet (Table 3). tion of selenium was 91.6% for selenome-
thionine as compared to 64.0% for sodium
TABLE3. Percentage improvement in ner absorption
of mineralsfi-om organic sources over that from in-
selenite; this represents a superiority of che-
organic sources.' lated Se over inorganic Se of 43.1 % which
is very close to that found with channel cat-
Mineral Egg-white diet Soybean meal diet fish of 44.6% (Table 3). This close agree-
Copper 40.2 124.9 ment suggests that both Atlantic salmon and
Iron 34.5 63.0 channel catfish have similar ability to retain
Manganese 39.8 116.1
Se from chelated sources.
Selenium 44.6 27.5
Zinc 37.5 74.2 Several factors can impede intestinal ab-
Net absorption of chelated- sorption of inorganic nutrients. Phytic acid,
Net absorption of inorganic which complexes many inorganic nutrients
I Calculation: x 100 (Lo et al. 1981; Spinelli et al. 1983), appar-
Net absorption of inorganic
66 PARIPATANANONT AND LOVELL
ently is responsible to some degree for the
lower net retention of the minerals in the
practical diet. The soybean meal diet con-
tained 1.2% phytic acid as determined by
J. Eya (Auburn University, personal com-
munication) in this laboratory using the pro-
cedure described by Huag and Lantzsch
(1983); whereas, the egg white diet is as-
sumed to contain a negligible amount. Net
absorption of all minerals except Se was
higher in the egg white-based diet than in
the soybean meal-based diet. Gatlin and
Wilson (1984) using zinc sulfate also found
that dietary zinc requirement for channel
catfish increased from 20 m&g in a casein-
based purified diet to over 100 m@g in a
soybean meal-based practical diet. Interac-
tion with other minerals or nutrients can de-
crease intestinal absorption of inorganic nu-
trients. Excess dietary Zn has been shown
to decrease Cu absorption in chicks (South-
ern and Baker 1983) and swine (Hill et al.
1983); excess Cu impaired absorption of
iron in swine (Bradley et al. 1983); and ex-
cess Fe decreased absorption of manganese
in rats (Johnson and Korynta 1992). Excess
calcium, in high-ash diets, decreased avail-
ability of Zn and caused Zn deficiency in
rainbow trout (NRC 1993). Aoyagi and
Baker (1994) found that addition of ascor-
bic acid in the diet decreased Cu absorption
in chicks. When the mineral is bound in
chelated form, those interactions among
minerals and other compounds are pre-
vented. This partially explains the higher
net absorption rate of the chelated minerals
over nonchelated minerals, especially in the
practical diet. Another factor could be that
chelation facilitates transport of the element
across the intestinal wall (Ashmead 1992).
The improvement in net absorption of
minerals from organic sources over that of
minerals from inorganic sources was higher
in the practical diet than in the purified diet
for all minerals except Se. This is appar-
ently due to the higher concentration of
phytate and other ligands in the practical
diet which bind to the inorganic minerals.
The reason that chelation did not improve
net absorption of Se more in the practical
diet than in the purified diet is probably that
Se is not cationic as are the other minerals
and, thus, chelation does not provide as
much protection from interaction with the
various ligands in the practical diet.
Net absorption of mineral elements by an
animal can provide an estimate of their bio-
availability. However, it cannot explain all
of the differences in bioavailability between
chelated and inorganic trace minerals. Pari-
patananont and Love11 (1995) using weight
gain as a criterion found that bioavailability
of chelated zinc when compared with inor-
ganic zinc was 352% in a purified diet and
482% in a soybean meal diet. In the present
study the net absorption of Zn from the che-
lated source as compared to the inorganic
source was 138% in the purified diet and
174% in the practical diet. This suggests that
other factors besides net absorption are re-
sponsible for the higher bioavailability of
chelated elements compared to inorganic el-
ements. Ashmead (1992) proposes that che-
lation also enhances post-absorption trans-
port and metabolism of trace minerals.
Spears (1989) demonstrated that net absorp-
tion alone did not explain the superior zinc
retention in lambs fed zinc methionine com-
pared to lambs fed zinc oxide and assumed
that the two zinc sources were metabolized
differently after absorption.
This study indicates that chelated trace el-
ements are appreciably more absorbable
than inorganic sources, especially in practi-
cal type fish diets, and suggests that lower
amounts of the chelated forms may be used
as supplements in commercial feeds. Net
absorption alone apparently does not ac-
count for all of the difference in bioavail-
ability between organic and inorganic
sources of trace elements, thus, more direct
animal responses, such as weight gain,
should be used to maximize the accuracy
for comparisons of bioavailability between
chelated and inorganic minerals.
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